Cytotoxic potential of selected medicinal plants in northeast Brazil

Background Great biodiversity is a highlight of Brazilian flora. In contrast, the therapeutic potentialities of most species used in folk medicine remain unknown. Several of these species are commonly used to treat cancer. In this study, we investigated the cytotoxic activity of 18 plants from 16 families that are found in the northeast region of Brazil. Methods The following species were studied: Byrsonima sericea DC. (Malpighiaceae), Cupania impressinervia Acev. Rodr. var. (revoluta) Radlk (Sapindaceae), Duranta repens Linn. (Verbenaceae), Helicostylis tomentosa (Poepp. & Endl) Rusby (Moraceae), Himatanthus bracteatus (A.DC.) Woodson (Apocynaceae), Ipomoea purga (Wender.) Hayne (Convolvulaceae), Ixora coccinea Linn. (Rubiaceae), Mabea piriri Aubl. (Euphorbiaceae), Miconia minutiflora (Melastomataceae), Momordica charantia L. (Cucurbitaceae), Ocotea glomerata (Nees) Mez (Lauraceae), Ocotea longifolia Kunth (Oreodaphne opifera Mart. Nees) (Lauraceae), Pavonia fruticosa (Mill.) Fawc. & Rendle (Malvaceae), Psychotria capitata Ruiz & Pav. (Rubiaceae), Schefflera morototoni (Aubl.) Maguire, Steyerm. & Frodin (Araliaceae), Solanum paludosum Moric. (Solanaceae), Xylopia frutescens Aubl. (Annonaceae) and Zanthoxylum rhoifolium Lam. (Rutaceae). Their dried leaves, stems, flowers or fruits were submitted to different solvent extractions, resulting in 55 extracts. After incubating for 72 h, the cytotoxicity of each extract was tested against tumor cell lines using the alamar blue assay. Results The B. sericea, D. repens, H. bracteatus, I. purga, I. coccinea, M. piriri, O. longifolia and P. capitata extracts demonstrated the most potent cytotoxic activity. The chloroform soluble fractions of D. repens flowers and the hexane extract of I. coccinea flowers led to the isolation of quercetin and a mixture of α- and β-amyrin, respectively, and quercetin showed moderate cytotoxic activity. Conclusion The B. sericea, D. repens, H. bracteatus, I. purga, I. coccinea, M. piriri, O. longifolia and P. capitata plants were identified as having potent cytotoxic effects. Further investigations are required to determine the mechanisms of cytotoxicity exhibited and their in vivo activities. This work reinforces the need to understand the therapeutics potentialities of Brazilian medicinal plants.


Background
In Brazil, the estimated number of described plant species (terrestrial and algae) ranges from 50,542 to 60,042 [1]; they are found in the following six different biomes: Amazônia (the Amazon rainforest in northern and central-western Brazil), Cerrado (the central Brazilian savanna), Mata Atlântica (the Atlantic rainforest ranging from sea level to the eastern highlands of Brazil), Caatinga (a xerophilous thorny forest found in northeastern Brazil), Pampa (the grasslands in southern Brazil) and Pantanal (periodically flooded grasslands by the Paraná and Paraguay rivers in central-western Brazil). Thus, Brazil is one of the most biodiverse nations in the world [1][2][3].
The cytotoxic potential of some Brazilian medicinal plants has been previously investigated [4][5][6][7]; however, due to the country's great biodiversity, the therapeutic potentialities of most species remain unknown. In this work, the cytotoxic activities of 18 plants belonging to 16 Table 1.

Plant extractions
The dried leaves, stems, flowers or fruits were triturated and extracted at room temperature with ethanol or hexane. The solvent was removed to produce the correspondent crude extracts. The extraction steps for each sample were repeated three times with one-week intervals. Part of leaves, fruits or flowers ethanol extract was suspended in methanol:water (9:1) solution and extracted successively with ethyl acetate, chloroform, hexane or hexane:ethyl acetate (1:1) to produce the corresponding partitioned extracts.

Chemical fractionation General procedures
Purification processes were performed through column chromatography (CC) using silica gel 60 (0.063-0.200 mm) as the stationary phase. Organic solvents or mixtures of increasing polarity were used as mobile phases. Silica gel 60 GF (Merck, Darmstadt, Germany) was used to perform analytical (0.25 mm) or preparative (0.75 mm) thin layer chromatographic (TLC) processes.
The 1 H (400 MHz) and 13 C (100 MHz) nuclear magnetic resonance (NMR) spectra were obtained on a Bruker Avance DRX-400 spectrometer (Bruker, Rheinstetten, Germany), operating at 300 K. The chemical shifts (δ) were expressed in units of ppm, using tetramethylsilane (TMS) as a reference (δ H = δ c = 0), and the coupling constants (J) were expressed in Hz. Chloroform-d was used as the solvent for the samples.

Isolation of compounds
The chloroform extract of D. repens flowers (7 g) was submitted to silica gel CC eluted with hexane and ethyl acetate (either pure or a mixture of increasing polarity), providing 50 fractions (5 mL each) that were grouped with hexane and ethyl acetate. After solvent evaporation, fractions 30-50 were purified with Sephadex-L water:methanol, yielding the compound quercetin (56, 11.0 mg). The compound structure was identified through a series of spectrometric data, such as NMR, as well as by comparison with data reported in the literature.
A hexane extract of I. coccinea flowers (26 g) was submitted to silica gel CC eluted with hexane, ethyl acetate and methanol (either pure or a mixture of increasing polarity), providing 100 fractions (5 mL each) that were grouped with hexane, ethyl acetate and methanol. After solvent evaporation from fractions 30 to 45 (eluted with hexane/ethyl acetate 8:2), a white solid (in the form of flakes) was obtained (57, 14.0 mg). This solid was identified as a mixture of αand β-amyrin. The compounds structures were identified through a series of spectrometric data, such as NMR, as well as by comparison with data reported in the literature.

Lauraceae
Canela de cheiro Fruit oil is used to treat arthralgia and rheumatic diseases [33] Pavonia Matatauba, Marupá Its fruit is used as a nutritional source and to make local crafts [28] Solanum paludosum Moric. Solanaceae Jurubeba-brava, jurubebaroxa An infusion of the roots is used to treat hepatic diseases. The fruit is used as a poison [26] Xylopia frutescens Aubl. Annonaceae Embira, semente-deembira Its seeds and fruits are used as a digestive aid (a decoction of a teaspoon in a cup of water). It is used as a tea after meals [26] Zanthoxylum rhoifolium Lam. Rutaceae Mamica de cadela, mamica de porca The roots are used as a febrifuge, digestant and tonic. The stem bark is used to treat flatulence, colic, dyspepsia, ear aches, toothaches and snake bites [34] 16:199 with cells in the exponential growth phase. All cell lines were tested for mycoplasma with a Mycoplasma Stain Kit (Sigma-Aldrich, St Louis, MO, USA) and found to be free from contamination. Heparinized blood (from healthy, 20-to 35-year-old, non-smoking donors who had not taken any drug for at least 15 days prior to the sampling) was collected, and peripheral blood mononuclear cells (PBMC) were isolated using a standard protocol, with Ficoll (Ficoll-Paque Plus, GE Healthcare Bio-Sciences AB, Sweden) density gradient centrifugation. PBMC were washed and resuspended at a concentration of 0.3 × 10 6 cells/mL in RPMI 1640 medium supplemented with 20 % fetal bovine serum, 2 mM L-glutamine, and 50 μg/mL gentamycin at 37°C with 5 % CO 2 . In addition, concanavalin A (ConA, Sigma Chemical Co. St Louis, MO, USA) was used as a mitogen to trigger cell division in T-lymphocytes. ConA (10 μg/mL) was added at the beginning of the culture, and after 24 h, the cells were treated with the test drugs.
For all experiments, cell viability was examined using Trypan blue exclusion assays. Over 90 % of the cells were viable at the beginning of the culture.

Cytotoxicity assay
Cell viability was quantified using alamar blue assay, as previously described [8]. For all experiments, cells were seeded in 96-well plates (0.7 × 10 5 cells/mL for adherent cells or 0.3 × 10 6 cells/mL for suspended cells in 100 μL of medium). After 24 h (or immediately for the suspended cells), the samples, which were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St Louis, MO, USA) at a final concentration of 50 μg/mL, were added to each well and incubated for 72 h. Doxorubicin (purity >95 %, Laboratórios IMA S.A.I.C., Buenos Aires, Argentina) was used as the positive control. The negative control received the vehicle used to dilute the tested samples (0.5 % DMSO). Four (for cell lines) or 24 h (for PBMC) before the end of the incubation, 20 μL of stock solution (0.312 mg/mL) of the alamar blue (resazurin, Sigma-Aldrich, St Louis, MO, USA) were added to each well. The absorbance was measured using a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, USA), and the drug effect was quantified as the percentage of control absorbance at 570 and 600 nm. The extracts that caused more than 75 % cell growth inhibition were tested again at concentrations varying from 0.39 to 50 μg/mL to determine the 50 % inhibitory concentration (IC 50 ). Isolated compounds were tested at concentrations varying from 0.19 to 25 μg/mL.

Statistical analysis
Data are presented as the mean ± S.E.M. The IC 50 values were obtained through nonlinear regression using the GraphPad program (Intuitive Software for Science, San Diego, USA).

Results and discussion
Fifty-five extracts were obtained from the plants studied, and their cytotoxicities were tested using the alamar blue assay after 72 h of incubation. The cytotoxicity of each  Table 2.
Concentration-response curves were generated and IC 50 values were calculated against four tumor cell lines (B16-F10, HepG2, K562 and HL-60) and against one non-tumor cells (PBMC) for the extracts that caused more than 75 % cell growth inhibition (Table 3). In addition, the chloroform soluble fractions of D. repens flowers and the hexane extract I. coccinea flowers isolated quercetin and the mixture of αand β-amyrin (Fig. 1), respectively, which were also tested for their cytotoxic activity (Table 3).  In the preclinical cytotoxic drug screening program used in this work, which is based in the United States National Cancer Institute program, only those extracts that present IC 50 values below 30 μg/mL and pure compounds with IC 50 values below 4 μg/mL in tumor cell line assays are considered to be promising for anticancer drug development [9][10][11]. Therefore, the B. sericea, D. repens, H. bracteatus, I. purga, I. coccinea, M. piriri, O. longifolia and P. capitata extracts demonstrated promising cytotoxic activity. Doxorubicin, a clinical useful chemotherapy drug, was used as the positive control and presented IC 50 values ranged from 0.02 to 0.08 μg/mL for HL-60 and B16-F10, respectively.
The pharmacology activity of B. sericea has been previously reported; the ethanolic extract from its leaves has gastroprotective properties that are mediated by nitric oxide and the K + ATP channel. Phytochemical studies have revealed the presence of flavonoids (i.e., rutin, isoquercitrin, kaempferol 3-O-rutinoside and quercetin). However, its cytotoxic activity has not been previously reported [12]. Previous studies of D. repens have reported the presence of triterpenes, flavonoids, steroids, C-alkylated flavonoids and acetosides, as well as some alkaloids. Interestingly, durantanin IV and V and E/Z acteoside isolated from the leaves of D. repens have demonstrated significant cytotoxic activity against a HepG2 cell line [13]. Jalapinoside, a macrocyclic bisdesmoside resin glycoside, was recently isolated from the roots of I. purga, and its cytotoxic activity was evaluated. Jalapinoside was not cytotoxic (IC 50 > 10 μg/mL), but reversal of multidrug resistance was observed using vinblastineresistant human breast carcinoma cells [14]. In this study, we isolated the flavonol quercetin from the flowers of D. repens, which demonstrated a cytotoxic effect (with IC 50 values ranging from 8.81 to 14.93 μg/mL on tumor cell lines HL-60 and K562, respectively). Quercetin has been previously reported as a cytotoxic agent because of its ability to induce apoptosis [15,16].
The antitumor activity of flowers of I. coccinea was previously studied in murine models. Inhibited tumor growth and increased life spans were observed in Dalton's lymphoma and Ehrlich ascites carcinoma-bearing mice [17]. Moreover, ixorapeptide I, a peptide derived from I. coccinea, displayed cytotoxicity against the Hep3B liver tumor cell line, with an IC 50 value of 3.36 μg/mL [18]. Interestedly, although the use of I. coccinea is currently only ornamental in Brazil, its flowers are an ingredient in an Ayurvedic cancer formulation in India [19,20]. The flowers of I. coccinea are used to treat cancer, leucorrhoea, dysentery, dysmenorrhea, hemoptysis and hypertension; its leaves are used to pacify vitiated pitta, skin diseases, colic, flatulence, diarrhea, indigestion, ulcers and wounds, and they are also used as an antiseptic [20,21]. The roots of I. coccinea are used as an astringent and antiseptic against scabies and other skin  [20]. In this study, the fractionation of the extract of I. coccinea flowers resulted in the mixture of αand βamyrin, which demonstrated weak cytotoxic activity toward tumor cell lines. In fact, no potent cytotoxic activity has been reported for these compounds.
Prieto et al. [22] have reported that essential oil from the leaves of O. longifolia, of which α-terpinolene and αphellandrene are the primary ingredients, demonstrates antifungal activity against Sitophilus zeamais. However, the cytotoxic activity of the O. longifolia plant has not been previously reported. No previous biological effects have been reported for the medicinal plants H. bracteatus, M. piriri and P. capitata.
The extracts from C. impressinervia, H. tomentosa, M. minutiflora, M. charantia, O. glomerata, P. fruticosa, S. morototoni, S. paludosum, X. frutescens and Z. rhoifolium showed no pronounced cytotoxic effects. Among these plants, essential oil from the leaves of X. frutescens has been previously assessed for its cytotoxic and antitumor effects; the oil demonstrated potent in vitro cytotoxic activity against tumor cell lines from different histotypes and in vivo antitumor activity in sarcoma 180 murine model [23]. Although essential oil from Z. rhoifolium leaves and M. charantia fruit extract have been previously assessed for cytotoxicity, they demonstrated no cytotoxic effects [24,25]. The cytotoxic potential of C. impressinervia, H. tomentosa, M. minutiflora, O. glomerata, P. fruticose, S. morototoni and S. paludosum plants has never been investigated.

Conclusion
In conclusion, we screened the cytotoxic potential of 55 extracts from 18 plants found in northeast Brazil, and extracts from B. sericea, D. repens, H. bracteatus, I. purga, I. coccinea, M. piriri, O. longifolia and P. capitata demonstrated potent cytotoxic effects. The fractionation of D. repens and I. coccinea extracts led to the isolation of quercetin and the mixture of αand β-amyrin, respectively, and quercetin showed moderate cytotoxic activity. Fractionation of the other plants should be performed to identify their active constituents, and further investigations are required to determine their mechanisms of cytotoxicity and in vivo activities. This work reinforces the need to understand the therapeutic potentialities of Brazilian medicinal plants.