Phytochemical composition and biological activities of extracts from ten species of the family Melastomataceae Juss

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Plants possess a renewable source of metabolites with enormous chemical structural diversity, which may have potential therapeutic relevance. Furthermore, this chemical diversity favors the possibility of finding new and different chemical constituents with antimicrobial, antioxidant and anti-tumor activities. This work analyzed preliminary phytochemical profiles and evaluated the antimicrobial, antioxidant and cytotoxic activities of hexane extracts of leaves of ten species of the family Melastomataceae. Phytochemical screening was performed using staining methods while total phenols and flavonoids were quantified by spectrophotometry. Antimicrobial activity was evaluated using the disk diffusion method. Antioxidant activity was determined by the 2,2-diphenyl-1picrylhydrazil (DPPH) method. Toxicity was recorded using the lethality test with Artemia salina Leach (1819). Cytotoxic activity of the extracts was assessed in vitro with acute monocytic leukemia cells (THP-1). Phytochemical analysis detected the presence of tannins, terpenes, steroids, polyphenols and flavonoids and the absence of alkaloids. Clidemia capitellata (Bonpl.) D. Don had the greatest amount of polyphenols (205.95 mg/g ± 4.14) while Clidemia hirta (L.) D. Don had the highest content of total flavonoids (143.99 mg/g ± 4.18). The hexane extracts did not show antimicrobial activity nor toxicity against Artemia salina. The extract of Tibouchina francavillana Cogn. was the most active in sequestering the DPPH radical. The extracts showed cytotoxicity in THP-1 cells with the appearance of apoptotic bodies and cell death. The extracts of Miconia amoena, Clidemia sericea and Clidemia capitellata are non-toxic against Artemia salina and induce the formation of apoptotic bodies and cell death of the THP-1 lineage.


Introduction
The increased incidence of bacterial resistance, which has been intensified by the extensive and indiscriminate use of broad-spectrum antibiotics, has raised an urgent need for antimicrobial screening (Eloff and McGaw, 2006). Targeted investigations of plant products increase the chances of finding new and different chemical constituents with antimicrobial activities since plants have a renewable source of metabolites with enormous structural chemical diversity, which may have potential therapeutic relevance (França, 2017).
On the other hand, deleterious effects to DNA, proteins, lipids and other tissue biomolecules resulting from the excess production of free radicals in the human body have been indicated as being responsible for the development of chronic and degenerative diseases such as cancer and premature aging . Antioxidants of plant origin have been established as an important exogenous factor in combating the cumulative effects of free radicals and, thus, reducing damage to the body (Sies, 1993).
The family Melastomataceae of the order Myrtales Juss. ExBercht. & J. Presl, possesses about 5,000 species distributed among 170 genera (Almeda et al., 2016). In Brazil, the country with the greatest number of flowering plant species in the world, Melastomataceae is the fifth most represented group of angiosperms, with about 73 genera and 1,481 species (JBRJ, 2020).
Studies of the biological potential of the family Melastomataceae have demonstrated that its species have analgesic, antioxidant, antimicrobial, anti-inflammatory activities (Vasconcelos et al., 2006;Andreo et al., 2006;Sobrinho et al., 2017;Bomfim et al., 2020). These activities may be associated with the presence of several secondary metabolites such as flavonoids, tannins, terpenes and anthocyanidins (Calderón et al., 2002;Serna and Martínez, 2015;Bomfim et al., 2020) which are synthesized by plants in response to environmental conditions and often during specific stages of their development. Polyphenols can act by damaging and altering the permeability of the bacterial cell membrane (Cushnie and Lamb, 2005) and combating oxidative stress. Terpenes (Trombetta et al., 2005) and tannins (Scalbert, 1991) inactivate bacterial enzymes. Alkaloids, saponins, lignoids have antitumor and antimutagenic activities (Marques and Lopes, 2015;Lee and Hwangb, 2004;Ardalani et al., 2017).

Plant material
Aerial parts of the studied plant species were collected in a remnant of Atlantic Forest in the municipality of Alagoinhas, state of Bahia, Brazil (12°10'42.62" S, 38°24'39.52' W). The leaves were dried in an oven at 48-50 °C for 24 horas, manually pulverized and macerated with hexane. The solvent was in contact with the plant material for 72 hours until first filtration, which was repeated three times. The filtrate was then evaporated at room temperature and the extracts stored at 4 °C until time of analysis.

Phytochemical profile
The samples were analyzed for the presence of alkaloids (Petruczynik, 2012), terpenes and steroids (Harbone, 1998) using thin layer chromatography (CCD). Total phenolic compounds were quantified spectrophotometrically using the Folin-Ciocalteu reaction (Singleton et al., 1999). The absorbance reading of the samples was performed at 620 nm with a UV-Vis spectrophotometer. The same preparation conditions were used to construct the calibration curve for gallic acid (y = 3.8883x + 0.11141; R 2 = 0.9954) (Bomfim et al., 2020). The results were expressed in milligrams of gallic acid per gram of crude extract (mg EAG/g of extract). Total flavonoids was determined according to the method described by Arvouet-Grand et al. (1994). The content of total flavonoids was measured at 492 nm with a UV-Vis spectrophotometer and expressed in milligrams of quercetin per gram of crude extract (mg EQ/g of extract). The standard quercetin curve was obtained under the same sample preparation conditions (y = 1.2078x -0.1175; R 2 = 0.9889) (Bomfim et al., 2020).

In vitro evaluation of antimicrobial activity
Antimicrobial activity was evaluated by the agar discdiffusion technique (CLSI, 2003), against the bacteria Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 15442), Escherichia coli (ATCC 94863), Micrococus luteus (ATCC 10240) and Bacillus subtilis (ATCC 6633). Sterile 6-mm filter paper discs impregnated with 10 μL of plant extract (100.0 mg/mL) were used and chloramphenicol (0.1%) and DMSO (4%) served as controls. The plates were incubated in an oven at 37 °C for 24 hours. The results were read by measuring the inhibition halos of bacterial growth (in mm) and comparing them to their respective controls. The test was performed in triplicate and repeated. The final result was the average of the halo measurements.

In vitro evaluation of antioxidant activity
Antioxidant activity of the extracts was evaluated according to Brand-Willians et al. (1995), through capture assay of the free radical 2,2-diphenyl-1-picrylhydrazil (DPPH). 50 µL of the samples diluted in ethanol a volume of 1 mL of complete RPMI medium. The cells were incubated for 24 hours at 37 °C. After the cell culture period, cell morphology was evaluated using an inverted microscope (20x). Each test was accompanied by a control group containing cells with RPMI and wells containing cells with RPMI and DMSO (0.5%).

Statistical analysis
The results were expressed as the mean of three repetitions ± the standard deviation. The distribution of the groups was tested by the Kolmogorov-Smirnov and compared by One-way ANOVA followed by Duncan test at a 5% significance variable, using GraphPadPrism 5.0, IBM SPSS statistics and Minitab 18. The statistical analysis of the sample mean was performed in separate groups of total phenols, total flavonoids and percentage of sequestered free radicals.

Results
Phytochemical analysis of the extracts detected tannins, terpenes and steroids. The extract of Clidemia capitellata had a higher content of total phenols with a value of 205.95 mg EAG/g. For quantification of total flavonoids, the extract of Clidemia hirta showed higher content with 143.99 mg EQ/g, as shown in Table 1.
The hexane extracts of the studied plant species did not show antimicrobial activity, under the conditions of the test used. The results of the antioxidant activity, shown in Table 1, reveal that 60% of the evaluated extracts exhibit free radical scavenging activity equal to or greater than 25%, which was considered significant. The most active extracts in DPPH radical sequestration were Tibouchina francavillana (80.64% ± 0.90), followed by Miconia alborufescens (68.33% ± 1.01).
In the statistical analysis, the means of the samples didn't show significant difference for the groups of total phenols, total flavonoids and percentage of free radicals.
(6.0 mg / mL), were transferred to microplates containing 150 µL of ethanol. Subsequently, serial dilutions were performed and. 100 µL of the DPPH solution (0.5 mM) was added to each well. The plates were kept protected from light at room temperature for 1 hour, after which absorbance readings were performed at 492 nm with a UV-Vis spectrophotometer. The percentage of free radical scavenging (% SRL) was determined using the equation used by Moreira et al. (2005).

Toxicological test with Artemia salina Leach (1819)
The toxicity assay of the extracts was performed according to the methodology proposed by Meyer et al. (1982), with adaptations. The extracts were tested in final concentrations of 10 μg/mL, 100 μg/mL and 1000 μg/mL. Saline water and DMSO (1.0%) were used as control groups. All wells were subjected to artificial lighting. Individuals killed after 24 and 48 hours of exposure to the different concentrations were counted. The program GraphPadPrism 5.0 was used to obtain the average lethal concentration (CL 50 ).

Lineages and cell culture
Acute monocytic leukemia cell lineage (THP-1) was cultured in RPMI (5.0 mL) supplemented with 10% v/v fetal bovine serum (PBS) and 20 µg/mL penicillin/ streptomycin (1%), obtained from Cultilab. The cultures were replicated, sown and cultivated until confluence in a 25 cm 2 culture bottles at 37 °C in a 5% CO 2 atmosphere and controlled humidity. The THP-1 lineage was donated by Laboratório de Imunologia of Instituto de Ciências da Saúde (ICS) of Universidade Federal da Bahia.

Preliminary in vitro evaluation of cytotoxicity of plant extract to THP-1
Plant extracts were diluted in DMSO (100 μg/mL) and added to plates containing THP-1 cells (1x10 6 /well) in Table 1. Phytochemical profile and in vitro free radical scavenging activity of leaf extracts from ten species of the family Melastomataceae.

Sample
Total phenols (mg EAG/g) and Tibouchina urvilleana by Amzar and Iqbal (2017), showed anti-inflammatory activity. Amzar and Iqbal (2017) reported a hepatoprotective effect of the aqueous extract of Clidemia hirta, a property that was attributed to the antioxidant effect and the capacity for scavenging free radicals observed for the extract. The hexane extracts of the plant species studied here did not demonstrate toxicity towards Artemia salina at the tested concentrations. A similar result, although with methanolic extract, was recorded by Alves et al. (2000) for leaves of Miconia albicans. On the other hand, Coe et al. (2010) recorded activity for the aqueous extract of Tibouchina aspera. According to the literature, the negative results suggest the absence of cytotoxic compounds in sufficient concentrations to allow trypanomicidal and anti-tumor activities (Mclaughlin et al., 1991), among others. However, the hexane extracts from Miconia amoena, Clidemia sericea and C. capitellata were found here to induce the formation of apoptotic bodies and cell death in the THP-1 lineage, which is an important feature in the treatment of leukemias (Portt et al., 2011). This, therefore, evidences the antitumor potential of species of Melastomataceae, as also shown by Narasimham et al. (2017) for ethanol extract of Clidemia hirta against tumor cells of the Dalton's lymphoma ascites lineage; by Silva et al. (2016) for ethanol extract of Miconia munutiflora against cancerous cells of human liver (HepG2) and human leukemia (HL60) lineages, in a study of plants form Northeast Brazil; and by Cunha et al. (2008) for ethanol extract of Miconia fallax on tumor cells of the human uterine adenocarcinoma (HeLa) lineage.

Conclusion
This study provides important results regarding the extracts of Miconia amoena, Clidemia sericea and Clidemia capitellata for being atoxic against Artemia salina and for inducing the formation of apoptotic bodies and cell death of the THP-1 lineage. In addition, most of the extracts showed significant antioxidant capacities and can be considered good natural antioxidants. Nonetheless, future trials using fractions and substances isolated from the extracts should be conducted to corroborate and ensure the results presented here.
The hexane extracts did not show toxicity towards Artemia salina in the tested concentrations. Only extracts form Miconia amoena, Clidemia sericea and Clidemia capitellata induced the formation of apoptotic bodies and cell death in the THP-1 lineage.

Discussion
Plants are a renewable source of chemical compounds with great structural diversity (Kreis et al., 2017), with many constituting therapeutic components for various diseases or serving as a basis for the development of alternative pharmacological products (França, 2017). Despite the different biological applications attributed to products originating from plants, they still represent an exhaustive opportunity for further investigations (Atanasov et al., 2015).
Phytochemical evaluations of species of Melastomataceae have revealed the presence of phenolic compounds, triterpenes, steroids, anthocyanins, saponins, organic acids, tannins, anthraquinones and benzoquinones among their constituents as secondary metabolites (Jones et al., 1980;Santos and Tavares, 2003;Serna and Martínez, 2015;Bomfim et al., 2020). The solvent and extractive method used can influence variation in the levels of total phenols and total flavonoids present in extracts (Oliveira et al., 2016), as well as in biological activity. Dianita et al. (2011) evaluated the antimicrobial activity of hexane, ethyl acetate and methanol extracts of Clidemia hirta, and found that only the hexane extract did not have activity. Santos et al. (2012) showed promising antimicrobial results for ursolic acid and oleonic acid, isolated from dichloromethane extract of Tibouchina candoleana, against the endodontic bacterium Bacteroides flagilis. Celotto et al. (2003) evaluated the antimicrobial activity of hexane, dichloromethane and ethanol extracts from three species of Miconias and found the ethanol extracts of Miconia albicans and M. rubiginosa to be active against five of the ten species of bacteria evaluated and against Candida albicans; Miconia stenostachia only showed activity for C. albicans.
The present work also did not observe any antimicrobial activity for the hexane extracts of the ten evaluated species. Analyses with the ethyl acetate extract of Clidemia hirta and C. capitellata, however, demonstrated antibacterial activity against M. luteus, S. aureus, P. aeruginosa and B. subtilis (Bomfim et al., 2020). These results corroborate those of other studies (Dianita et al., 2011;Celotto et al., 2003).
The extracts that showed greater antioxidant activity had higher phenolic contents, corroborating previous studies for the family Melastomataceae (Bomfim et al., 2020). Research has demonstrated the direct correlation between the presence of phenolic compounds and antioxidant activity (Zielinski et al., 2014;Rani et al., 2018). During the capture and neutralization of oxidizing species, phenolic compounds form stable intermediates without causing damage to cell structures due to the resonance of the aromatic ring present in these compounds (Soares, 2002). Other studies, with different types of metabolites, triterpenes, flavonoids and steroids, isolates of Tibouchina granulosa for Pérez-Castorena (2014)