Phytochemical analysis and antimicrobial and antioxidant activities of Henriettea succosa (Aubl.) DC. leaves

Keyworks biological activity natural products secondary metabolites Henriettea succosa is a tree species consumed in abundance by birds, however, there is no report on its phytochemical profile and biological activity. This study performed the phytochemical screening and the antimicrobial and antioxidant potential of H. succosa leaves. The hexane (Hex), ethyl acetate (AcOEt) and methanol (MeOH) extracts of the leaves were evaluated for chemical composition by Thin Layer Chromatography and spectrophotometric analysis; the antimicrobial activity was determined by the Minimum Inhibitory Concentration (MIC) and Minimum Microbicide Concentration (MMC); antioxidant activity was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging, determination of the reducing power and the phosphomolybdenum complex reduction assay. The photoprotor action of the extracts was also evaluated. The results showed a higher content of phenolic compounds (444.08 ± 0.020 mg EAG/g) and tannins (414.37 ± 0.16 mg EAG/g) in the MeOH extract, which was effective against Staphylococcus aureus and Serratia marcescens, with MIC of 1 mg/ ml and CMM of 2 mg/ml. The MIC and MMC of AcOEt for Micrococcus luteus was 1 mg/mL, this was also considered the minimum concentration necessary for the Hex extract to act on the S. aureus strain. The MeOH extract showed greater antioxidant activity by the DPPH (79.09%) and reducing power (327.2 ± 0.00 mg EAA/g) methods, while the AcOEt extract showed greater activity by the phosphomolybdenum method (40.5%). However, none of the extracts showed a photoprotective effect against UV radiation. In summary, this study revealed that the leaves of H. succosa have secondary metabolites with bactericidal potential, in addition to antioxidant action.


INTRODUCTION
Human culture has always been influenced by plant biodiversity, mainly due to the medicinal properties it provides, thus boosting the search for bioactive compounds for the synthesis of new drugs, with secondary metabolites considered to be leading molecules both in natural form and in models for medicinal chemistry (Valli et al., 2012).
In this scenario, the Melastomataceae family stands out, which has more than 4,800 species distributed in different regions of the world, predominantly in the Neotropical area. In Brazil, it is considered the sixth largest family of Angiosperms, with 68 genera and more than 1500 species, and present in the most diverse plant formations. Among its genera is Henriettea, composed of 22 species (Arantes and Monteiro, 2002).
Henriettea succosa (Aubl.) DC., popularly known as mundururu-meloso in Brazil, is a tree species that can reach 5 to 13 m in height. It is a fruit tree used as food for birds frugivores and predominates in the Brazilian Atlantic Forest (Baumgratz, 2015;Cazetta et al., 2019;Costa et al., 2006). Most studies found evaluated the floristic biodiversity, structure and diversity of tree species and fungal systematics (Bonilla-Mata & Acosta-Vargas, 2020;Farnum, 2019;Hernández-Restrepo et al., 2020;Machado et al., 2016;Silva et al., 2021). Given absent works in the literature about the biological activities of the referred species, this research aimed to perform the phytochemical analysis and determine the antimicrobial and antioxidant effect of the H. succosa leaves.

MATERIAL AND METHODS
This study was conducted at the Microbiology and Bromatology Laboratories located at the Federal Institute of Pernambuco (IFPE) -Campus Barreiros, Barreiros/PE, Brazil.

Plant material
The H. succosa leaves were identified by the botanist M.Sc. Earl Celestino de Oliveira Chagas, and the exsiccatae deposited in the herbarium of the Environmental Institute of the State of Alagoas (IMA-AL) under number MAC 0050941.

Obtaining leaf extracts from H. succosa
The leaves (2000 g) of H. succosa were dried for four days, in an oven with controlled temperature (40 °C) and constant renewal of air, ground, and stored in an airtight container before extraction. The extracts were obtained through exhaustive maceration with hexane (Hex), or ethyl acetate (AcOEt), or methanol (MeOH), in the proportion of 1:10 (w/v) for seven days at room temperature. Subsequently, the material was filtered and the solvent was removed from the extract under reduced pressure, using a rotary evaporator at 65 °C.

Phytochemical profile by Thin Layer Chromatography (TLC)
Phytochemical analysis of H. succosa leaf extracts was performed by Thin Layer Chromatography (TLC), using silica gel plates precoated by F254 (Wagner and Bladt, 2001). In the mobile phase of the column, 5 mg / mL of hexane extracts (Hex: AcOEt -7:3), ethyl acetate extract (Hex: AcOEt -6:4), and methanolic extract were applied (Hex: AcOEt: MeOH -2:5:3). The eluents were used to fraction the compounds present in the extracts. Standard developers were cochromatographed for each class of phytochemical constituent. The plates were observed in an ultraviolet chamber at the wavelengths of 254 nm and 365 nm.

Quantification of total phenolic compounds
Total phenolics quantify in H. succosa leaf extracts was performed according to Silva et al. (2006), with modifications. In a test tube 0.2 ml of the extract (500 mg / ml), 0.5 ml of 10% Folin-Ciocalteu reagent (v/v) and 1 ml of sodium carbonate solution (Na2CO3) were added to a 7.5% (w/v). After stirring, the samples remained for 30 minutes in the dark and at room temperature. At the end of this time, 3 ml of distilled water were added and the reading was made at 760 nm. The standard curve was prepared with gallic acid, and the phenol content was expressed in milligrams of Gallic Acid Equivalent per gram of extract (mg GAE/g).

Determination of total tannins
This analysis was carried out according to Shad et al. (2012) method. 500 µL of each H. succosa leaf extracts (500 mg / mL) and 2.5 of the reagent Folin-Ciocalteu 10% (v/v) were added in a test tube. After stirring for 3 min, 2 mL of 20% (w/v) sodium carbonate (Na2CO3) was added, and then, after standing for 2 h in the dark, the reading was made at 725 nm. A standard curve was drawn up using tannic acid, and the tannin content was expressed in milligrams tannic acid equivalent per gram of sample (mg TAE/g).

Determination of total flavonoids
The analysis was carried out following the methodology of Barroso et al. (2011), where 1 ml of each H. succosa leaf extracts (500 mg/ml), 4 ml of distilled water, and 300 μl of sodium nitrite (NaNO2) at 25% (w/v) were added in a test tube. After standing for 5 min, 300 µL of aluminum chloride (AlCl3) 10% (w/v), 2 mL of sodium hydroxide (NaOH) at 1 mol/L, and 2,4 mL of distilled water were added and, then, the reading was made at 510 nm. A standard curve was drawn up using rutin, and the flavonoid content was expressed as milligrams rutin equivalent per gram of sample (mg RE/g).

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Microbicide Concentration (MMC)
The MIC of H. succosa leaf extracts in the strains used was determined by the broth microdilution method (Ingroff et al., 2002), where 90 µL of Muller Hinton Broth (MHB) were transferred to the wells of a 96-well microdilution plate with U-shaped bottom (Alamar, Diadema, SP, Brazil). Then, 90 μL of the product emulsion was inoculated from the third column of the plate (A3). Serial dilutions were performed, where a 90μL aliquot was taken from the most concentrated well to the next, producing concentrations of 0.03 mg / mL in the last column (A12). Por fim, 10 μL das suspensões bacterianas ou fúngicas foram adicionados em cada poço. The plates were incubated at 35 ± 2 °C for 24 h for bacteria, and 28 ± 2 °C for 48 h for the fungal strain. After that time, 30 μL of rezasurin (0.1 mg/mL) was added for quantitative analysis of microbial growth in the wells and determination of the relative antimicrobial activity. To determine the MMC, aliquots of 5 μL of the concentrations of the extracts that presented MIC were subcultured in Petri dishes containing CMH. After 24 h of incubation for bacteria (35 ± 2 °C) and 48 h for the fungus (28 ± 2 °C), a reading was performed to evaluate the MMC based on the controls. MMC was defined as the lowest concentration of the product capable of inhibiting bacterial or fungal growth or allowing growth of less than three CFU, resulting in a 99.9% bactericidal activity. The tests for antimicrobial activity were performed in duplicate and the results expressed by the arithmetic mean of the MIC or MMC.

Determination of reducing power
The reducing power of H. succosa leaf extracts was determined using the method proposed by Waterman and Mole (1994). 100 µL of the extracts diluted in methanol were used, with a final concentration of 0.5 mg/mL. Afterward, 8.5 ml of distilled water, 1 ml of the FeCl3 solution (0.1 M) were added and, after 3 min, 1 ml of the potassium ferricyanide solution (0.08 M) was added. After 15 min, the reading was performed at 720 nm. A standard curve was drawn up using ascorbic acid, and the results obtained were expressed in milligram equivalent to ascorbic acid per gram of the extract (mg EAA/g).

Sequestering activity of the radical 2,2diphenyl-1-picrilhhydrazi (DPPH)
The test to determine the ability of H. succosa leaf extracts to sequester the free radical DPPH (2,2-diphenyl-1-picrilhidrazi) was carried out according to Cavin et al., (1998), with modifications, where 0.1 ml aliquot of each extract was added to 3.9 ml of a solution of DPPH (0.004%, w/v) and, after resting for 30 min in the dark, the reading was performed at 517 nm. Ascorbic acid was used as a standard and the percentage of the sequestering activity (% SA) of the DPPH radical was calculated using the equation: Where: Abscontrol is the absorbance of DPPH + ethanol and Abssample is the absorbance of radical DPPH + sample (sample or standard). The antiradical efficiency was established using linear regression analysis and the results were expressed through the concentration of the sample necessary to obtain half of the sequestering activity of the DPPH radicals ± average standard error (EC50 ± A.S.E.).

Reduction test for the phosphomolybdenum complex
The antioxidant capacity of H. succosa leaf extracts was evaluated using the phosphomolybdenum method (Prieto et al., 1999). Briefly, 0.3 mL of different concentrations (25, 50, 100 and 150 µg/mL) of the extracts were combined with 3 mL of the reactive solution (0.6 mol/L sulfuric acid, 28 mmol/L sodium phosphate, and 4 mmol/L ammonium molybdate) in test tubes, which were incubated at 95 °C for 90 min. The absorbance of the solution was measured at 695 nm against a control (0.3 mL of distilled water and 3 mL of the reagent). For calculation purposes, the rutin pattern was considered to be 100% antioxidant activity.

Photoprotective activity
The evaluation of the photoprotective activity of H. succosa leaf extracts was performed in vitro according to Mansur et al. (1986). This test aims to evaluate whether the samples provide protection against UVA and UVB radiation. For that, each extract was diluted in ethanol until a concentration of 100 µg / mL was obtained. The absorbance reading was performed between 290 and 320 nm. The Sun Protection Factor (SPF) was calculated using the equation: SPF (spectrometry) = FC x 290∑320 ЕE(λ) x l (λ) x abs (λ)

Statistical analysis
The results of the tests were subjected to Tukey test through the Sisvar statistical program at the level of 5% probability.

Phytochemical profile
The phytochemical profile of Hex, AcOEt and MeOH extracts from the leaves of H. succosa revealed the presence of different compounds (Table 1). Hex extract showed curmarins, triterpenes and steroids, AcOEt, flavonoids, tannins and triterpenes, and in contrast, MeOH presented only two of the nine compounds analyzed: flavonoids and tannins.
Due to the nonpolar nature of the hexane extract, there is a trend in lipophilic compounds extraction, such as methyl esters of fatty acids, traces of triterpenes, and coumarins (Figueiredo et al., 2008). Thus, the chemical composition of the Hex extract of the leaves of H. succosa adds fat-soluble compounds driven by the lipophilicity of the solvent. Also, the variation in these components can be influenced by genetic and environmental factors of the plant, a condition that distinguishes the metabolites between species of the same family.
Coumarins can be found in the roots, flowers, fruits, and leaves of vegetables, and their presence may be related, among other activities, to the antimicrobial and antioxidant actions of plant extracts (Detsi et al., 2017), activities reported in the present study. Generally, more complex terpenes, such as triterpenes are abundant in plants and confer bioactive properties in the prevention and treatment of malignant tumors, antiinflammatory, antimicrobial, antioxidant effect, among others (Cháirez-Ranpirez et al., 2016;Isah et al., 2016).
The presence of triterpenes in plant extracts is also related to the synthesis of plant phytosterols and steroid hormones, such as plant brassinosteroids (Bishop and Koncz, 2002), a common peculiarity in the phytochemical findings of the present study, since the presence of triterpenes and steroids in the hexane extract of H. succosa leaves.
The flavonoids in the AcOEt and MeOH extracts may be responsible for the pharmacological properties of the species, including antimicrobial, anti-inflammatory, and antioxidant action (Tsuchiya, 2010). Thus, they can contribute to the possible effects of the extracts on dermatological problems. Table 2 shows the results of the determination of total phenolics, tannins and flavonoids from H. succosa leaf extracts. There was a significant increase in these classes of metabolites in the MeOH extract, with a predominance of phenolic compounds (444.08 mg EAG/g) and tannins (414.37 mg EAT/g). The hexane extract showed concentrations below the minimum required for quantification (data not shown).

Measurement of total phenolics, tannins and flavonoids
Methanol is a polar solvent capable of promoting greater solubility and interaction between the molecules present in the extract (Felhi et al., 2017), a condition that may be related to the increase in the content of phenolic compounds, tannins, and flavonoids verified in this study, when compared to the AcOEt extract and the hexane.
In plants, phenolic compounds (flavonoids, simple phenols, tannins, stilbenes) are components of the electron transport chain in mitochondria and chloroplasts, in addition to being involved in oxidation-reduction processes, growth regulation, and plant development, a mechanism related to its significant increase in quantification tests (Babenko et al., 2019). Also, they are bioactive metabolites that perform antimicrobial, antioxidant, anti-tumor, anti-inflammatory, anti-aging, and chemopreventive action (Metsämuuronen and Sirén, 2019).
However, as studies with species of the family Melastomataceae are scarce, comparative analyzes related to the phytochemical profile are difficult. However, chemical investigations with the genus Miconia, from the same family, revealed the presence of flavonoids, triterpenes, steroids, phenolic acids, quinones, tannins, and lignans (Sabbag Cunha et al., 2019), similar findings, from the phytochemical point of view to those found in the present study.

Antimicrobial activity
For the antimicrobial evaluation of the H. succosa leaf extracts (Table 3) against the tested microorganisms, the MIC was determined, which refers to the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. Already MMC refers to the lowest concentration of antimicrobial that will prevent the growth of an organism after subculture on to antibiotic/extractsfree media (Andrews, 2001). The AcOEt extract at a concentration of 0.5 mg/mL had significant bacteriostatic and fungistatic activities, inhibiting gram-positive bacteria: S. aureus, E. faecallis, gram-negative: S. marcencens, and the fungal strain C. albicans. In contrast, doses of 4 mg/mL of this extract were needed to inhibit the growth of B. subtilis and P. aeruginosa. The effective antimicrobial effect of the MeOH extract was against the M. luteus strain, with MIC of 0.25 mg/mL and MMC of 1 mg/mL. However, the Hex extract did not demonstrate significant bactericidal and fungicidal activities against S. marcencens and C. albicans, respectively, with MMC of 8 mg/mL. According to Rios and Recio (2005), an extract can be considered promising when it is active at a concentration below 0.1 mg/mL. However, the effect of the product capable of causing the eventual death of a microorganism occurs when the MIC/MMC ratio is between 1 and 2 (Hafidh et al., 2011). Therefore, the three extracts were bactericidal against most of the tested strains, however, none of the extracts showed activity against C. albicans. Low antifungal efficacy may be related to chemical decomposition or microbial degradation of organic compounds by target microorganisms (Rongai et al., 2017). Gilbert et al. (2014) observed the antibacterial effect of methanolic extract from the leaves of Dissotis thollonii Cogn against strains of E. coli, a condition favored by the significant presence of polyphenols and flavonoids in species of the family Melastomataceae. Such secondary metabolites can promote rupture of membrane lipopolysaccharides contained in gram-negative bacteria such as E. coli, thus facilitating the entry of antimicrobial compounds to target sites within the cell (Zakaria et al., 2011). In addition, bioactive products can inhibit the synthesis of essential bacterial proteins or act on cell wall receptors, and thus prevent the growth of the pathogen (Lavigne, 2009). Thus, possibly the antibacterial potential of H. succosa leaf extracts was amplified by the presence of triterpenes in the sample, especially in the AcOEt extract, in which there was an increase in the number of inhibited microorganisms (Saleem et al., 2010).

Antioxidant activity
The antioxidant capacity of the Hex, AcOEt, and MeOH extracts from the leaves of H. succosa was determined by the tests: 1 -Sequestering activity of the DPPH radical; 2 -Determination of the reducing power; 3 -Phosphomolybdenum complex formation method, summarized in figures 1 and 2. The free radical DPPH can be reduced in the presence of antioxidant compounds, capable of donating hydrogen atoms and, thus, making it a stable diamagnetic molecule. The MeOH extract exhibited a greater capacity to reduce the DPPH radical (79.09 ± 0.001%) when compared to the AcOEt extract (43.53 ± 0.050%).
The polarity of the extractive solvents contributes to the increase in the percentage of bioactive molecules, a condition related to the content of phenolic and flavonoid compounds present in the MeOH extract, however, the discrepancy in the water-solvent ratio can influence the solubility of the phytochemicals and thus alter the possible biological effects (Rahaiee et al., 2015), a phenomenon observed in this study, since, although the AcOEt extract contains greater varieties of metabolites when related to MeOH extract, it presented a low capacity for eliminating the DPPH radical. Nzogong et al. (2018) evaluated the antioxidant capacity of two plants of the Melastomataceae family and observed low elimination of the DPPH radical by ethanolic extracts. The discrepancy in the antioxidant percentage of crude extracts from plants inserted in the same family may be related to variations in the content of antioxidant compounds, such as phenolic derivatives, therefore, the existence of such compounds could explain the antioxidant activity found in the species studied in this study.
The reducing activity of H. succosa leaf extracts was also carried out using the metal ion reducing potency method, a reaction that promotes the reduction of Fe +3 in Fe +2 by antioxidant compounds (Waterman and Mole, 1994). It was inferred that the MeOH extract had a greater capacity to reduce Fe +3 (327.2 ± 0.00 mg EAA/g) when compared to the AcOEt extract (227.8 ± 0.01 mg EAA/g). The reducing properties of plant extracts are mostly attributed to the cleavage of the free radical and therefore the donation of hydrogen molecules, besides, they prevent the formation of peroxide precursors (Rumzhum et al., 2012).
In the phosphomolybdenum complex formation test, the phosphate/molybdenum complex is formed at acid pH, and the subsequent reduction of molybdenum to molybdenum by the plant extract (Prieto et al., 1999). The results showed that the AcOEt extract promoted a greater reduction of molybdenum (40.5 ± 0.462%), an effect that may be related to the content of chemical compounds present in the extract, with synergism between them.

Photoprotective activity
The method described by Mansur is widely used to measure the sun protection factor (SPF) in vitro, its principle is to relate the absorbance of the test product with the erythematous effect of radiation and the intensity of light at wavelengths from 290 to 320 nm (UVB region in the spectrum) (Violante et al., 2009). Table 6 shows the SPF of Hex, AcOEt, and MeOH extracts from H. succosa leaves.
At a concentration of 100 µg/mL, the MeOH extract was shown to have a higher absorbance of ultraviolet light B (2.34 ± 4.13), followed by AcOEt (2.14 ± 3.02) and Hex (1.98 ± 3.45). According to Anvisa (2012), photoprotective products must have a minimum SPF value of 6, thus, the results obtained for the extracts used do not meet the limit required.
The increase in the protection factor of the MeOH extract is probably due to higher concentrations of photoprotective compounds in the sample, in general, phenolic compounds induce defense against UV radiation, since similar substances in plant species perform such functions (Rozema et al., 2001) Sun Protection Factor (SPF); Hex: hexane; AcOEt: ethyl acetate; MeOH: Methanol.

CONCLUSIONS
H. succosa leaf are a source of secondary metabolites, especially coumarins, flavonoids, tannins and steroids, and MeOH has a greater capacity to extract such compounds in significant concentrations. In addition, the tested products showed antibacterial and fungistatic activity, with emphasis on AcOEt, effective against most strains, except for S. aureus, E. faecallis and C. albicans. In addition, the extracts prevented and/or controlled oxidative stress, being potentially antioxidant, however, they were unable to exercise photoprotection in vitro against UV-B-induced damage. Our results are pioneering in the phytochemical and biological exploration of H. succosa leaves, inferring that the species has pharmacological and chemical potential for future studies, however, a more in-depth approach to the species is necessary in order to identify specific compounds and other bioactivities.