Bioactive Indole Alkaloids from Croton echioides

Bioguided fractionation of a hydroethanolic extract from the stem bark of Croton echioides Baill. led to isolation of the new indole alkaloid N-trans-feruloyl-3,5-dihydroxyindolin-2-one as a stereoisomeric mixture and the known alkaloids N-trans-p-coumaroyl-tryptamine, N-trans-pcoumaroyl-5-hydroxytryptamine, N-trans-4-methoxy-cinnamoyl-5-hydroxytryptamine, N-transferuloyl-5-hydroxytryptamine (moschamine), from the ethyl acetate fraction. The flavonoids 3-o-methyl kaempferol, 3-o-methyl quercetin, 3,7-di-o-methyl quercetin and 3,3’-di-o-methyl quercetin, together with the benzoic acid derivatives 4-hydroxybenzoic acid, 4-hydroxy-3methoxybenzoic acid and 4-hydroxy-3,5-dimethoxybenzoic acid were also isolated. Alkaloids and the flavonoids showed strong antioxidant properties in vitro radical scavenging assay (2,2-diphenyl1-picrylhydrazyl, DPPH), with IC50 values ranging from 9.2 to 17.5 μmol L, lower than those of the positive control Trolox (IC50 = 17.9 μmol L). Alkaloids showed cytotoxic activity against the HCT-116 human cancer cell line, with IC50 values ranging from 86.8 to 210.7 μmol L. Compound N-trans-4-methoxy-cinnamoyl-5-hydroxytryptamine was the most active, with an IC50 value of 86.8 μmol L.


Introduction
][3] This genus is a rich source of special metabolites, mainly clerodane diterpenes, flavonoids, and different classes of alkaloids. 4Alkaloids related to benzylisoquinolines, such as morphinandienones and tetrahydroprotoberberine alkaloids are the most frequent class found in members of the genus Croton. 5Glutarimide alkaloids and a new class of sesquiterpene guaiane-type alkaloids have recently been reported from Croton species. 6roton L. is the second-largest genus in the family Euphorbiaceae, with about 1,200 species, 350 of which occur in Brazil. 5Croton echioides Baill., popularly known as "quebra-faca", "caatinga branca", "velame" and "canela-de-velho", is a small native tree found in the North-eastern region of Brazil. 7,8The stem bark of this plant has been widely marketed as an aphrodisiac and tonic, as a substitute for the roots of Amazon Marapuama, Ptychopetalum olacoides Benth.(Olacaceae). 9To our knowledge, no chemical or biological study of this species has been reported.

HSCCC separations
The elution systems used in HSCCC separations were optimized by determination of the partition coefficient (Kp) of target compounds in a series of biphasic liquid systems by UV-Vis determinations.Sets of biphasic solvent systems composed of hexane, petroleum ether, ethyl acetate, methanol, ethanol and water was examined at different arrangements and volume ratios.The biphasic liquid system was prepared by adding portions of the solvents in a separatory funnel and, after the equilibrium was reached, 1 mL of each phase (upper and lower) were added in a test tube.After that, 1 mg of the solute was introduced.The test tube was shaken manually until equilibrium was reached.The system was equilibrated for at least 1 h and the concentration of the solute in the upper and the lower phase was analyzed by UV-Vis.The Kp was calculated as the ratio of the solute concentration in the upper and the lower phase.For the samples purification process, the most suitable solvent system (H 2 O/EtOH/EtOAc/hexane; 2.5:1:1.5:1.5 v/v) was prepared and thoroughly equilibrated in a separatory funnel at room temperature and the two phases were separated shortly before use.The sample solution was prepared by dissolving the crude sample in 10 mL of the stationary lower phase of the solvent system.The multilayer coiled column was first entirely filled with the lower phase.The upper mobile phase was then pumped into the inlet of the column on tail to head option at the flow rate of 1.0 mL min -1 , while the apparatus was run at 800 rpm.After hydrodynamic equilibrium was reached, a sample was injected.The eluent was continuously monitored by TLC and the fractions were collected according to the chromatographic profile.After target compounds were eluted, the HSCCC apparatus was stopped and the column contents were fractionated.

Plant material
T h e a e r i a l p a r t s o f f l ow e r i n g p l a n t s o f

Cytotoxic assays
The cytotoxicity activity against HCT-116 cells was determined by in vitro sulforhodamine B test (SRB-test). 18CT-116 cell lines, originated from a human colorectal carcinoma, were cultured and maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 50 µg mL -1 gentamycin, in incubator set at 37 °C, 5% CO 2 and 95% relative humidity.The cells were expanded when monolayer reached confluence after 3.After reaching 80% confluence, cells were digested by using 0.25% trypsin (Gibco) containing 1 mmol EDTA (ethylenediamine tetraacetic acid).The cells were seeded in 96-well tissue plates (TPP-Techno Plastic Products, Trasadingen, Switzerland) at a density of 8 × 10 5 cell mL -1 in 100 µL of medium for 24 h in the CO 2 incubator.The sample solutions with different concentrations (3.12, 6.25, 12.5, 25, 50 and 100 µg mL -1 ) were prepared and added to the culture media.After incubation for 48 h, the cell monolayers were washed with 100 µL phosphate buffered saline (PBS) sterile, fixed with trichloroacetic acid and stained for 30 min with SRB (7.16 mmol L -1 ) (Sigma Chemical Co., St. Louis, MO, USA) in acetic acid solution.The dye was removed by four washes with acetic acid solution (175 mmol L -1 ), and protein-bound dye was extracted with 10 mmol L -1 unbuffered tris base [tris(hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader (Power Wave XS, BIO-TEK, Winooski, VT, USA).The IC 50 values were determined from a percentage of cells relative to untreated control and the half-life of the HTC cells in the presence and absence of each tested sample was estimated as the time point at which the cell viability decreased to 50% in relation to the beginning of the test.All the tests were performed in triplicate.

DPPH assay
The free radical scavenging activity was determined by in vitro DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. 19ample stock solutions (1.0 mg mL -1 ) of analyte and Trolox (positive control) were diluted to final concentrations of 250, 125, 50, 25, 10 and 5 µmol L -1 , in methanol.1 mL of a 0.3 mmol L -1 DPPH solution in methanol was added to 2.5 mL of sample solutions of different concentrations, and allowed to react at room temperature.After 30 min the absorbance values were measured at 518 nm and converted into the percentage antioxidant activity. 1 mL of methanol DPPH solution 0.3 mmol L -1 plus methanol (2.5 mL) was used as a negative control.The IC 50 values were calculated by linear regression.All the tests were performed in triplicate.

Results and Discussion
The ethanol-aqueous extract of stem bark of C. echioides was successively partitioned into hexane, dichloromethane, ethyl acetate and butanol.The resulting fractions were subjected to free radical DPPH scavenging assay, which demonstrated that the ethyl acetate fraction was the most active, with IC 50 = 48.9± 0.2 µg mL -1 .In order to isolate bioactive compounds, this fraction was submitted to fractionation on a chromatographic column and the subfractions were also assayed in the DPPH.From that results, the most active subfractions were purified by chromatographic methods affording indolinone and N-substituted tryptamine alkaloid type compounds (1-5) which chemical structures are shown in Figure 1.In addition, were isolated flavonoids derived from kaempferol and quercetin (6-9), as well was benzoic acid derivatives (10-12).The structures of the isolated compounds were elucidated by analysis of their NMR spectroscopic data (including NMR 2D) and HR-ESIMS, and by comparison with those reported.
Compound 1 was isolated as a stereoisomeric mixture at C-3 position due to the absence of optical rotation deviation.The IR spectrum showed mainly two strong bands at 1711 and 1650 cm -1 corresponding to the carbonyl stretching vibrations of (γ) lactam and NHCO, respectively.A broad band was found at 3390 cm -1 due to N−H and OH stretching.The positive HR-ESIMS spectrum gave the ion peak at m/z 407.1220 [M + Na] + (calculated 407.1219), corresponding to the molecular formula C 20 H 20 N 2 O 6 .The NMR spectrum showed signals for oxindole and trans-feruloyl moieties which were confirmed by 1 H and 13 C NMR data assignments (Table 1).Specifically, from the 1 H NMR spectra three aromatic hydrogens at d 6.87 (d, J 2.1 Hz, H-4), 6.68 (dd, J 8. Compound 4 has previously been synthesized, but no complete NMR assignments were reported. 12Here, we report its isolation and characterization from a natural source. The alkaloids (1-5), flavonoids (6-9) and benzoic acid derivatives (10-12) were evaluated for their antioxidant activity by the free scavenging DPPH method.The results for compounds 1-5, expressed by IC 50 , are shown in Table 2. Compounds 3-5 and the flavonoids 7 and 8 showed prominent antioxidant properties, with IC 50 values ranging from 9.2 to 17.5 µmol L -1 , lower than those of the positive control Trolox (IC 50 = 17.9 µmol L -1 ).Comparison of the IC 50 value of compounds 2 (203.7 µmol L -1 ) and 3 (10.7 µmol L -1 ) showed that the hydroxy group at the C-5 of the tryptamine moiety is fundamental for the antioxidant activity of these indole alkaloids.
The cytotoxic activity of isolated indole alkaloids (1-5) was tested in vitro against the HCT-116 human cancer cell line, and the results are expressed in Table 2, with IC 50 values ranging from 86.8 to 210.7 µmol L -1 .Compound 4 was the most bioactive, with an IC 50 value of 86.8 µmol L -1 .According to the texted protocol, the alkaloid 1 was not bioactive (IC 50 > 250 µmol L -1 ), which suggests that the indole unit present in alkaloids (2-5) is important for the cytotoxic activity.To our knowledge, this is the first report of indolinone alkaloid from a species of Croton, which is an important contribution to chemotaxonomic knowledge of this genus.1][22] Also, anti-inflammatory activity (compounds 2 and 3), 23 growth-promoting activity for fibroblasts (compound 3) 24,25 and in vitro cytotoxic activity against the CaCo2 colon cancer cells (compound 5) 21 were previously reported.This is the first report on the cytotoxicity of compounds 1-5 against the HCT-116 human cancer cell line.

Conclusions
The present report is the first chemical study of C. echioides Baill., despite this plant species being widely marketed as a substitution to the traditional Amazon Marapuama, Ptychopetalum olacoides.Five indole alkaloids were isolated and characterized by NMR data.One of them (N-trans-feruloyl-3,5dihydroxyindolin-2-one) is a new natural product from Croton species.Their antioxidant and cytotoxicity activity were reported together with other phenolic compound types.These results represent a relevant contribution to the chemical and biological knowledge of this vegetal species and can support its correct and safe medicinal uses.

Table 1 .
13,13C NMR and HMBC data of the compound 1 in CD 3 OD