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Mikania laevigata: chemical characterization and selective cytotoxic activity of extracts on tumor cell lines.

ARTICLE INFO

Keywords:

Asteraceae

Mikania laevigata

Hep-2

HeLa

MRC-5

Cytotoxic activity

Chemical composition

ABSTRACT

Cancer is the second major cause of mortality worldwide, losing only to cardiovascular disease. Nowadays, around 50% of antineoplastic drugs were discovered and isolated by indications of plants in folk medicine. In Brazilian flora there are many species of plants which have great therapeutic importance, highlighting the Mikania laevigata (Asteraceae) that has been used for their valuable properties, especially in the respiratory tract. In the present study, the compounds of M. laevigata extracts were characterized by High Resolution Mass Spectrometry (HRMS) and Gas Chromatography with Mass analysis (GC/MS-EI). Therefore, the presence of some compounds with promising biological properties as antitumor activity was detected. Coumarin (1,2-benzopyrone) was previously reported as responsible for some biological activities of this plant species. Here, the extracts were evaluated by their cytotoxic activity against tumor (Hep-2, HeLa) and non tumor (MRC-5) cell lines, presenting significant inhibitory activity of cell growth in all extracts analyzed, chloroform, ethyl acetate, hexane, ethanol, which is related to its chemical composition. From the four different extracts here tested, two of them, hexane and ethanol, presented a clear selectivity against both tumor cells lines investigated. This can be explained by variances and increase of phenolic compounds in the ethanol fraction and an association of molecules with coumarin found in the hexane fraction.

[c] 2013 Elsevier GmbH. All rights reserved.

Introduction

The great diversity of vegetal species with therapeutic potential in Brazilian ecosystems provides material for specialized studies to research new drugs against different diseases. Compounds isolated or derivatives from plants have contributed to treatment of different neoplasias. A notorious example concerns to vinblastine and vincristine alkaloids, which were isolated from Catharanthus roseus and used in medical practice as antineoplastic agents (Cragg and Newman 2005).

The use of plants compounds as prototypes of new drugs has a historical and economic importance. Some plants extracts were defined as effective in treating cancer, whose action is attributed to additional or synergistic effect of compounds present in the extract (Li et al. 2000). In consequence, the cytostatic effect observed in tumor cells seems to be more effective than the effect of isolated and biologically active compounds (Vickers 2002).

The Mikania genus, Asteraceae family, has a successful therapeutic importance (Rufatto et al. 2012). Mikania laevigata is a subarbustive plant from South America localized in Southeast and South Brazil, from Sao Paulo to Rio Grande do Sul. It has been initially used for the treatment of respiratory disorders including asthma, bronchitis, chronic lungs diseases and for cough (Santos et al. 2006; Bolina et al. 2009). Its antiulcer, anti-inflammatory, analgesic, antispasmodic and antimicrobial (Bighetti et al. 2005; Suyenaga et al. 2002; Yatsuda et al. 2005) activities also have been investigated, but so far no reports suggested selectivity of extracts against tumor cell lines.

The pharmacological effects of Mikania genus are attributed mainly to the presence of coumarin (1,2-benzopyrone) and derivatives. However, other metabolites showed to produce significant pharmacological effects. Recent studies report the presence of coumarin. triterpenes/steroids, flavonoid glycosides, dihydro-coumarin, o-coumaric acid, kaurenoic acid, cinnamoylgrandifloric acid, stigmasterol, cupressenic acid, isopropiloxi-granclifloric acid, isobutiloxi-grandifloric acid, kaurenol, spathulenol, caryophyllene oxide, syringaldehyde, saponins, tannins (Bolina et al. 2009; Bighetti et al. 2005; Yatsuda et al. 2005; Santos et al. 2006). Ferreira and Oliveira (2010) demonstrated new constituents, which were isolated from the leaves of M. laevigata: taraxerol, lupeol, trans-melilotoside, cis-melilotoside, adenosine, patuletin 3-0-[beta]-D-glucopyranosicle, kaempferol 3-0-[beta]-D-glucopyranoside, quercetin 3-0-[beta]-D-glucopyranoside, methyl-3,5-di-O-caffeoyl quinate and 3,3',5-trihydroxy-4',6,7-trimethoxyflavone.

Few studies have reported cytotoxic activity of isolated compounds or total extracts obtained from the Mikania genus. This study aims to characterize the chemical composition and evaluate the cytotoxic activity in a selective manner of different extracts obtained from leaves of Mikania laevigata against the tumor cell lines Hep-2 (human laryngeal epidermoid carcinoma cells) and HeLa (human cervical adenocarcinoma), and non tumor MRC-5 (human lung fibroblast) cell line.

Materials and methods

Chemicals

All the reagents were of ultrapure grade. Water was treated in a Milli-Q water purification system (TGI Pure Water Systems, USA). Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were acquired from Hyclone Lab Inc. (USA). Folin-Ciocalteu's phenol reagent (Merck) was used for the determination of total phenols.

Plant material

The leaves of Mikania laevigata Sch. Bip. ex Baker were collected in October 2011, in Garibaldi city (29 13'32.28" Sand 51 32'11.13" W) located in Rio Grande do Sul state, southern Brazil. The species was identified based on the literature and its voucher specimen deposited in the Herbarium of the University of Caxias do Sul (HUCS 38180).

Mikania extracts

The extracts were obtained by maceration under sonication of the crushed fresh leaves (50g), at room temperature, for 20 min, using the following solvents: hexane, chloroform, ethyl acetate, ethanol and ethanol/water (1:1, v/v). After extraction, the mixture was filtered and the solvent evaporated. The five dried extracts obtained were dissolved in 50% ethanol.

Chemical composition of the extracts

For chemical analysis by High Resolution Mass Spectrometry (HRMS), the extracts of M. laevigata were diluted in specific solutions according with the analysis mode (positive or negative). In positive mode, 0.1 ml of each extract was diluted in 1 ml of a solution of chromatographic grade acetonitrile/deionized water (1:1, v/v) and 0.1% formic acid. In the negative mode, 0.1 ml of each extract was diluted into 1 ml of a solution of chromatographic grade acetonitrile/deionized water (1:1, v/v) and 0.1% ammonium hydroxide. The solutions were infused directly into the ESI source by means of a syringe pump (Harvard Apparatus) at a flow rate of 10 [micro]l [min.sup.-1]. ESI(+)-MS, ESI(-)-MS and tandem ESI-MS/MS were acquired using a hybrid high-resolution and high accuracy (5 WM Orbitrap mass spectrometer (Thermo Fisher Scientific) with the conditions: capillary and cone voltages were set to +3500V and +40 V. respectively, with a de-solvation temperature of 100 C. For MS/MS, the energy for the collision induced dissociations (CID) was optimized for each component. Diagnostic ions in the Mikania extracts were identified by the comparison of their MS/MS dissociation patterns with compounds identified in previous studies. For data acquisition and processing, Xcalibur software (Thermo Fisher Scientific) was used. The data were collected in the m/z range of 50-700 at the speed of two scans per second, providing the resolution of 50,000 (FWHM) at m/z 200. No important ions were observed below m/z 50 or above m/z 450, therefore ESI(+)-MS and ESI(-)-MS data is shown in the m/z 50-450 range.

In addition, extracts of M. laevigata were evaluated qualitatively and quantitatively by Gas Chromatograph coupled with Mass Spectrometer with ionization by Electron Impact (GC/MS-EI) in Hewlett Packard 6890/MSD 5973, equipped with HP-Chemstation software and Wiley 275 library spectra. Analyses were performed on a column HP-5MS 5% phenylmethylsiloxane (30 m x 250 [micro]m), 0.25 [micro]m thick film (Hewlett Packard, California, USA) with the following temperature program: 40 [degrees]C (4 min), 310 [degrees]C at 8 [degrees]C/min (35 min), 280 [degrees]C interface, splitless, carrier gas He (43 cm/s), acquisition mass range 45-550 and 1 [micro]l injection.

Phenolic compounds

The quantitation of phenolic compounds was performed by the Folin-Ciocalteu colorimetric method, which involves the reduction of the reagent by the phenolic compounds of the samples with concomitant formation of a blue complex. The total phenolics content of each extract was quantified using a standard curve prepared with gallic acid (5-120 [micro]g/ml: Chem. Service, Inc., USA) and expressed as [micro]g gallic acid equivalents (GAE)/mL of extract.

Cytotoxic assay

Tumor (Hep-2, HeLa) and non tumor (MRC-5) cell lines were cultured in DMEM supplemented with antibiotics and 10% Fetal Bovine Serum (FBS) at 5% C[O.sub.2] and 37 [degrees]C. For the assessment of the cytotoxic activities of hexane, chloroform, ethyl acetate, ethanol and ethanol/water Mikania extracts, cells were seeded in 96-well flat-bottomed microplates at a density of 7 x [10.sup.4] cells/m1 in 10% FBS DMEM. After cell attachment, serial dilutions (10-800 [micro]g/m1) of the extracts in culture medium were added and incubated for 1 h, subsequently incubated without extract for 24 h. Cell proliferation was determined by the Tetrazolium salt method (MIT) (Denizot and Lang 1986). At the end of the incubation period, following incubation with MTT solution for 2 h, the media was removed, the formazan crystals were solubilized in 100 [micro]l dimethyl sulfoxide (DMSO)/well and the absorbance values were determined at 540 nm. At least three independent experiments were taken for each experimental cell line and [IC.sub.50] (dose causing 50% cell death) calculated using mean and standard deviation.

Statistical analysis

Results were expressed as mean [+ or -] standard deviation obtained from three independent experiments. Statistical significance was evaluated using analysis of variance ANOVA and Tukey test. P-values less than 0.05 were considered significant by the SPSS 20.0 program.

Results and discussion

The genus Mikania is among the list of best-selling natural products in the world because its biological activity. Different classes of compounds were previously isolated from Mikania, which can be associated to the pharmacological potential observed and related to the plant. Different compounds were search in extracts using a variation of solvent polarity (hexane, chloroform, ethyl acetate, ethanol and ethanol/water (1:1)). We selected High Resolution Mass Spectrometry (HRMS) and Gas Chromatography with Mass (GC/MS-EI) to evaluate the complex constituents. Some differences between the techniques are: HRMS detects volatile and nonvolatile compounds, confirms the chemical structure by its molecular ion and tandem MS/MS; the GC/MS-EI is able to analyze only volatile compounds giving fragmentation pathways dates. They are complementary techniques.The literature reports different information about compounds identification using GC/MS-EI and HRMS.

The HRMS is new and has increased in importance to characterize and identify metabolites. This technique has been successfully used for the analysis of natural products (Vessecchi et al. 2011; Yang et al. 2007). Since the widely accepted accuracy threshold for confirmation of elemental compositions was established as 5 ppm (Lacorte and Fernandez-Alba 2006), which usually provides highly reliable identification of the compounds. On the other hand, the GC/MS-EI has broad application and sensibility, which has been used to identify hundreds of components present in natural and biological systems (e.g. water pollutants, drug metabolites, secondary metabolites in vegetables, etc).

The analysis of extracts of M. laevigata by HRMS identified a series of compounds with important biological activities (Table 1). Fig. 1 shows the chemical structures of compounds identified by HRMS.

Table 1

Chemical compounds identified in M. laevigata extracts by high
resolution mass spectrometry, in positive and negative mode.

Entry  Precursor ion m/z  Extract (%)        Identification

1               104.1066  D(10O); E(55)      4-Amino-l-pentanol

2               137.1317  C(20); D(8);       [beta]-pinene
                          E(14)

3               147.0436  A(100l; B(58);     Coumarin
                          C(7); D(31);
                          E(35)

4               149.0625  E(40)              Dihydrocoumarin

5               193.0509  C(2)               Scopoletin

6               203.1810  D(17); E(2)        Curcumene

7               163.0396  D(20); E(43)       o-Coumaric acid

8               185.0245  C(20)              Psoralen

9               191.0353  E(61)              Scopoletin

10              301.2172  A(60); C(4)        Kaurenoic acid

11              325.1815  A(7); B(6); C(5);  o-Ceranylscopoletin
                          D(6); E(16)

12              447.2525  A(31); B(8)        Cmnanioylgrandiflonc acitl

Entry  Elem. corn p.                  Diff. ppm

1      [C.sub.5][H.sub.13]ON              0.474

2      [C.sub.10][H.sub.16]              -4.709

3      [C.sub.9][H.sub.6][O.sub.2]       -2.421

4      [C.sub.9][H.sub.8][O.sub.2]        1.801

5      [C.sub.10][H.sub.8][O.sub.4]       4.228

6      [C.sub.15][H.sub.22]               5.041

7      [C.sub.9][H.sub.8][O.sub.3]        0.496

8      [C.sub.11][H.sub.6][O.sub.3]       3.410

9      [C.sub.10][H.sub.8][O.sub.4].      2.466

10     [C.sub.20][H.sub.30][O.sub.2]      1.477

11     [C.sub.21][H.sub.26][O.sub.3]      3.476

12     [C.sub.29][H.sub.36][O.sub.4]      2.313

Entry  Fragmentations pathways

1

2      109.1009 [M-[C.sub.2][H.sub.3]] *;

       95.0854 [M-[C.sub.3][H.sub.5]] *;

       81.0697 [C.sub.4][H.sub.7]] *

3      103.0541 [M-C[O.sub.2]] *

4      121.0285 [M-C[O.sub.2]] *

5

6

7      119.0500 [M-COOH] *

8

9      172.0454 [M-[H.sub.2]]O *

10

11

12     299.1997 [M-ph[C.sub.2][H.sub.2]COO] *;

       147.0444 [[C.sub.11][H.sub.15]] *

Entry  Ref.

1      Caiqin et al. (2007)

2      Alves et al. (1995)

3      Vidalet al. (2006)

4      Vidal et al. (2006)

5      Hens and Kulanthaivel (1985)

6      Chowdhury et al. (2007)

7      Vidal et al. (2006)

8      Wang et al. (2011)

9      Herz and Kulanthaivel (1985)

10     Alves et al. (1995)

11     Herz and Kulanthaivel (1985)

12     Vichnewski et al. (1977)


The full mass spectrum of hexane extract in positive mode (A), which presents as the main constituent the Coumarin with m/z 147.0436: and negative mode (B), with kaurenoic acid (m/z 301.2172) and cinnamoylgranclifloric acid (m/z 447.2525), were shows in Fig. 2. It is possible to see the MS/MS of Coumarin, with carbon dioxide loss (m/z 103.0541). The mass measurement accuracy is also easily obtained for all the characteristic fragment ions, thus providing important information for unequivocal identification, being able to differentiate also isobaric interferences. However, the structural elucidation of some compounds by its FIRMS spectrum is not trivial and the deduction needs more studies for confirmation.

In GC/MS-EI analysis, it was also possible to characterize some volatile compounds which are summarized in Table 2.

Table 2

Chemical compounds identified in M. laevigata extracts
by gas chromatograph with mass detector (GC/MS-EI).

Extracts     R.T.  Compounds                         Area
            (min)                                     (%)

Hexane      21.30  2H-1-benzopyran-2-one(coumarin)   7.46

Chloroform  10.43  [alpha]-Pinene                   21.71

            12.92  1.8-Cineol                        3.72

            21.29  Benzofuran (coumarone)             729

Extracts    Molecular formula            Ref.

Hexane      [C.sub.9][H.sub.6][O.sub.2]  Vidal et
                                         al.(200ti)

Chloroform  [C.sub10][H.sub.16]          Rehder et al.
                                         (2006). Duarte
                                         et al. (2005)

            [C.sub.10][H.sub.18]O

            [C.sub.8][H.sub.6]O


Therefore, all compounds identified in extracts of M. laevigata by both techniques are important because many have proven to induce antitumor activity and may also act synergistically. Coumarin was identified in all extracts. Studies in various tumor cell lines have pointed to coumarin and its derivatives as potential substances for cancer treatment (Egan et al. 1997; Fatima et al. 2005; Lin et al. 1996; Weber et al. 1998; Lacy and O'kennedy 2004). Other compounds (Fig. 1) with antitumor activity were identified in the extracts analyzed: curcumene, psoralen, kaurenoic acid, scopoletin, o-coumaric acid, [beta]-pinene (Mazza and Oomah 2000; Wang et al. 2011; Costa-Lotufo et al. 2002; Cassady et al. 1979; Szliszka et al. 2009; Silva et al. 2007).

The phenolic compounds are substances that offer protection against oxidative damage (Duthie et al. 1997). They are related to several activities, including antioxidant effect (Coimbra et al. 2006), protective effect against DNA damage (Morley et al. 2005) and apoptosis induction in transformed cells or tumors (Chen et al. 2003). In this context, studies with medicinal plants which assess these characteristics are important. Until now, no report in literature showed presence of phenolic content in M. laevigata. In this study, the extracts presented different amounts of total phenolic compounds (Table 3). Here, polar solvents showed a high phenolic content that should be a factor to consider in the cytotoxic activity here observed.

Table 3

Total phenolics determined by Folin-Ciocalteu method in M.
laevigata extracts.

Mikania extracts  Total phenolics ([micro]g GAE/ml) (a)

Chloroform                     81.666 [+ or -] 1.25 (b)
Hexane                         85.291 [+ or -] 2.95 (b)
Ethyl acetate                 101.125 [+ or -] 3.33 (a)
Ethanol                       105.041 [+ or -] 0.81 (a)
Ethanol/water                 106.250 [+ or -] 1.02 (a)

(a) Results presented as mean [+ or -] SD. Different letters
indicate statistical difference by analysis of variance ANOVA
and post-hoc Tukey (p < 0.05).


The effects on the proliferation of tumor and non tumor cells after extract treatment varied not just according to type of extract used, but also according to extract concentration, as can be seen in the Fig. 3. Extracts as hexane and ethanol showed clear selective cytotoxic activity to tumor cells, while chloroform and ethyl acetate extracts presented no statistical differences between tumor and non tumor cell lines. In addition, the results showed that the extracts of Mikania were cytotoxic in all cell lines tested, presenting different [IC.sub.50] (dose causing 50% cell death), as shown in Table 4.

Table 4

Cell viability IC50 of Mikania extracts on the proliferation
of tumor (Hep-2. HeLa) and non tumor (MRC-5) cells.

Cell viability [IC.sub.50] *

Mikania             Chloroform         Ethyl       Hexane       Ethanol
                    ([mu]g/ml)       acetate   ([mu]g/ml)    ([mu]g/ml)
                                  ([mu]g/ml)

         Hep-2     80 [+ or -]   50 [+ or -]  75 [+ or -]  600 [+ or -]
                       0.1 (a)        0.1(a)      0.4 (a)        0.1(b)

         HeLa      40 [+ or -]  300 [+ or -]    160 [+ or  800 [+ or -]
                       0.5 (a)       0.2 (b)    -]0.1 (b,       0.7 (c)
                                                       c)

         MRC-5 #  101.62 [+ or    92.5 [+ or         >500          >800
                     -]3.6 (a)     -]2.1 (a)

Results represent the average of 3 independent experiments
performed in triplicate. Same line with different superscript
lower-case letter are significantly different at p < 0.05 by
analysis of variance ANOVA and pos-hoc Tukey. Results presented
 as mean [+ or -] SD.

# Non-tumor cells.

* Cell viability was assessed by (MIT) assay and dose-response
curves calculated the [IC.sub.50] values (dose causing 50% cell
survival) for each extract and treatment.


Fig. 3 highlights a decrease in the cell viability according with extracts concentration. The [IC.sub.50] values in Table 4 clearly indicate that the chloroform extract showed higher cytotoxicity (p < 0.05) against the HeLa cell line. However, ethyl acetate and hexane extracts were more cytotoxic (p < 0.05) in the Hep-2 line.

In comparison, reports present cytotoxic activity of isolated pure substances like saponins in HeLa cells (Herrmann and Wink, 2011) with [IC.sub.50] of 41.3 [micro]g/ml values. This same study revealed that a combinations of isolated saponins with thymol and menthol presented [IC.sub.50] values in HeLa models between 4.9 [micro]g/ml and 35.7 [micro]g/ml. Cao et al. (2010) investigated the cytotoxic activity of a novel isolated substance, a polysaccharide APS-1d, and showed that the growth of HeLa cells were inhibited in a time-and concentration-dependent manner and exhibited significant inhibition at concentrations range of 30-300 [micro]g/ml. Examples of cytotoxic activity using crude extracts in Hep-2 cells can reveal even higher [IC.sub.50] levels as mentioned above. Crude extracts of different Euphorbia species showed a variance in [IC.sub.50] of 85.39-853.91 [micro]g/ml, decreasing cellular viability in Hep-2 cells (Whelan and Ryan 2003). Recently, our group reported that red propolis crude extracts showed selectivity against tumor cells and inhibitory growth concentration levels above the NCI values, presenting cytotoxic activity at [IC.sub.50] 128.12 [micro]g/m1 for Hep-2 and 85.77 [micro]g/ml for HeLa (Frozza et al. 2013).

In this way, our results are significant when it is considered that cervix (cervical) cancer is the second most frequent tumor in the female population and the fourth cause of death by cancer in Brazil. The infection by human papillomavirus (HPV) can lead to this type of cancer and the changes in the cells are easily discovered on Pap smear. No less important, the laryngeal cancer, the most common among those who reach the head and neck, occurs predominantly in men and represents about 25% of malignant tumors that affect this area of the body.

In conclusion, these results suggest that the extracts from M. laevigata have important antitumor activity against the cells tested since they were able to inhibit their proliferation. A clear selectivity was observed for tumor cell lines in the hexane and ethanol extracts of M. laevigata. This can be explained by variances and increase of phenolic compounds in the ethanol fraction and an association of molecules with coumarin found in the hexane fraction. Further studies in future including other tumor cell lines should better elucidate variances in selectivity here observed.

Acknowledgments

The authors thank the FAPERGS, FINE, CAPES and CNPq for financial support.

* Corresponding author at: Technology Department, Biothecnology Institute. University of Caxias do Sul. R. Francisco Getulio Vargas 1130, 95070-560 Caxias do Sul. Brazil. Tel.: +5554 3218 2149x2668.

E-mail addresses: sidnei.moura@ucs.br. siclmoura@gmail.com (S. Moura).

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L.C. Rufatto (a), T.C. Finimundy (b), M. Roesch-Ely (b), S. Moura (a), (*)

(a) Laboratory of Biotechnology of Natural and Synthetics Products, Technology Department, Biotechnology Institute, University of Caxias do Sul, Brazil

(b) Laboratory of Genomics. Proteomics and DNA Repair. Biotechnology Institute. University of Caxias do Sul, Brazil
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Author:Rufatto, L.C.; Finimundy, T.C.; Roesch-Ely, M.; Moura, S.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3BRAZ
Date:Jul 15, 2013
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