Antitumor , Antibiotic and Antileishmanial Properties of the Pyranonaphthoquinone Psychorubrin from Mitracarpus frigidus

The bioactivity guided fractionation of the dichloromethane extract of Mitracarpus frigidus afforded the pyranonaphthoquinone psychorubrin. This compound, hitherto unknown in the genus Mitracarpus, had its biological activity evaluated against one panel of bacteria and two fungi, three tumor cell lines (HL60, Jurkat and MCF-7) and four Leishmania species. Its identity was confirmed unambiguously by 1H, 13C, 1H-COSY, IR and UV-Vis spectroscopy and mass spectrometry. Psychorubrin displayed a very promising antitumor with IC50 of 4.5, 5.6 and 1.1 μM for HL60, Jurkat and MCF-7 cell lines, respectively. Antimicrobial activity, mainly against Cryptococcus neoformans (MIC of 87.3 μM) was observed. A pronounced antileishmanial potential was also verified with IC50 varying from 1.7 to 2.7 μM for the Leishmania species tested. This is the first report of the presence of pyranonapthoquinones in the Mitracarpus genus, which may serve as a chemotaxonomical marker.

Psychorubrin (1) (Figure 1) was first described in Psychotria rubra (Rubiaceae), a Chinese folk medicine which demonstrated activity against KB cells (Hayashi et al. 1987).Other authors erroneously attributed a different structure for psychorubrin to a napthoquinone isolated from P. camponutans which showed strong in vitro activity against brine shrimp, KB cells, and chloroquine-resistant P. falciparum (Jacobs et al. 2008).This compound was later shown to be psychorubrin by means of total synthesis.Three total syntheses of psychorubrin have also been reported (Kesteleyn et al. 1999, Bulbule et al. 2003, Nguyen and Kimpe 2004).
been subjected to phytochemical investigation, except a recently published paper (Fabri et al. 2009).In that research article, we describe the antitumor, antimicrobial and antileishmanial properties of crude extracts of M. frigidus.Upon the discovery of strong biological activity of the dichloromethane (CH 2 Cl 2 ) extract, the authors sought to isolate the compound or compounds responsible for this activity.

PLANT MATERIAL
Mitracarpus frigidus aerial parts were collected in Juiz de Fora, state of Minas Gerais, Brazil, in May, 2009.The plant was identified by Dra.Tatiana Konno from the Ecological and Socioenvironmental Core of Macaé/UFRJ.A voucher specimen (CESJ 46076) was deposited at the Leopoldo Krieger Herbarium at the Universidade Federal de Juiz de Fora.

EXTRACTION AND ISOLATION
Preparation of the crude CH 2 Cl 2 extract was previously described in detail (Fabri et al. 2009).Purification of this extract was performed in the following manner: the CH 2 Cl 2 extract (4.8 g) was chromatographed on a 74 x 4 cm column of silica gel (70-230 mesh) with a gradient of increasing polarity (CH 2 Cl 2 , CH 2 Cl 2 -EtOAc, EtOAc, EtOAc-MeOH, MeOH) to obtain a total of twenty fractions.The fractions obtained were analyzed by thin layer chromatography on silica gel 60 F 254 (Merck) using hexane: EtOAc, 60:40, v/v and hexane: EtOAc, 30:70 v/v as the mobile phase for fractions F 1 to F 10 and fractions F 11 to F 20 , respectively.Identification was made with a UV lamp (254 and 365 nm) and with vanillin: sulfuric acid followed by heating.The fractions were pooled and concentrated on a rotary film evaporator under reduced pressure.While preparing the fractions for HPLC analysis (vide infra), we proceeded to dissolve the samples in methanol.Upon addition of methanol to fraction To the best of our knowledge, Mitracarpus frigidus (Willd.ex Roem.& Schult.)K. Shum, an annual shrub commonly found in Brazil, has never 1083 BIOLOGICAL PROPERTIES OF PSYCHORUBRIN 7, an off-white solid precipitated.This was filtered and dried under vacuum to yield 100 mg (2.08% w/w) of the title compound (1).The sample was homogeneous by TLC with an Rf of 0.57 (hexane:EtOAc 7:3) and revealed as a brownishorange spot with vanillin-sulfuric acid.

STRUCTURAL ELUCIDATION
1 H-NMR and COSY (300 MHz) and 13 C-NMR (75 MHz) spectra were recorded on a Bruker DRX spectrometer using the residual solvent peak (CHCl 3 ) as reference.The IR spectrum was recorded as a KBr pellet on a Bomem B102 FT spectrophotometer.The UV spectrum was acquired in MeOH on a Shimadzu UV160 spectrophotometer.The EI mass spectrum was obtained on a Hewlett-Packard 5973 MSD spectrometer by direct insertion in the positive ion mode (70 eV).

HPLC ANALYSIS
During the course of our investigations of the crude extract, it was necessary to develop an HPLC method suitable for determining the purity of the various column fractions which were obtained.These analyses were performed on an Agilent Technologies 1200 Series, with a PDA detector and automatic injector.The column employed was a Zorbax SB-18; 250 x 4.6 mm, 5 μm particle size.A linear gradient of a binary solvent system, A:B, which varied from 0 to 100% B ran at a flow rate of 1 mL.min -1 over 30 minutes where A consists of acetonitrile: H 2 O, 5:95, pH adjusted to 4.0 with H 3 PO 4 and B consists of acetonitrile: H 2 O, 90:10, pH adjusted to 4.0 with H 3 PO 4 .The mobile phase was returned to its original composition over the course of 30 minutes, and additional 10 minutes were allowed for the column to re-equilibrate before injection of the next sample.The sample volume was 10 μL at a concentration of 1 mg.mL -1 and the temperature was maintained at 25°C during the analysis.Detection was performed simultaneously at 210, 230, 254 and 280 nm.

Human Tumor Cells
Three human tumor cell lines were used, Jurkat (human immortalized line of T lymphocyte), HL60 (human promyelocytic leukemia) and MCF-7 (breast cancer).HL60 and Jurkat cells were kindly furnished by Dr. Gustavo Amarante-Mendes (Universidade de São Paulo, Brazil).MCF-7 was generously provided by Alfredo Goes (Universidade Federal de Minas Gerais, Brazil).All lineages were maintained in the logarithmic phase of growth in RPMI 1640 supplemented with 100 IU.mL -1 penicillin and 100 μg.mL -1 streptomycin enriched with 2mM of L-glutamine and 10% of fetal bovine serum.All cultures were maintained at 37°C in a humidified incubator with 5% CO 2 and 95% air.The media were changed twice weekly and they were regularly examined.All cell lines were used between 16 and 30 passages.

Antitumor and Antileishmanial Assays
Cell viability was estimated by measuring the rate of mitochondrial reduction of tetrazoliumdye (MTT).MTT is a standard colorimetric assay, in which mitochondrial activity was measured by splitting tetrazolium salts with mitochondrial dehydrogenases in viable cells only (Mosmann 1983).To evaluate the antitumor activity, the tumor cell lines (Jurkat, HL60 and MCF-7) were inoculated at 50,000 cells per well.The plates were pre-incubated for 24 h at 37°C to allow cells adaptation prior to the addition of the test compounds.The half maximal inhibitory concentration (IC 50 ) was determined over a range of concentrations of a freshly prepared solution of (1) (100.0;50.0; 25.0; 12.5; 6.25; 3.12; 1.56; 0.78; 0.39 µM) tested for 48 h in an atmosphere of 5% CO 2 and 100% relative humidity.Control groups included treatment with 0.1% DMSO (negative control) and etoposide 14 µM (positive control).All compounds were tested in triplicate, in three independent experiments (Monks et al. 1991).The antileishmanial activity was performed according to a previously described method (M'Bongo et al. 1997, Braga et al. 2007).Briefly, promastigotes from a logarithmic phase culture were suspended to yield 2 million cells.mL - (L.amazonensis) or 3 million cells.mL - (L.chagasi, L. braziliensis and L. major) after Neubauer chamber counting.The test was performed in 96-well microtiter plates maintained at 24°C.The assays were performed in triplicate.The parasites were exposed to increasing concentrations of (1) (100.0;50.0; 25.0; 12.5; 6.25; 3.12; 1.56; 0.78; 0.39 µM) for 72 h at 24°C, for the determination of IC50 values.Controls with DMSO were also performed.Amphotericin B was used as the reference drug.
For both assays, after 4 h of the end of incubation of cells with different compounds, 20 μL of MTT solution (5 mg.mL -1 in phosphatebuffered saline) were added to each well, the supernatant was removed and 200 μL of 0.04M HCl in isopropyl alcohol were added to dissolve the formazan crystal.The optical densities (ODs) were evaluated in a spectrophotometer at 590 nm (antitumor assay) and 570 nm (antileishmanial assay).Controls included drug-containing medium (background) and drug-free complete medium.Drug-free complete medium was used as control (blank) and was treated in the same way as the drug-containing media.Interactions between compounds and media were estimated on the basis of the variations between drug-containing medium and drug-free medium to avoid false-positive or false-negative results.

DNA Fragmentation Assay
Cell cycle status and quantification of DNA fragmentation (hypodiploid DNA-content) were performed by propidium iodide (PI) staining according to Nicoletti et al. (1991) Cells were treated with psychorubrin (1) 43 µM in a 5% CO 2 /95% air-humidified atmosphere at 37°C for 24 h.After drug incubation, the cells were centrifuged and resuspended in hypotonic fluorochrome solution -HFS (50 µg.mL -1 PI in 0.1% sodium citrate plus 0.1% Triton X-100).The samples in HFS were incubated at 4°C during 4 h and immediately analyzed by flow cytometry.The PI fluorescence of 20,000 individual nuclei was measured using a FACScalibur flow cytometer (Becton Dickinson Immunocytometry Systems).The DNA content of cell lines was analyzed by FlowJo software (TreeStar Inc).

Serial Dilution Assay for Determination of the Minimal Inhibitory Concentration (MIC)
The minimal inhibitory concentration (MIC) of each extract was determined by using broth microdilution techniques as described previously for bacteria and yeasts, respectively (Perez et al. 1990, NCCLS 2002).MIC values were determined in RPMI 1640 buffered to a pH 7.0 with MOPS for yeasts and Mueller Hinton broth (MHB) for bacteria.Yeasts were cultured at 30°C for 48 h in SDA and bacteria were cultured overnight at 37°C for 24 h in MHA.Stock solution of (1) was diluted two-fold from 2170 to 10.9 μM (final volume = 80 μL) and a final DMSO concentration ≤ 1%.Then, 100 μL of RPMI or MHB were added onto microplates.Finally, 20 μL of 10 6 CFU.mL -1 (according to McFarland turbidity standards) of standardized yeasts and bacterial suspensions were inoculated onto microplates and the test was performed in a volume of 200 μL.Plates were incubated at 30°C for 48 h for yeasts and 37°C for 24 h for bacteria.The same tests were performed simultaneously for growth control (RPMI + yeast and MHB + bacteria) and sterility control (RPMI or MHB + psychorubrin).The MIC values were calculated as the highest dilution showing complete inhibition of tested strain.Chloramphenicol and amphotericin B were used as standard antimicrobial agents for bacteria and yeast, respectively.

STATISTICAL ANALYSIS
The IC 50 values for antitumor and antileishmanial activities were determined using Prism 5.0 (GraphPad Software Inc.).Data were presented as median and 95% confidence interval.Statistical differences between the treatments and the control were evaluated by ANOVA test.To express the DNA content, each data point represented mean ± SD from at least two independent experiments performed in duplicate.Statistical differences between the treatments and the control were evaluated by ANOVA test followed by Bonferroni (P > 0.05).

RESULTS AND DISCUSSION
Under the conditions used in the HPLC method, psychorubrin eluted as a narrow peak at 12.6 minutes and had a chromatographic purity of 98%.
Some of these presented antimicrobial, antiparasitic and anticancer properties (Lee et al. 1996, Wang et al. 1998, Krishnan and Bastow 2000).Psychorubrin (1) were also isolated from Psychotria rubra and P. camponutans that also belong to the Rubiaceae family (Hayashi et al. 1987).The fact that it was not reported outside this genus is an indication of its chemotaxonomical importance.
A literature search revealed few reports regarding to its biological activity although significant cytotoxicity against KB tumor cells has been previously reported (Hayashi et al. 1987).
In our study, we demonstrated (Table I) that this compound is toxic against some tumor cells, including leukemia (HL60 and Jurkat), and solid tumors such as breast cancer (MCF-7).The antitumor activity observed in this study is supported by previous scientific reports on biological activities of other pyranonaphthoquinone derivatives as eleutherin, β-lapachone, and its structural isomer, α-lapachone.The mechanism of topoisomerase II inhibition by those three derivatives was examined systematically with respect to the steps of the catalytic cycle of the enzyme.Etoposide, a topoisomerase inhibitor, was used as a control (Krishnan and Bastow 2000).
We investigated if the cytotoxic effects of psychorubrin (1) observed in our experiments were associated with its potential to induce cell death by apoptosis.We used the method described by Nicoletti et al. (1991) which is based on the principle that the apoptotic cells, among other typical features, are characterized by DNA fragmentation and, consequently, loss of nuclear DNA content.Apoptotic nuclei appeared as a broad hypodiploid DNA peak which was easily discriminable from the narrow peak of cells with normal (diploid) DNA content.This simple and reproducible method should prove useful for assessing apoptosis of specific cell populations in heterogeneous tissues.Our results (Figure 2) clearly demonstrated that psychorubrin induced an increase of subdiploid DNA content (DNA fragmentation) in HL60 and MCF-7 cell when compared with their respective control (DMSO 0.05%).This effect was cell type dependent.To the resistant lineage MCF-7, psychorubrin (1) (43 µM) demonstrated a proapoptotic potential better than etoposide (14 µM), the clinical drug used as positive control in our experiments, possessing pro-apoptotic activities against several cancers.
In vitro cytotoxicity assays can be used to predict human toxicity and for the general screening of chemicals.Different cytotoxicity assays can give different results depending on the test agent used and the cytotoxicity assay employed (Fotakis and Timbrell 2006).In this context, the DNA content analysis revealed a different toxicity profile for psychorubrin compared with the MTT method.This effect can be attributed to the fact that MTT evaluates the energetic function of mitochondria that is preserved during the course of apoptosis.When the damage level to mitochondria is mild or moderate, mitochondria can still generate sufficient energy for initiation and execution of the apoptotic process
Data are presented as median and 95% confidence interval (in parentheses).b,c Positive controls.BIOLOGICAL PROPERTIES OF PSYCHORUBRIN (Yang et al. 2010).The increase of subdiploid DNA content (DNA fragmentation) observed in HL60 and MCF-7 cells after psychorubrin treatment suggested that this compound activates the apoptosis pathway.Many chemotherapeutic strategies interfere with important steps in cell cycle progression such as DNA replication and repair.They act as DNA polymerase inhibitors, topoisomerase inhibitors, or DNA alkylating agents to impede cancer progression and induction of apoptosis as a secondary effect (Li et al. 2006).Apoptosis is accompanied by the shrinkage and fragmentation of both cells and their nuclei, loss of microvilli, and extensive degradation of chromosomal DNA.Degradation of nuclear DNA into nucleosomal units is one of the hallmarks of apoptotic cell death.Molecular characterization of this process demonstrated that the chromosomal DNA cleavage is caspase-dependent, in particular caspase 3 cleaves ICAD to dissociate the CAD:ICAD complex, allowing CAD to cleave chromosomal DNA (Nagata 2000).Despite the cell mechanism involved on DNA fragmentation induced by psychorubrin in HL60 and MCF-7 cells were not described in the present work, our results suggest that this compound has a potential antitumoral activity, inducing the apoptotic pathway.
Psychorubrin (1) also presented an interesting antileishmanial activity with IC 50 values above 3 μM against promastigote forms of different Leishmania species (Table I).Leishmaniasis is a debilitating disease caused by protozoan parasites of the genus Leishmania, which affects an estimated 12 million people worldwide.The discovery of new lead compounds for leishmaniasis is therefore a pressing concern for global health programs (Sanchez et al. 2010).
We also demonstrated that psychorubrin (1) displayed a broader spectrum of antimicrobial activity, most particularly against C. neoformans (MIC of 10 μM), (Table II), an opportunistic yeast and etiological agent of cryptococcosis.Cryptococcosis is an opportunistic systemic mycosis that involves the central nervous system, especially in immunocompromised patients (Perfect and Casadevall 2002).Cryptococcosis is currently treated with amphotericin B/azoles as the main therapeutic choice (Van der Horst et al. 1997).Nevertheless, the high lethality rate among immunocompromised patients indicates that new therapeutic options are necessary.

Figure 2 -
Figure 2 -Effect of psychorubrin (1) on DNA content of tumor cell lines.HL-60, Jurkat e MCF-7 cells were incubated with 43 µM psychorubrin or with 0.05% DMSO for 24 h.DNA content was assayed by PI stain and flow cytometry analysis.Etoposide (14µM), positive control is demonstrated.Each data point represents mean ± SD from at least two independent experiments performed in duplicate.a Statistically different