Antiproliferative activity in tumor cell lines , antioxidant capacity and total phenolic , flavonoid and tannin contents of Myrciaria floribunda

Myrciaria floribunda (H. West ex Willd.) O. Berg, Myrtaceae, is a native plant species of the Atlantic Rain Forest, from north to south of Brazil. The lyophilized ethyl acetate extract from the leaves of M. floribunda was investigated for its antiproliferative activity in tumor cell lines, antioxidant capacity and its total phenolic, flavonoid and tannin contents. Antiproliferative activity was tested in vitro against seven human cancer cells and against immortalized human skin keratinocytes line (HaCat, no cancer cell). Antioxidant activity was determined using 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and oxygen radical absorbing capacity (ORAC) assays and total phenolic, flavonoid and tannin contents were determined by spectrophotometric techniques. Ethyl acetate extract of M. floribunda exhibited antiproliferative activity against cancer cell lines with total growth inhibition (TGI) between 69.70 and 172.10 μg/mL. For HaCat cell, TGI value was 213.60 μg/mL. M. floribunda showed a strong antioxidant potential: EC50 of 45.89±0.42 μg/mL and 0.55±0.05 mmol TE/g for DPPH and ORAC, respectively. Total phenolic content was 0.23±0.013g gallic acid equivalents (GAE)/g extract and exhibited 13.10±1.60% of tannins content. The content of flavonoid was 24.08±0.44% expressed as rutin equivalents. These results provide a direction for further researches about the antitumoral potential of M. floribunda.


INTRODUCTION
WHO's International Agency for Research on Cancer (IARC) estimates that 14.1 million new cancer cases and 8.2 million cancer-related deaths occurred in 2012 (IARC 2013).The 2016 and 2017 annual estimation regarding Brazil foresees the emergence of approximately 600,000 new cancer cases (INCA 2015).LUIS A.C. TIETBOHL et al.According to Newman and Cragg (2016), the utilization of natural products and/or their novel structures, in order to discover and develop anticancer drugs is still alive and over 50% of the drugs in clinical trials for anticancer activity were isolated from natural sources or are related to them.Among the natural products, plant-derived compounds have been an important source of several clinically useful anti-cancer agents (Cragg and Newman 2005).
The Myrtaceae family has species typically of many Brazilian biomes with their floristic diversity.A great diversity of plants from this family has been employed for medicinal purposes by Brazilian's population (Cruz and Kaplan 2004).Some genus of this family were studied for its antiproliferative activity (Apel et al. 2006, Levy and Carley 2012, Annadurai et al. 2012, Kiruthiga et al. 2011).The Myrciaria genus was reported to show antiproliferative activity against tumor cell lines (Leite- Legatti et al. 2012, Wang et al. 2014).
Despite studies about essential oils, chemical analysis and biological activities of the leaves of M. floribunda remain scare in the literature.Only one previous study with M. floribunda leaves reported that its ethyl acetate extract has phenolic compounds and the flavonoid myricetin-3-galoctoside was identified as one of the main compounds (Tietbohl 2012).As flavonoids are known to be anticancer and antioxidant agents (Batra andSharma 2013, Weng andYen 2012), this prompted us to evaluate the antiproliferative activity in tumor cell lines and to determine the antioxidant capacity and the contents of total phenolic, flavonoids and tannins of the ethyl acetate extract from the leaves of M. floribunda.

OXYGEN RADICAL ABSORBING CAPACITY ASSAY (ORAC)
The evaluation was performed using a 96well microplate reader (Fluostar Optima -Fluoroluminometer, BMG Labtech).The antioxidant capacity of the ethyl acetate extract from leaves was measured by fluorescence decay of fluorescein (Sigma), induced by 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH).Measurements were done by following time course of the fluorescence decay, in order to estimate antioxidant capacity.Using Trolox as standard, a calibration curve was generated using the net area under the curve AUC (AUCTrolox -AUCblank).ORAC values were calculated using the linear regression and expressed as Trolox Equivalent (TE).TE = slope regression curve (sample) / slope regression curve (Trolox) (Huang et al. 2002).The experiments were realized in triplicate and acceptable R 2 was ≥ 0.95.

DPPH RADICAL SCAVENGING ASSAY
Sample stock solution of the ethyl acetate extract (1.0 mg/mL) was diluted to final concentrations of 250, 125, 50, 25, 12.5 and 6.25 µg/mL in methanol.One mL of a 0.3 mM DPPH ethanol solution 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 wavelength and converted into the percentage antioxidant capacity (AA) using the following formula: AA% = 100 -[(Abs sample -Abs blank) x 100 / Abs control].Methanol (1.0 mL) plus extract solution (2.5 mL) was used as a blank.DPPH solution (1.0 mL; 0.3 mM) plus methanol (2.5 mL) was used as a negative control.The EC 50 value was calculated by linear regression of plots where the abscissa represented the concentration of evaluated extract and the ordinate was related to average percent of antioxidant capacity (Mensor et al. 2001).The LUIS A.C. TIETBOHL et al.
experiments were realized in triplicate with R 2 = 0.99 and Trolox was used as an antioxidant control.

DETERMINATION OF TOTAL FLAVONOIDS
An ultraviolet spectrophotometric method was used for total flavonoid quantitation of the ethyl acetate extract of Myrciaria floribunda, as rutin equivalents, according to an adaptation of the method developed by Rolim et al. (2005).The concentration of total flavonoids was determined spectrophotometrically as rutin equivalents compared to the standard curve of rutin.Standard rutin was dissolved in 95.0% ethanol and 0.02M acetic acid (99:1) to concentrations of 12.5; 25.0; 50.0; 75.0 and 85.0 mg/mL.The absorbance measurements were obtained at 360.0 nm.The mixture of ethanol 95% and acetic acid 0.02M (99:1) was used as a solvent for the preparation of the sample solution.Analyses were performed in triplicates and the results are presented as mean ± standard deviation (% w/w, ± s.d.).

DETERMINATION OF TOTAL POLYPHENOLIC CONTENTS
The total polyphenol content was determined by spectrophotometry, using gallic acid as a standard, according to the method described by Anesini et al. (2008).Briefly, 1.0 mL of the diluted sample extract in methanol (200 μg/mL) was transferred in duplicate to separate tubes containing 5.0 mL of a 1/10 dilution of Folin-Ciocalteu's reagent in water.Then, 4.0 mL of a sodium carbonate solution (7.5% w/v) was added.The tubes were then allowed to stand at room temperature for 60 min before the absorbance at 765 nm was measured against water.Results were expressed as grams of gallic acid equivalents (GAE) per gram of extract (g of GAE/g).The concentration of polyphenols in samples was derived from a standard curve of gallic acid ranging from 10 to 50 μg/mL.

DETERMINATION OF TANNINS
Tannins were measured by protein precipitation assay with the use of hide powder method described for Eugenia uniflora (Myrtaceae) in the Brazilian Pharmacopoeia (Brasil 2010).The total tannin concentration was assayed by measuring at 715 nm the absorbance of the product formed with Folin-Denis reagent.Polyphenols unadsorbed on hide powder was determined from 10 mL of methanol solution of the ethyl acetate extract (200 μg/mL) with addition of 0.1 g hide powder (Sigma) vigorously mixed for 60 min and filtrated.The polyphenol adsorved in hide powder, calculated from the difference among polyphenol total and polyphenol unadsorbed, was derived from a five-point standard curve of pyrogallol (Sigma) ranging from 3 to 100 µg/mL.The curves showed satisfactory linearity within the analyzed range (R 2 > 0.99).Tannin content (%) was determined as the ratio between the adsorbed polyphenol content and the initial concentration of sample (Macedo et al. 2013).Analyses were performed in triplicates and the results are presented as mean ± standard deviation (% w/w, ± s.d.).

RESULTS AND DISCUSSION
A range of M. floribunda extract concentration (from 0.25, 2.5, 25.0 to 250.0 µg/mL) was used to investigate the relative degree of growth inhibition against a series of cancer cell lines.Figure 1 demonstrates that M. floribunda showed a marked inhibition effect on cancer cells growth and it was not in a concentration-dependent manner.
mL), NCI-H460 (lung, TGI = 81.90µg/mL), PC-3 (prostate, TGI = 100.90µg/mL), OVCAR-03 (ovary, TGI = 131.20 µg/mL) and HT29 (colon, TGI = 172.10µg/mL).Although an excellent growth inhibiter of cancer cells, doxorubicin also affects the growth of non-tumor cells, for example, the ones from the HaCat cell line (Table I).In this regard, the assayed M. floribunda extract seems to be safer than since the toxicity to healthy cells was reached only at high concentrations.For example, the extract reaches a TGI higher than 210 µg/mL for the HaCat cell line.Some species from Myrciaria genus have been reported to show antiproliferative activity.The non-polar extract from the fruit peels of Myrciaria jaboticaba (Vell.)Berg demonstrated antiproliferative effects against leukemia (K-562) and prostate cancer cell (PC-3) (Leite- Legatti et al. 2012).Although the polar extract (ethanol) of M. jaboticaba peel, rich in polyphenol compounds, was not active against prostate cancer cell (PC-3) and ovary (OVCAR-3) and has shown concentrations that produce 50% of growth inhibition higher than 250.0 µg/mL (Leite-Legatti et al. 2012).In contrast, the polar extract of M. floribunda also rich in polyphenols has inhibited prostate (PC-3) and ovary (OVCAR-3) cells growth by 100% at concentrations of 100.90 µg/mL and 131.20 µg/ mL, respectively.It has been reported that the deep purple of M. jaboticaba peel contains high concentrations of total and free ellagic acid (Abe et al. 2012), a polyphenol that has a powerful potential to suppress the proliferation of leukemia cells (Mertens-Talcott et al. 2003).In addition, M. jaboticaba also contains anthocyanins capable of suppressing neoplastic cell lines (Song et al. 2012).
A study with the polar extract (ethanol) of M. dubia leaves and pericarp showed antiproliferative activity against liver carcinoma cell line (Hep-G2) at 149.5 ± 23.8 and 124.0 ± 12.3 μg/mL, respectively (Tauchen et al. 2016).However, the polar extracts of M. dubia leaves and pericarp were not active against colon cell (HT29) and inhibited 50% of the cell growth at a concentration higher 500.0µg/mL.
Flavonoid compounds extracted from M. cauliflora exhibited antiproliferative effect against HT29 and HCT116 colon cell lines (Reynertson et al. 2006).Several studies revealed flavonoids as potential antiproliferative and antioxidant agents (Batra and Sharma 2013, Weng and Yen 2012).Since a previous study with the ethyl acetate extract of M. floribunda leaves reported the flavonoid myricetin-3-galoctoside as one of the main compounds (Tietbohl 2012), this prompted us to determine the total phenolic, flavonoid and tannin contents and the antioxidant capacity of M. floribunda leaves in order to correlate chemical composition with antiproliferative effect.Table II presents the total phenolic, flavonoid and tannin contents determined by spectrophotometric techniques and the antioxidant capacity of the ethyl acetate extract of M. floribunda leaves by DPPH and ORAC assays.
The concentration of phenolic compounds in samples was derived from a standard curve of gallic acid (y = 0.0105x + 0.1136) with R 2 = 0.997 (Table II).Total phenolic content of the ethyl acetate extract of M. floribunda leaves was 0.230 ± 0.0136 g gallic acid equivalents (GAE)/g extract.This value was higher than those reported in previous studies for Myrciaria spp., such as M. cauliflora fruits contains 0.031 ± 0.1 g GAE/g (Reynertson 2007), M. jaboticaba fresh peel has 0.114 g GAE/g and the whole fruit shows 0.032 g GAE/g (Leite-Legatti et al. 2012) and M. dubia fruits fresh and dried exhibit 0.01176 and 0.01161 g GAE/g, respectively (Akter et al. 2011, Costa et al. 2013).
Tannin content of the ethyl acetate extract of M. floribunda leaves was 13.10 ± 1.6% calculated from a calibration curve of pyrogallol (y = 0.0131x + 0.039) with R 2 = 0.999 (Table II).The value obtained in this work was higher than those reported by Duarte et al. (2010) that showed a range of to 6.0% of tannin content for the ethanolic extract of M. cauliflora leaves cultivated in six sampling sites.
The ORAC value of the ethyl acetate extract was 0.55 ± 0.05 mmolTE/g (Table II).Leite-Legatti et al. (2012) found ORAC values of 25.51 mmolTE/g, 5.23 mmolTE/g and 1.51 mmolTE/g for the freezedried powdered extract, the fresh fruit peel and the whole fresh fruit of M. jaboticaba, respectively.The ORAC value found for M. floribunda leaves in the present study indicates a lower antioxidant potential compared to M. jaboticaba fruit peel and whole fresh fruit, although the differences in plant organs, extraction methods, solvents used and species could explain such variations.
The EC 50 of the ethyl acetate extract in the DPPH assay was 45.89 ± 0.42 μg/mL and the positive control Trolox was 2.95 ± 0.05 µg/mL The best adjustment correlation was obtained using a linear model (y = 0.762x + 15.03; R 2 = 0.998).Wang et al. ( 2014) worked with M. cauliflora and reported the EC 50 of the ethanol extracts of peel, stem and seeds of 49.00, 17.00 and 27.00 µg/mL, respectively, and the water extract of seed was 5.30 µg/mL.Einbond et al. (2004) found EC 50 value of 6.2 ± 0.7 μg/mL for semi-purified aqueous fraction of M. cauliflora fruits by DPPH assay.These reported data indicate that Myrciaria spp.water extracts show a higher potent antioxidant activity compared to ethyl acetate extract.However, the chemical composition of aqueous extract is different than the alcohol extracts.Aqueous plant extracts often contain water-soluble antioxidant vitamins and sugars that may enhance the antioxidant activity (Degenhardt et al. 2000).
The ethyl acetate extract of M. floribunda leaves demonstrated antioxidant capacity and antiproliferative activity at the present study.Chemical assays of the extract revealed a high amount of total phenolic compounds, tannin (13%) and flavonoids (24%).Therefore, it is possible to assert that antioxidant capacity and antiproliferative activity of the extract are in suitable correlation with the chemical compound contents (total phenolic compounds, tannin and flavonoids), once these classes of compounds are known as potent antioxidants and agents (Einbond et al. 2004, Batra and Sharma 2013, Weng and Yen 2012, Wang et al. 1997).Moreover, Tietbohl (2012) reported that the main constituent of the ethyl acetate extract of M. floribunda leaves is myricetin-3-galoctoside, which has showed an antitumor-promoting activity, and could be related to the results observed in this study (Gao et al. 2002, Hayder et al. 2008).

CONCLUSIONS
Data obtained indicate an antiproliferative activity against tumor cell lines and antioxidant capacity in vitro of M. floribunda leaves extract.The cell line MCF-7 (breast cancer) was more sensitive to M. floribunda, while normal cells were less growthinhibited.The present study is the first report of total phenolic, flavonoid and tannin contents of M. floribunda leaves.Thus, this work contributes to the chemical and pharmacological knowledge of M. floribunda.Despite the promising results found here, further biological studies should be performed, including in vivo investigations, looking toward a medicinal use of this bioactive natural product.

TABLE I Antiproliferative activity of the ethyl acetate extract from the leaves of Myrciaria floribunda. Cells TGI (µg/mL) a Doxorrubicin b M. floribunda c
b Positive control.c Ethyl acetate extract from the leaves of Myrciaria floribunda.

TABLE II Total phenolic, flavonoid and tannin contents and antioxidant capacity by the DPPH and ORAC assays of the ethyl acetate extract from the leaves of Myrciaria
floribunda.Total phenolics data expressed as grams of gallic acid equivalents per gram (g of GAE/g) of extract.DPPH assay data expressed as EC50 (concentration that inhibited 50% of the DPPH radical) in micrograms per milliliters (µg/ mL).ORAC data expressed as milimol of Trolox equivalents per gram (mmol of TE/g) of extract.Total flavonoids data expressed as equivalent in rutin.LUIS A.C. TIETBOHL et al.
b c d e