Antitumor screening of crude extracts of ten medicinal plants of Polygala genus from Southern Brazil

Article history: Received on: 19/09/2016 Accepted on: 10/05/2017 Available online: 30/10/2017 In this study crude extracts from 10 species among of the 39 Polygala L. genus found in the South region of Brazil, were subjected to screening for antitumor activity against the tumor cell lines, HL-60 (myeloid leukemia), Jurkat (lymphoblastic leukemia), THP-1 (monocytic leukemia), MCF-7 and MDA-MB-231 (breast carcinoma), HCT-116 (colorectal carcinoma) and Vero (kidney of green monkey, used as a non-tumor cell model). The evaluation was carried out using the MTT assay. Only the extracts of Polygala densiracemosa, Polygala campestris and Polygala molluginifolia inhibited the tumor cell viability by more than 50%. The best results were observed for P. molluginifolia, with inhibition of the cell viability of HL-60 (83%), Jurkat (78%), MDA-MB-231 (75%), MCF-7 (70%) and HCT-116 (64%), however this species also inhibited the viability of the Vero cells (32%). The P. campestris species showed selectivity for Jurkat (66%) and HL-60 (78%) cells whilst P. densiracemosa showed selectivity only for HL-60 cells (66%). In addition, the toxicity toward nontumor cells was low (11% for P. campestris and 12% P. densiracemosa), showing that these species are promising sources for obtaining candidates for chemotherapy drugs.


INTRODUCTION
The Polygala L. genus is the most abundant of the Polygalaceae family, with around 725 species distributed worldwide in many different environments.In southern Brazil 39 species of this genus have been identified (Ludtke et al., 2013).The Polygala genus is characterized by the accumulation of cumarins, flavonoids, polysaccharides, sterols, styrylpyrones, triterpenic saponins and xanthones (Klein Júnior et al., 2012).Several in vitro and in vivo studies have highlighted the important biological effects of species of the genus Polygala, for instance: the anti-inflammatory activity of sterols from Polygala sabulosa (Borges et al., 2014), isoflavones from Polygala molluginifolia (Arruda-Silva et al., 2014) and triterpenic saponins from Polygala tenuifolia (Cheong et al., 2011); the antitumor activity of polysaccharides from Polygala tenuifolia (Xin et al., 2012;Zhang et al., 2015) and xanthones from Polygala hongkongensis (Wu et al., 2011); the antidepressant activity of styrylpyrones from Polygala sabulosa (Capra et al., 2010); and the antinociceptive activity of flavonoids from Polygala paniculata (Lapa et al., 2009).Currently, chemotherapy is the main procedure for the treatment of cancer and it induces serious side effects on patients.Extensive studies seeking new active plant extracts that can be used in the treatment of cancer have been carried out in the search for drug candidates that have high efficacy and safety (Gao et al., 2005;Ni et al., 2010).This paper describes the screening of extracts of Polygala L. spp.for antitumor activity against cell lineages of different types of leukemias and carcinomas using the MTT cell viability assay.
with 2 mM of L-glutamine and 10% (leukemic cells) or 5% (adherent cells) of fetal bovine serum.All cultures were maintained at 37 ºC in a humidified incubator with 5% CO 2 and 95% air.The media were changed twice weekly and they were regularly examined.

Evaluation of the cytotoxic effect against human tumor cell lines
Cells were seeded at densities/well of 50,000 for HL-60 cells and 100,000 for Jurkat and THP-1 cells.Adherent cells were inoculated at 10,000 cells/well.The plates were pre-incubated for 24 h at 37 ºC to allow adaptation of the cells prior to the addition of the extracts.Freshly prepared solutions of the different extracts were screened at 50 μg mL -1 in DMSO.Subsequently, the plates were inoculated for 48 h in an atmosphere of 5% CO 2 and 100% relative humidity.Control groups included treatment with 0.5% DMSO (negative control) and etoposide (10 μM, positive control).Cell viability was estimated by measuring the rate of mitochondrial reduction of MTT.All substances were dissolved in dimethylsulfoxide (DMSO) prior to dilution, with a final cell concentration of 0.5%.All extracts were tested in triplicate, in two independent experiments.

In vitro cell viability assay -MTT assay
Cell viability was evaluated using the MTT assay as described by Mosmann (1983).Briefly, after 4 h of incubation of the cells with the different extracts, 20 μL of MTT solution (5 mg mL -1 in phosphate-buffered saline) were added to each well, the supernatant was removed and 200 μL of 0.04 M HCl in isopropyl alcohol were added to dissolve the formazan crystals.The optical densities (OD) were evaluated using a spectrophotometer at 595 nm.Controls included drug-containing medium (background) and extract-free complete medium.Extract-free complete medium was used as the control (blank) and was treated in the same way as the extract-containing media.Results were expressed as percentage of cell proliferation, compared with the 0.5% DMSO control and were calculated as follows: viability (%) = (mean OD treated culturemean OD background)/(mean OD untreated culture, i.e. 0.5% DMSOmean OD blank wells) x 100.Interaction of the extract with the medium was estimated on the basis of the variations between the drug-containing medium and the extractfree medium to avoid false-positive or false-negative results in relation to the control (Ulukaya et al., 2004).

RESULTS AND DISCUSSION
In the experiments six tumor cell lines and one line of non-tumor cells (Vero) were evaluated.This latter lineage has been used in toxicity studies as a predictive model of normal cells (Freire et al., 2008).Three cell lines were derived from colorectal carcinoma (HCT-116) and two from breast cancer cells: an estrogen-dependent lineage represented by MCF-7 (responsive to chemotherapy) and an aggressive and unresponsive estrogenindependent lineage MDA-MB-231 (Srisawat et al., 2014).
To represent models of relevant leukemias with an impact on public health, cells derived from the human promyelocytic leukemic (HL-60 cells), acute T-lymphocytic leukemia (Jurkat) and acute monocytic leukemia (THP-1) were used.The cell lines were evaluated by the MTT method in which the tetrazolium salt is reduced to purple formazan crystals mainly by mitochondrial succinate dehydrogenase and the color intensity of the formazan dye is correlated to the number of viable cells (Mosmann, 1983).The results demonstrate that only P. densiracemosa, P. campestris and P. molluginifolia by more than 50% the proliferation of tumor cells and presented low inhibition of the proliferation of non-tumor cells (Figure 1 and 2), which is a parameter for antitumor screening (Senthilraja et al., 2015).The P. densiracemosa sample was active only in the inhibition of the HL-60 strain (66%) and Vero cell viability (12%), suggesting a possible selectivity for myeloid leukemia cells and low toxicity against non-tumor cells.The P. campestris sample inhibited the viability of Jurkat (66%) and HL-60 (78%) cells, with low toxicity toward Vero cells (11%).The P. molluginifolia sample inhibited by over 50% the proliferation in 5 of the 6 tumor cell lines tested: HL-60 (83%), Jurkat (78%), MDA-MB-231 (75%), MCF-7 (70%) and HCT-116 (64%).However, the Vero cell viability was inhibited by 32%, demonstrating cytotoxicity also toward non-tumor cells (Figure 1 and Table 1).Isoflavones are the main secondary metabolites of P. molluginifolia (Venzke et al., 2013), which may be related to the pronounced anti-tumor activity of this species (De la Parra et al., 2016).
Polygala campestris and P. densiracemosa, in preliminary studies, showed the accumulation of xanthones (unpublished data), which may explain the cell inhibition profile (HL-60 and Jurkat) and low toxicity against non-tumor cells.Previous studies have demonstrated the potent activity of xanthones isolated from P. hongkongensis against three tumor cell lines (HepG2 human hepatocellular carcinoma cells; GLC-82 human lung carcinoma cells and MCF-7 human breast carcinoma cells), using the MTT assay (Wu et al., 2011).In another study, the antitumor activity of a root extract of Polygala senega was demonstrated (Paul et al., 2010).
One of the important criteria for a candidate antitumor drug is to have minimal or no side effects on the normal cells of patients undergoing chemotherapy.One way to achieve this is by employing lower doses of the drug.This invariably means that the drug should not only have potent activity, but should also display a high degree of selectivity (Badisa et al., 2009).The P. campestris sample showed selectivity for cell lines of lymphoid and myeloid leukemia while P. densiracemosa showed selectivity only for myeloid cells, and both species presented low toxicity toward non-tumor cells.The species P. molluginifolia demonstrated the highest inhibition values for tumor cells, as can be seen in Figure 2, where 83% cell proliferation can be observed for HL-60 cells, suggesting a potent antitumor activity.However, this species also inhibited the viability of non-tumor cells lines by 32%.

CONCLUSIONS
The best results found for antitumor screening were observed for the P. molluginifolia sample, but this species also inhibited the viability of Vero cells.
The species P. campestris showed selectivity for Jurkat and HL-60 cells whilst P. densiracemosa showed selectivity only for HL-60 cells, with low toxicity toward non-tumor cells.These results demonstrate that P. densiracemosa and P. campestris are promising sources for obtaining candidates for chemotherapy drugs.

Fig. 1 :
Fig.1: Effect of crude plant extracts on proliferation/ viability of non-tumor cells(Vero).Data representative of two independent experiments performed in triplicate.Control groups included treatment with 0.5% DMSO (negative control) and etoposide (10 μM, positive control).

Table 1 :
Effect of crude plant extracts on proliferation/ viability of tumor cells and non-tumor cells(Vero).