Preliminary phytochemical analysis and evaluation of the antioxidant and anti-proliferative effects of Plinia peruviana leaves: an in vitro approach

Abstract Plinia peruviana (Poir.) Govaerts (Myrtaceae) occurs from Pará to Rio Grande do Sul states, Brazil. The leaf extract of this species was investigated. The total of polyphenols and flavonoid contents were determined by spectrophotometric techniques. Antioxidant activity by the oxygen radical absorbance capacity (ORAC) and anti-proliferative activity was tested against the MCF-7 and HeLa lines. The amount of polyphenols from the lyophilized extract was 944 ± 0.0856 mg GAE/g, moreover, total flavonoid content of the extract of 531.8 ± 0.0040 mg RE/g extract. This study performed the first pharmacological exploration of the leaves of P. peruviana. Our results demonstrated the antioxidant and anti-proliferative effects of this specie on MCF-7 and HeLa cell lines. This makes this specie an interesting medicinal plant for human epithelial cervical cancer and human breast cancer anti-proliferative approach.


Introduction
In recent years, it has been observed that the number of studies reporting antioxidant activities of natural products has increased considerably since phenolic compounds inhibit the formation of free radicals and maintain normal redox regulation of the human body (Gill and Tuteja 2010).Such facts drive the search for antioxidant and anticancer compounds from natural sources (Ghasemzadeh et al. 2018;Galasso et al. 2018;Abubakar et al. 2018).
In this context, the family Myrtaceae is considered one of the most important in Brazil and the dominant woody family in southeastern Atlantic Forest (Citadin et al. 2010;Costa et al. 2013).The leaves of various species of this family have been used in folk medicine for their interesting bioactivities such as antifungal, antibacterial, anti-inflammatory and antioxidant properties.These activities were mainly related to the presence of flavonoids (Dias et al. 2018;Falcão et al. 2018;Souza-Moreira et al. 2018).
The species Plinia peruviana (Poir.)Govaerts (Jabuticabeira), belonging to the family Myrtaceae, occurs from the state of Par a to Rio Grande do Sul state, Brazil (Citadin et al. 2010;Costa et al. 2013).The fruits of this species have traditionally been used for their medicinal values, including antidiarrheal effects, as well as for respiratory problems, asthma and chronic inflammation of the tonsils (Morton 1987).
Despite the established phytochemical values for the fruits, the biological activities of the leaves of this species remain largely unknown.In this sense, this report is the first phytochemical investigation of hydroalcoholic extract obtained from the leaves of P. peruviana.In addition, this work was set to determine the in vitro antioxidant activity, total phenols and flavonoids and anticancer activities on breast (MCF-7) and cervical (HeLa) cancer cell lines.

Plant material
The leaves of the plant were collected in Santa Cruz do Sul, Rio Grande do Sul state, Brazil (29S 44 0 47.5 00 /52 W 21 0 24.2 00 ), in October 2017.Dr. Renato Aquino Z achia identified the plant material and a sample was deposited (voucher 17.753) at the Herbarium of the Universidade Federal de Santa Maria (UFSM).

Preparation of extract
The extraction process was performed according to the method of Strzemski et al. (2017).The plant was dried in an air circulation oven and then reduced to powder.The hydroalcoholic extract of P. peruviana was prepared in 70% ethanol (1/10 weight/ volume ratio) by ultrasonic bath (KQ-3200DE, 300 Â 150 Â 150 mm, SHUMEI, Kunshan, China) for 30 minutes at room temperature and its residue extracted again.After that, it was concentrated on a rotary evaporator (Buchi RII) under reduced pressure at 40 C, lyophilized and stored under refrigeration.

Total phenolics content
Total phenolic content of P. peruviana samples was determined by Folin-Ciocalteu assay as reported by Chandra and de Mejia (2004) with slight modification.The results were expressed as milligrams of gallic acid equivalents per gram of dried extract (mg GAE/g).

Total flavonoids content
The flavonoid contents of P. peruviana were estimated by the method described by Zhishen et al. (1999).The determination of total flavonoids was performed with the colorimetric aluminum chloride assay.The results were expressed as milligrams of rutin equivalents per gram of dried extract (mg RE/g).

Analysis of phenolic compounds high efficiency liquid chromatography coupled to diode array detector (HPLC-DAD)
The phenolic compounds were identified by HPLC/DAD analysis according to (Evaristo and Leitão, 2001), with minor modifications.For the separation of the compounds, the liquid chromatograph (SHIMADZU, Kyoto/Japan) was used, model pump LC -20AT, automatic injector SIL -20 A, DAD SPD -M20A detector and CBM 20 A communicator and controlled by LC SP1 software.The analytical column used was Shim-pack CLC-ODS (M), particle with 5 lm in diameter, dimensions of 4.6 mm X 150 mm.The elution occurred by gradient in a flow of 0.9 mL/min using two mobile phases: A, composed of 2% acetic acid in water and B, only methanol UV/HPLC.The injection volume of the samples was 40 lL, and the detection was monitored in the photodiode system at wavelengths between 230 and 400 nm for 55 minutes.
Identification was performed by integrating the peaks using the standard external method, at 270 nm for gallic acid; 278 nm for catechin and epicatechin; and 365 nm for rutin.Chromatographic peaks were confirmed by comparing the retention time with the reference standards and by the DAD spectrum (200-600 nm).All chromatographic operations were performed at room temperature.

Assay of the absorption capacity of the oxygen radical (ORAC)
The ORAC assay was performed, according to Ou et al. (2001) using a black 96-well microplate.The method consists in verifying the ability of the sample to sequester free radicals against the peroxyl radical, induced by 2,2 0 -azobis-(2-methylpropanoamidine) (AAPH).The sample (10 ppm) and the Trolox standard were diluted in phosphate buffer (75 mM, pH 7.4) and mixed with fluorescein (81 nM) and AAPH (152 mM).The reaction occurred at 37 C and fluorescence (k excitation ¼ 485 nm and k emission ¼ 528 nm) was monitored (SpectraMaxV R M3) every minute for 91 minutes.The antioxidant capacity was determined using area under the curve (AUC) and the results were compared with a Trolox analytical curve (2 to 96 lmol/L) and expressed in lmol Trolox Equivalent (TE) per gram of dry extract.The analyzes were done in triplicates and on alternate days.
2.5.In vitro antiproliferative activity 2.5.1.Cell culture, treatments and cytotoxicity assay The tumor cell lines HeLa (human epithelial cervical cancer) and MCF-7 (human breast cancer) were used as in vitro models to study the antitumor activity of the extract.All cells were grown in DMEM medium (4.5 g/l glucose), supplemented by 10% (v/v) FBS, at 37 C with 5% CO2.They were routinely cultured in 75 cm2 culture flasks and harvested using trypsin-EDTA, when the cells reached approximately 80% of confluence.As a positive control, it was used vinblastine at the concentration of 10 mg/mL.HeLa (8.5 Â 104 cells/ml) and MCF-7 (1 Â 105 cells/ml) cell lines were seeded into the 60 central wells of 96-well cell culture plates in 100 ml of complete culture medium.Cells were incubated for 24 hours under 5% CO 2 at 37 C and the medium was then replaced with 100 ml of fresh medium, supplemented by 5% (v/v) FBS, containing the extract at the concentration range of 25-500 lg/ml.Untreated control cells were exposed to medium with 5% (v/v) FBS only.The cell lines were exposed for 24 hours to each treatment, and their viability was assessed by the MTT assay.
The MTT endpoint is based on the protocol first described by (Mosmann, 1983), which is a measurement of cell metabolic activity.After complete the cell treatment time, the medium was removed, and 100 ml of MTT in PBS (5 mg/ml) diluted 1:10 in medium without FBS was then added to each well.The microplates were further incubated for 3 h under 5% CO2 at 37 C, after which the medium was removed.Thereafter, 100 ml of DMSO was added to each well to dissolve the purple formazan product.Plates were then placed on a microtiter-plate shaker for 10 min at room temperature, and the absorbance of the resulting solutions was measured at 550 nm using a Multiskan FC (Thermo Scientific, San Jose, CA, USA) microplate reader.Cell viability was calculated as the percentage of tetrazolium salt reduced by viable cells in each sample.
The cytotoxicity of each sample in each cell line was expressed as percentage of viability with regard to untreated control cells (the mean optical density of untreated cells was set at 100% viability), and in terms of its IC 50 (concentration causing 50% death of the cell population), calculated for each cell line by curve fitting of percent cell survival against the extract concentrations.

Statistical analysis
The data were analyzed using one-way ANOVA followed by Tukey's post hoc.The results were expressed as mean ± SD and p < 0.05 was considered as statistically significant.Total phenolics 1 (mg of GAE/g ± SD) 2 944 ± 0.0856 y ¼ 0.0027x þ 0.0082 0.9985 Total Flavonoids 1 (mg of RE/g ± SD) 3  531.8± 0.0040 y ¼ 0.0022x þ 0.019 0.9975 1 Values presented in mean ± standard deviation of triplicate assays. 2 Total phenolic compounds expressed as mg EAG per g extract. 3Total flavonoids expressed as mg RE per g extract.

Total phenolic and flavonoid contents
Total phenolic content of P. peruviana was carried out based on the reaction of the samples with Folin-Ciocalteu reagent, which results in a blue colored solution, which intensity was directly proportional to the amount of phenolic compounds present.Total phenolic (TP) and flavonoid contents (FC) are reported in Table 1.TP and FC were 944 ± 0.0856 mg GAE/g and 531.8 ± 0.0040 mg RE/g, respectively.Previous studies reported that TP for leaves of the Myrtaceae family, such as Plinia edulis, was 54.34 ± 0.02 mg/g extract (Carvalho et al. 2012).In relation to plants of the same species, only fruit studies were found, reporting 320 mg GAE/g extract (Leite-Legatti et al. 2012).
The FC found in our study was lower than the fruits of the same species according to the previous study that presented FC ¼ 642.99 ± 25.75 mg/g (Batista et al., 2017), but was higher than the leaves of the same family (Plinia edulis), reporting FC of 35.16 ± 0.01 mg/g (Carvalho et al. 2012).

Analysis of the phenolic compounds of the extract by HPLC-DAD
In the present work, the HPLC profile of P. peruviana was also acquired (Figure 1).We identified the presence of polyphenols gallic acid, catechin, epicatechin and rutin (Figure 1).The retention times (Rts) of the identified compounds were: gallic acid (Rt ¼ 6.55 min, peak 1), catechin (Rt ¼ 13.97 min, peak 2), epicatechin (Rt ¼ 17.89 min, peak 3) and rutin (Rt ¼ 27.86 min, peak 4).The amount of each compound is shown in Table 2.
Data from the literature indicate the presence of phenolic compounds such as quercetin, isoquercitrin, quercitrin, myricetin, rutin, cinnamic acid, camarinoic acid, gallic acid and ellagic acid in fruits of the same species (Reynertson et al. 2006;Pereira et al., 2017;Neves et al., 2018).Some of these compounds were not identified in the leaves, but we also reported the presence of catechin and epicatechin.Gallic acid (peak 1), catechin (peak 2), epicatechin (peak 3), and rutin (peak 4).

Antioxidant activity
Natural products, due to their composition of phenolic compounds, are reported as potential anti-proliferative and antioxidant agents (Abdul Wahab et al. 2018).The extract prepared from the leaves of P. peruviana presented promising antioxidant activity by the ORAC assay.The relative fluorescence measured over time defines the antioxidant potential of the compounds over the Trolox standard (Figure 2).P. peruviana showed a fall in fluorescence that remained between the values measured by AUC.
Through the equation of the line (y ¼ 0.484x þ 5.0528), a value of 1941.1 ± 717.65 lM TE/g of P. peruviana was obtained.To date, no studies have reported the antioxidant activity of leaves of this species, but, Leite-Legatti et al. (2012) evaluated the antioxidant activity of the freeze-dried dry extract of Jabuticaba hulls and for whole fresh fruits (5230.4lM ET/g and 1511 lM TE/g, respectively).
Similarly, the in vitro antioxidant activity of leaves of Calyptranthes tricona (Myrtaceae), found ORAC values of 56165.79 ± 1212.84 lM TE/g and 2021.51 ± 127.60 lM TE/g for ethanolic and hexanic extracts, respectively (Kich et al. 2017).This property is related to the concentration of phenolic compounds present in  the extract, since these compounds are considered excellent natural antioxidants (Afrin et al. 2018).

Antiproliferative activity
One of the strategies for the discovery of new anticancer substances is the search for natural products with antioxidant properties and cytotoxic effects (El Abed et al. 2018).
Chemotherapy is generally employed in the cancer treatment.However, a large number of side effects is its limitation.Thus, it is interesting to seek for the alternative treatments for cancer that are no side effects and not so expensive cost (Tungmunnithum et al. 2018).
Considering the antioxidant activity of the extract of this species, we tested the antiproliferative activity against the MCF-7 and HeLa cell lines (Figure 3).To date, no studies have been found that evaluated the anti-proliferative effect of leaf extract of this species.
After a 24 hr treatment, the IC 50 of Jabuticaba leaves extract was 263.88 and 462.77lg/mL (HeLa and MCF-7 respectively).Previous studies using the aqueous extract of the seed of this plant demonstrated promising anti-proliferative effects in lineage of mouth cancer cells (HSC-3) (Wang et al. 2014).In addition, anti-proliferative effects of Jabuticaba fruits were also verified against leukemic lineage (K-562) and prostate cancer (PC-3) (Leite- Legatti et al. 2012).Previous evidence describes some flavonoid compounds extracted from this species with anti-proliferative effects against HT29 and HCT116 colon cell lines (Reynertson et al. 2006).Purification and the identification of the active compounds in the extract of Jabuticaba leaves are required for a better understanding of the cell death mechanisms involved and to deduce possible future applications.

Conclusion
The results of this report indicate that the hydroalcoholic extract of leaves of P. peruviana presents antioxidant activities and anti-proliferative potential against MCF-7 and HeLa.The anticancer activity may be related to the presence of polyphenols, flavonoids and the high antioxidant capacity detected by the ORAC assay.This study is the first to report the biological potentials of this species.Thus, contributing to the pharmacological knowledge of P. peruviana.More studies will be needed to establish the safe use of this plant by the population, since there are no references in this literature about this species.

Figure 2 .
Figure 2. Antioxidant activity of P. peruviana in the ORAC assay.

Figure 3 .
Figure3.The cell rate of cells after 24 hours of treatment with extracts was extracted from more significant cytotoxic activity in the HeLa line.Control ¼ Vinblastine 10 mg/mL.Cell viability was assessed using the MTT assay.Ã p < 0.05 compared with MCF-7.

Table 1 .
Polyphenols and flavonoids contents of the lyophilized hydroalcoholic extract of P. peruviana leaves.

Table 2 .
Components of the lyophilized hydroalcoholic extract of P. peruviana leaves.