Universidade Federal de Santa Maria
Ci. e Nat., Santa Maria, v. 43, e78, 2021
DOI: 10.5902/2179460X65941
ISSN 2179460X
Submitted: 25/05/2021 • Approved: 29/07/2021 • Published: 30/08/2021
Chemistry
Phenolic compounds, antimicrobial activity, cytotoxicity and identification of phytochemicals present in Inga marginata Willd seeds
Compostos fenólicos, atividade antimicrobiana, citotoxicidade e identificação de fitoquímicos presentes em sementes de Inga marginata Willd
Déborah Cristina Barcelos FloresI
Caroline Pagnossim BoeiraII
Clarice Madalena Bueno RolimIII
Daniele Rubert Nogueira LibrelottoIV
Frederico Luiz ReisV
Liziane Maria Barassuol MorandiniVI
Ademir Farias MorelVII
Claudia Severo da RosaVIII
I Master in Science and Technology in Food, Federal University of Santa Maria, Department of Technology in Food Science, Santa Maria, RS, Brazil
https://orcid.org/0000-0003-1674-5210 – deborahbflores@gmail.com
II Master in Science and Technology in Food, Federal University of Santa Maria, Department of Technology in Food Science, Santa Maria, RS, Brazil
https://orcid.org/0000-0003-1674-5210 – carolinepagnossim@hotmail.com
III PhD in Pharmaceutical Science and Technology, Professor, Federal University of Santa Maria, Industrial Pharmacy Department, Santa Maria, RS, Brazil
https://orcid.org/0000-0002-9634-8970 – clarice.rolim@gmail.com
IV PhD em Research, Development and Control of Medicines, Professor, Federal University of Santa Maria, Industrial Pharmacy Department, Santa Maria, RS, Brazil
https://orcid.org/0000-0003-0570-5555 - daniele.rubert@gmail.com
V Master in Organic Chemistry, Federal University of Santa Maria, Chemistry Department, Santa Maria, RS, Brazil
https://orcid.org/0000-0001-5269-337X – reis.fredericoreis@gmail.com
VI PhD in Chemistry, Professor, Federal University of Santa Maria, Chemistry Department, Santa Maria, RS, Brazil
https://orcid.org/0000-0001-8257-8444 - lizianemorandini@gmail.com
VII PhD in Chemistry, Professor, Federal University of Santa Maria, Chemistry Department, Santa Maria, RS, Brazil
https://orcid.org/0000-0003-3536-2418 - ademirfariasm@gmail.com
VIII PhD in Food Science, Professor, Federal University of Santa Maria, Department of Technology in Food Science, Santa Maria, RS, Brazil
https://orcid.org/0000-0001-6931-7741 - claudiasr37@yahoo.com.br
ABSTRACT
Brazil has an invaluable source of plants and fruits rich in phenolic compounds important to health, many of which have yet to be investigated. Inga marginata Willd is a fruit that can be found throughout Brazil, and its seeds are rich in phenolic compounds and antimicrobial activity, thus making their extraction and characterization highly relevant. This study aimed to determine the total phenolic compounds, antioxidant capacity, antibacterial and antifungal activities, cytotoxicity evaluation, and characterize the phytochemical compounds present in the extract. This study demonstrated that extraction by agitation at 30 ºC presented the highest yield of total phenolic compounds (48.23 mg GAE g-1), antioxidant capacity (40.34 mg TEAC g-1), and IC50 (2.60 mg mL-1). The extracts at 30 and 60 ºC inhibited all microorganisms tested, and the temperature of 30 ºC acted as a bactericide and fungicide at low concentrations. Ten phytochemical compounds were found, mainly being antioxidants and antimicrobials. The cytotoxicity assays showed that Inga marginata seeds do not present cellular cytotoxicity up to the concentration of 250 µg mL-1, maintaining cell viability above 90%.
Keywords: Total phenolics; Extraction; Identification by ESI-ToF-MS; Toxicity; Antimicrobial activity
RESUMO
O Brasil possui uma fonte inestimável de plantas e frutos ricos em compostos fenólicos importantes à saúde, muitos dos quais ainda não foram investigados. O Inga marginata Willd é um fruto que pode ser encontrado em todo o território brasileiro, e suas sementes são ricas em compostos fenólicos e atividade antimicrobiana, tornando sua extração e caracterização altamente relevantes. Este estudo teve como objetivo, determinar os compostos fenólicos totais, a capacidade antioxidante, atividades antibacteriana e antifúngica, avaliação da citotoxicidade e caracterização dos compostos fitoquímicos presentes no extrato. O estudo demonstrou que a extração por agitação a 30 ºC, apresentou maior rendimento de compostos fenólicos totais (48.23 mg GAE g-1), capacidade antioxidante (40.34 mg TEAC g-1), e IC50 (2.60 mg mL-1). Os extratos a 30 e 60 ºC da semente de Ingá inibiram todos os micro-organismos testados, sendo que a temperatura de 30 ºC atuou como bactericida e fungicida em baixas concentrações. Foram encontrados dez compostos fitoquímicos, principalmente antioxidantes e antimicrobianos. Os ensaios de citotoxicidade mostraram que a semente de Inga marginata não apresentaram citotoxicidade celular até a concentração de 250 µg mL-1, mantendo a viabilidade celular acima de 90%.
Palavras-chave: Fenólicos totais; Extração; Identificação por ESI-ToF-MS; Toxicidade; Atividade antimicrobiana
1 INTRODUction
Brazil has a rich biodiversity, and the Amazon rainforest is an invaluable source of plants rich in bioactive compounds. With the growing search for a natural and healthy life, consumer interest in natural bioactive compounds has increased (SILVA; ROGEZ; LARONDELLE, 2007). Bioactive compounds constitute the majority of active ingredients present in plant parts, provide many benefits to human health, and act in food preservation (HARUNA; YAHAYA, 2021). The genus Inga belongs to the Fabaceae family, and plants of this family can be found throughout Brazil and South and Central America. The Inga fruit of the species Inga marginata Willd is popularly known as “Ingá-mirim” or “Ingá feijão” characterized as long yellow-green pods containing 2 to 15 seeds of green color and flaky-sweet white flesh (LIMA; SANTOS; LA PORTA, 2018; POSSETE; RODRIGUES, 2010). Previous studies with the genus Inga have shown that phenolic compounds are present in the leaves of Inga umbellifera and Inga goldmani (LOKVAM; COLEY; KURSAR, 2004), in addition to Inga edulis leaves (NASCIMENTO; SOBRINHO; SOUZA; De SOUZA; SOUSA, 2021). From the leaves and bark of the species Inga edulis, phenolic compounds and flavonoids have also been isolated and their antioxidant capacity evaluated (DIAS; SOUZA; ROGEZ, 2010; TCHUENMOGNE et al., 2013). In fruit and vegetable residues, for example, seeds are commonly discarded despite having excellent nutritional and functional characteristics. Studies have suggested that antioxidants in fruits and their residues may reduce the risk of cancer and related mortality. In addition, the antioxidant potential and functional properties have been investigated, especially to replace the use of synthetic antioxidants in food products that may represent a health risk (SUHAIL et al., 2012). Phenolic compounds are involved in the defense mechanism of plants and protect them against ultraviolet radiation and pathogens (DAI; MUMPER, 2010). Various biological effects have already been studied, including antioxidant, anti-inflammatory, antibacterial, and antiviral activities (TANASE et al., 2019). This demonstrates that numerous studies can be conducted with this class of compounds and underlines the importance of further research to discover and identify new compounds. Phenolic compounds are attractive due to their antioxidant activity that reduces oxidative stress and prevents or delays oxidation by eliminating free radicals, both in the human body and food preservation (KOOLEN; SILVA; GOZZO; SOUZA; SOUZA, 2013).
The use of antioxidants derived from natural resources has been gaining attention due to their health benefits, including preventing cardiovascular diseases, inflammation, and aging-related disorders (AQIL et al., 2012). In addition, the use of these plant extracts has become a promising alternative for preserving processed foods, reducing reliance on other chemical additives, and prolonging the shelf life of products (MUNIR et al., 2018). The search for substances that can help develop new drugs has increased the interest of the food industry and research institutions in natural products. In this sense, preliminary tests allow researchers to make inferences about the toxicity of such products. One model of toxicity evaluation that is gaining strength is cytotoxicity, which uses mammalian cells and evaluates the damage caused to cells, cell colony formation, and cell viability. Cell cultures have become a much-appreciated model because they are reproducible, sensitive, fast, and do not require animal testing (BEDNARCZUK; VERDAM; MIGUEL; MIGUEL, 2010; ROGERO; LUGÃO; IKEDA; CRUZ, 2003).
Given the above, this study aims to determine the phenolic compounds, antioxidant capacity by in vitro methods, evaluate antibacterial and antifungal activities, characterize the phytochemical compounds by electrospray ionization time-of-flight mass spectrometry (ESI-ToF-MS) in the extract, and the cytotoxicity of Inga marginata Willd seeds.
2 MATERIALS AND METHODS
2.1 Fruit collection and sample preparation
Inga marginata Willd fruits were collected in the Federal University of Santa Maria, Santa Maria (-29.7215°S and 53.7184°W), southern Brazil. The fruits were opened manually and the seeds were reserved. Pre-drying was performed at 45 ± 5 °C for 48 h in a forced air circulation oven (Marconi, MA-035/100, Piracicaba, Brazil) according to Djikeng et al., (2018), with modifications. After drying, the seeds were ground in a knife mill (Willy, SL-31), passed through a 20-mesh sieve, and stored at -18 °C. The species Inga marginata Willd was registered under number ACD526F in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SISGEN).
2.2 Obtaining the hydroalcoholic extracts
Following the method of Victório, Lage, and Kuster (2010) with modifications, the extracts were obtained through solid-liquid extraction using 2 g of pre-dried and ground sample, and 40 mL of 70% ethanol (1:10; p/v) was added to a beaker. The extraction time was set to 25 min and, and just the temperatures of 30 and 60 ºC were used according to Piovesan et al., (2017). The samples were then subjected to constant stirring with a mechanical stirrer (Marconi, MA-039, Piracicaba, Brazil). After extraction, each extract containing the solvent and ground material mixture was subjected to centrifugation (Centrilab, SH-120) at 3000 rpm for 10 min and filtered. The volume was adjusted and the samples were then stored in amber flasks in a freezer (-18 °C).
2.3 Total phenolic content
The total phenolic compounds (TPC) were determined by the Folin-Ciocalteu spectrophotometric method of Roesler, Malta, Carrasco, Holanda, Souza, and Pastore (2007), with modifications. The absorbance was read at 760 nm in a spectrophotometer (Biospectro, SP-220, São Paulo, Brazil). A standard curve was performed for quantification (y = 0.0012x + 0.021 R² = 0.9953) using gallic acid as a positive control, and the results were expressed as milligrams of gallic acid per gram of dry sample (mg GAE g-1).
2.4 Radical scavenging activity determination
For the antioxidant assays, the radical scavenging activity of the extracts was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Brand-Williams, Cuvelier, and Berset, 1995). Readings were performed in a spectrophotometer (Biospectro, SP-220) at a wavelength of 517 nm. The free radical scavenging capacity was calculated according to Equation 1 and expressed as percent inhibition of radical oxidation. The scavenging activity was measured by decreasing the absorbance of the samples compared to the DPPH standard.
|
|
(1) |
Where %DPPH Sca is the percentage of inhibited DPPH, Abssamp is the absorbance of the sample, AbsNC is the negative control, and AbsPC is the positive control of the measured calibrated curves. To determine the IC50, the equation of the line obtained from the absorbance values (AA%) of the increasing concentrations of the samples was used, replacing the value of Y by 50, and obtaining the value of X as the concentration of the sample with the capacity to reduce the DPPH by 50%. The result is expressed as Trolox Equivalent Antioxidant Capacity (µmol TEAC g-1).
2.5 Antibacterial activity and antifungal activity
Standard strains from the American Type Culture Collection (ATCC) were used for microbiological assays. For the extracts obtained at 30 and 60 ºC, the antibacterial and antifungal activities were evaluated by the broth microdilution method, and the 96-well plate analysis was performed by spectrophotometry at a wavelength of 620 nm (NCCLS, 2017, 2018). A collection of twenty-four microorganisms was used, including five Gram-positive bacteria: Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 11778), Enterococcus fecalis (ATCC 19433), Enterococcus ssp. (ATCC 6589), and Bacillus subtilis (ATCC 19659), and ten Gram-negative bacteria: Salmonella enteric serovar Typhimurium (ATCC 14028), Escherichia coli (ATCC 25922), Shigella sonnei (ATCC 25931), Enterobacter aerogenes (ATCC 13048), Salmonella enteritidis (ATCC 13076), Shigella flexneri (ATCC 12022), Pseudomonas aeroginosa (ATCC 27853), Morganella morganii (ATCC 25829), Proteus mirabilis (ATCC 25933), and Klebsiella pneumoniae (clinical isolate). Nine yeasts were also used: Candida parapsilosis (ATCC 22019), Candida tropicalis (ATCC 750), Candida albicans (ATCC 10231), Candida glabrata (ATCC 2001), Candida dubliniensis (ATCC MYA-577), Candida krusei (ATCC 6258), Cryptococcus gatti (ATCC 56990), Cryptococcus neoformans (ATCC 28952), and Saccharomyces cerevisiae (ATCC 2601). The minimum inhibitory concentration (MIC) was determined in 96-well culture plates by the microdilution method using a microorganism suspension at a density of 105 CFU mL-1 with Casein Soy Broth incubated for 24 h at 37 °C for bacteria, and Sabouraud Broth incubated for 48 h 25 °C for the yeast (NCCLS, 2017, 2018). Cultures that showed no growth were used to re-inoculate the plates (Casein Soy Broth and Sabouraud) and determine the minimum lethal concentration (MLC). The appropriate blanks were assayed simultaneously, and all samples were tested in triplicate. The results are expressed as a function of MIC and MLC.
2.6 Inga seed extract characterization by ESI-ToF MS
The extract at the temperature that obtained the highest phenolic and antioxidant content from the Inga seed was submitted to characterization by electrospray ionization time-of-flight mass spectrometry (ESI-ToF MS; Xevo G2 Qtof, Waters Inc., Milford, USA). Then, 50 μL of the extract was used, which was previously filtered through a syringe with a PTFE membrane (13 mm x 0.22 µm) and then diluted in 2 mL of acetonitrile (ACN)/H2O (1:1) with 0.1% formic acid in positive mode (BIZZI et al., 2019). Moreover, in negative mode, the extract was solubilized in 2 mL of methanol and 50 μL of ammonium hydroxide. Mass spectra were acquired from 50 to 1000 Dalton (Da), and detection by ESI-TOF MS was performed in positive ion and negative ion modes with a capillary voltage of 2.00 kV, cone voltage of 20 V, and extractor cone voltage of 3.0 V. The flow rates of the desolvation gas and gas cone were 500 and 10 L h-1, respectively. The desolvation temperature was set to 500 °C and the source temperature to 150 °C. As for the negative ion mode, the capillary voltage was 2.5 kV, cone voltage was 40 V, and the extractor cone voltage was 4.0 V. The flow rates of the desolvation gas and gas cone were 200 and 10 L h-1, respectively. The desolvation temperature was set to 450 °C and the source temperature to 150 °C. System control and data acquisition were performed by using MassLynx V 4.1 software.
2.7 Inga seed cytotoxicity
The Inga marginata Willd seeds were freeze-dried and dissolved in 1 mL of phosphate-buffered saline (PBS) without dimethylsulfoxide (DMSO). Swiss albino mouse fibroblast cell line (3T3) cultured in Dulbecco’s Modified Eagle Medium (DMEM) and supplemented with 10% (v/v) fetal bovine serum (FBS) at 37 °C and 5% CO2 was used. The cells were harvested using trypsin-EDTA when they reached a confluence of 80%, and the samples were seeded in 96-well plates at a density of 1 x 105 cells mL-1 and incubated for 24 h under 5% CO2 at 37 °C. Afterward, the medium was replaced with 100 μL of fresh medium supplemented with 5% (v/v) FBS with the samples (concentration from 15.6 to 500 μg mL-1), the control cells (only the medium containing 5% (v/v) FBS, the remaining cells were exposed to each treatment for 24 h, after which the medium containing the samples was removed, and 100 μL of MTT (0.5 mg mL-1) or NR dye (50 μg mL-1) was added to each well. Then, the plates were incubated for 3 h, the medium removed and single washing of the NRU assay wells carried out in PBS, followed by adding 100 µL of DMSO (MTT assay). For the NRU assay, 100 µL of a solution containing 50% absolute ethanol and 1% acetic acid in distilled water was added. After 10 min of stirring at room temperature, the absorbance of the solutions was measured at 550 nm using a microplate reader (Multiskan FC, Thermo Scientific, San Jose, CA, USA). The cytotoxic effects of the samples were evaluated by 2,5-diphenyl-3-(4,5-dimethyl-2-thiazolyl) tetrazolium bromide (MTT) as described by Mossmann (1983) and by neutral red uptake (NRU) viability assay according to Borenfreund and Puerner (1985). The cytotoxicity of the sample was expressed as percent viability relative to untreated control cells (average of untreated cells adjusted at 100% viability). In terms of IC50 (concentration required for 50% cell death), it was calculated by adjusting the percentage of the cell survival curve against the sample concentrations.
2.8 Statistical analysis
All experiments were performed in triplicate, and the results were expressed as mean values and standard deviation (SD), submitted to analysis of variance (ANOVA) and Tukey’s test with a significance level of 95% (p<0.05) using Statistica 10.0 software (Stat Soft, Inc., USA). For cytotoxicity analysis, the data were submitted to ANOVA, and the means were compared by Dunnet’s test in multiple comparisons, considering there was a significant difference when a level of p<0.05 was reached through the GraphPad Prism 6.0 software.
3 RESULTS AND DISCUSSION
3.1 Evaluation of total phenolic compound content, antioxidant capacity of Inga seeds, and IC50
The total phenolic content of the Inga seed extracts and radical scavenging activity are presented in Table 1.
Table 1 – Content of total phenolic compounds and radical scavenging activity (DPPH and IC50)
|
Temperature |
Total phenolics (mg GAE g-1) |
DPPH (µmol TEAC g-1) |
IC50 (mg mL-1) |
|
30ºC |
48.23a ± 1.90 |
40.34a ± 2.04 |
2.60b ± 1.48 |
|
60ºC |
36.89b ± 2.49 |
34.15b ± 2.75 |
3.80a ± 1.97 |
Source: Authors (2021)
In where: Results are expressed as mean ± SD. GAE = Gallic acid equivalent; DPPH = 2,2-diphenyl-1-picrylhydrazyl. IC 50 = inhibitory capacity. TEAC = Trolox equivalent antioxidant capacity. Values expressed as mean ± standard deviation with different letters on the same column indicating a significant difference (p<0.05) by Tukey’s test.
The extract obtained at 30 °C showed the highest total phenolic content (48.23 mg GAE g-1), which was statistically different from the temperature of 60 °C (36.89 mg GAE g-1) (Figure 1A). Phenolic compounds are important to human health due to their high antioxidant activity and their effects against anti-inflammatory, cardiovascular, and neurodegenerative diseases, including cancer (SCALBERT; JOHNSON; SALTMARSH, 2005).
Figure 1 – Yields of total phenolic compounds (A) and antioxidant capacity by the DPPH method (B) and inhibitory capacity (IC50) (C) of Inga seed extracts obtained at 30 and 60ºC
|
|
|
|
|
|
Source: Authors (2021)
The most proven mechanism of action of these compounds is eliminating free radicals, which reduces oxidative stress (SILVA; ROGEZ; LARONDELLE, 2007). Several external and internal factors can originate the degradation process of phenolic compounds, and temperature is the most important factor that promotes degradation reactions. During the extraction at the highest temperature, oxidative enzymes present in the sample are released during the extraction and promote degradation reactions (Liazid, Palma, Brigui, and Barroso, 2007). Mendoza, Martínez, Martínez, Barba, and De Ortiz (2016) found low phenolic compound content in Inga Paterno seeds (0.55 mg GAE g-1), and this low content may be due to not using extraction parameters (e.g., temperature) and use of extracting solvents to obtain the phenolic compounds.
The antioxidant capacity at 30 ºC (40.34 µmol TEAC g-1) was statistically higher compared to the extract obtained at 60 ºC (34.15 µmol TEAC g-1) (Table 1 and Figure 1B), which may be because the stability of the antioxidant compounds varies according to their composition and distribution in plants, fruits, or seeds. Some of these compounds are unstable at high temperatures, prone to oxidation, or thermolabile or volatile, thus decreasing the concentration of antioxidant compounds in the extracts (ALARA; ABDURAHMAN; UKAEGBU, 2021). Regarding the inhibitory capacity of the extract (IC50), the temperature of 30 ºC (2.60 mg mL-1) (Table 1 and Figure 1C), was the one with the lowest IC50 value, meaning the plant has high antioxidant power. The IC50 is a parameter used to determine the antioxidant potential of plants and demonstrates the amount of plant extract required to capture DPPH radicals by 50% (NEGRELLE; GOMES, 2007).
3.2 Antimicrobial activity of Inga seed extracts at 30 and 60 ºC
The antibacterial (Gram-positive and Gram-negative bacteria) and antifungal activity of the obtained extracts were analyzed (Tables 2 and 3). The MIC and MLC values of each extract were tested and compared to controls (antibiotics).
Table 2 – Antibacterial activity (MIC and MLC in μg/mL) of the extracts obtained at 30 and 60 ºC from the Inga marginata Willd seeds
|
Gram-positive bacteria |
Sample |
|||||||
|
Seed 30ºC |
Seed 60ºC |
Chloramphenicolª |
Ampicilianª |
|||||
|
MIC |
MLC |
MIC |
MLC |
MIC |
MLC |
MIC |
MLC |
|
|
Bs |
62.5 |
125 |
125 |
500 |
62.5 |
50 |
100 |
200 |
|
Bc |
62.5 |
- |
62.5 |
- |
3.12 |
12.5 |
200 |
200 |
|
Eph |
62.5 |
- |
62.5 |
- |
3.12 |
12.5 |
1.56 |
12.5 |
|
Sa |
31.2 |
500 |
62.5 |
250 |
1.56 |
6.25 |
200 |
200 |
|
Gram-negative bacteria |
Sample |
|||||||
|
St |
250 |
- |
250 |
- |
3.2 |
12.5 |
200 |
200 |
|
Sn |
125 |
500 |
125 |
500 |
1.56 |
12.5 |
3.12 |
100 |
|
Pm |
250 |
- |
250 |
- |
6.25 |
25 |
25 |
200 |
|
Ec |
250 |
- |
250 |
- |
3.12 |
100 |
200 |
200 |
|
Pa |
250 |
- |
250 |
- |
3.12 |
12.5 |
50 |
200 |
|
Sf |
62.5 |
500 |
62.5 |
500 |
1.56 |
3.12 |
12.5 |
200 |
|
Ss |
125 |
- |
125 |
- |
1.56 |
3.12 |
25 |
200 |
|
Kp |
125 |
500 |
125 |
500 |
6.25 |
200 |
100 |
200 |
|
Ea |
31.2 |
125 |
62.5 |
250 |
1.56 |
12.5 |
200 |
200 |
|
Mn |
62.5 |
250 |
125 |
- |
6.25 |
50 |
200 |
200 |
Source: Authors (2021)
In where: (a) Antibiotics. (-) There was no inhibition at the tested concentration. GRAM +: Bs: Bacillus subtilis; Bc: Bacillus cereus; Ef: Enterococcus fecalis; Sa: Staphylococcus aureus; GRAM-: St: Salmonella enterica serovar Typhimurium; Sn: Salmonella enteritidis; Pm: Proteus mirabilis; Ec: Escherichia coli; Pa: Pseudomonas aeruginosa; Sf: Shigella flexneri; Ss: Shigella sonnei; Kp: Klebsiella pneumonia; Ea: Enterobacter aerogenes; Mm: Morganela morgani.
The extracts obtained exhibited significant inhibitory activity against all bacteria analyzed at 30 ºC, and the MIC values for Gram-positive bacteria (Table 2) varied between 31.2 μg mL-1 (Staphylococcus aureus) and 62.5 μg mL-1 (Bacillus subtilis; Bacillus cereus; Enterococcus fecalis). As for the Gram-negative bacteria (Table 2), these values were between 31.2 μg mL-1 (Enterobacter aerogenes) and 250 μg mL-1 (Salmonella enterica serovar Typhimurium; Proteus mirabilis; Escherichia coli; Pseudomonas aeruginosa). For the extract at the temperature of 60 °C, the MIC values for the Gram-positive bacteria (Table 2) ranged from 62.5 μg mL-1 (Bacillus cereus; Enterococcus fecalis; Staphylococcus aureus) to 125 μg mL-1 (Bacillus subtilis). For the Gram-negative bacteria (Table 2), the values were between 62.5 μg mL-1 (Shigella flexneri; Enterobacter aerogenes) and 250 μg mL-1 (Salmonella enterica serovar Typhimurium; Proteus mirabilis; Escherichia coli; Pseudomonas aeruginosa). In both temperatures, the extracts showed antifungal potential (Table 3), with the temperature of 30 ºC inhibiting the growth of all fungi evaluated with MIC values of 62.5 μg mL-1 (Cryptococcus gatti; Cryptococcus neoformans) and 500 μg mL-1 (Candida glabrata; Candida krusei; Candida parapsilosis; Saccharomyces cerevisiae). For the extract at 60 °C, the values ranged from 62.5 μg mL-1 (Cryptococcus gatti; Cryptococcus neoformans) to 500 μg mL-1 (Candida glabrata; Candida krusei; Candida parapsilosis; Candida tropicalis; Saccharomyces cerevisiae). The extracts also exhibited bactericidal potential (Table 2), and at 30 ºC, the MLC was 125 μg mL-1 for Bacillus subtilis and Enterobacter aerogenes. At 60 ºC, the MLC was 250 μg mL-1 for Staphylococcus aureus and Enterobacter aerogenes. Regarding antifungal activity (Table 3), the minimum MLC value of the extract at 30 ºC was 250 μg mL-1 and 500 μg mL-1 at 60 ºC.
Table 3 – Antifungal activity (MIC and MLC in μg/mL) of the extracts obtained at 30 and 60 ºC from the Inga marginata Willd seeds
|
Microorganism and MIC/MLC (μg/mL) |
Fungi |
Sample |
|||||||
|
Seed 30 ºC |
Seed 60 ºC |
Fluconazolea |
Nystatinaa |
||||||
|
MIC |
MLC |
MIC |
MLC |
MIC |
MLC |
MIC |
MLC |
||
|
Ca |
250 |
- |
250 |
- |
25 |
100 |
50 |
100 |
|
|
Cd |
125 |
- |
250 |
- |
3.12 |
12.5 |
50 |
100 |
|
|
Cl |
500 |
- |
500 |
- |
3.12 |
200 |
50 |
100 |
|
|
Ck |
500 |
- |
500 |
- |
25 |
200 |
12.5 |
50 |
|
|
Cp |
500 |
- |
500 |
- |
1.56 |
25 |
1.56 |
100 |
|
|
Ct |
250 |
- |
500 |
- |
50 |
200 |
100 |
200 |
|
|
Cg |
62.5 |
500 |
62.5 |
500 |
3.12 |
25 |
25 |
100 |
|
|
Cn |
62.5 |
250 |
62.5 |
500 |
3.12 |
12.5 |
25 |
100 |
|
|
Sc |
500 |
- |
500 |
- |
1.56 |
25 |
1.56 |
3.12 |
|
Source: Authors (2021)
In where: (a) Antibiotics. (-)There was no inhibition at the tested concentration. Ca: Candida albicans; Cd: Candida dubliniensis; Cl: Candida glabrata; Ck: Candida krusei; Cp: Candida parapsilosis; Ct: Candida tropicalis; Cg: Cryptococcus gatti; Cn: Cryptococcus neoformans; Sc: Saccharomyces cerevisiae.
Nascimento, Sobrinho, Souza, Souza, and Sousa (2021) analyzed Inga edulis leaves by solid-liquid extraction and reported that MIC ranged from 125 to 500 µg mL-1 and from 12.5 to 200 µg mL-1 for Gram-negative and Gram-positive bacteria, respectively. Moreover, Macedo, Ribeiro, Taveira, Gomes, Barros, and Maria-Neto (2016) evaluated Inga laurina Willd seed extracts and noted that they significantly inhibited the growth of Candida tropicalis and Candida buinensis and the yeasts C. tropicalis and C. buinensis. The bacteria tested in this study can cause food spoilage, including physical damage and chemical changes, such as oxidation and color changes or the appearance of strange flavors and odors resulting from microbial growth and metabolism. There are several types of bacteria associated with the spoilage of fresh produce, and they are also toxic to human health. This demand could be met with the use of natural antimicrobials, that is, adding plant extracts with antimicrobial action to preserve food (ZHENG; BAE; JUNG; HEU; LEE, 2013). The antibacterial effects of phenolic compounds are mainly due to their chemical structure in hydroxyl groups in their molecules. In fact, the number and position of these hydroxyl groups (i.e., the hydroxylation pattern) on the phenolic ring seem to be associated with the inhibition exerted on the target fungal bacteria (SANHUEZA; MELO; MONTERO; MAISEY; MENDOZA; WILKENS, 2017).
3.3 Characterization of Inga marginata Willd seed extract by ESI-ToF MS
To characterize the compounds, only the extract obtained at 30 ºC was used, which exhibited high phenolic compound content, antioxidant capacity, and ability to inhibit microorganisms (Tables 2 and 3). The identification of the peaks was performed based on their exact mass, together with information previously reported in the literature, and the chemical structures were determined based on mass spectral data, and ten phytochemical compounds were identified (Table 4). Including neoesperidin dihydrocalcone, which has significant antioxidant properties and attracted increasing interest in recent years, exhibits a wide range of biological and antioxidant activities, such as anti-inflammatory and antimicrobial properties (FRATTARUOLO et al., 2019). Caffeic acid was also found, and several studies have proven its antibacterial, antiviral, antioxidant, anti-inflammatory, anti-atherosclerotic, immunostimulatory, antidiabetic, and cardioprotective activities, in addition to being widely found in vegetables, especially fruits (ESPÍNDOLA et al., 2019). Additionally, Dias, Souza, and Rogez (2010) also reported finding procyanidins in Inga edulis leaves, and in addition to their antioxidant properties, these compounds have been reported to exhibit anticancer, anti-infective, cardioprotective, anti-inflammatory, and anti-allergic properties (MARTIN; GOYA; RAMOS, 2013).
Gerberinol is a still poorly investigated compound and was also found. In the study by Lenta (2015), the authors identified and isolated this compound in the stem bark of the D. canaliculata plant, which exhibits antiprotozoal activity in vitro. The amino acid L-valine has been identified as part of a group of amino acids known as branched-chain amino acids, namely L-leucine, L-isoleucine, and L-valine. Together, these proteins promote normal growth, regulate blood sugar levels, energize the body, and are crucial in tissue repair (CRUZAT; KRAUSE; NEWSHOLME, 2014). This compound was also found in a ginger extract in the study by Alolga, Mais, and Onoja (2017).
Table 4 – Characterization of phytochemical and chemical compounds in the Inga marginata Willd seed extract by ESI-ToF MS in positive and negative modes
|
Nº |
Experimental mass (m/z) |
Theoretical mass (m/z) |
Pure error (p p m) |
Possible molecular structure |
Compounds |
Reference |
|
1 |
543.1331 |
543.1350 |
2 |
C23H26O15 |
Neohesperidin Dihydrochalcone |
(HU et al., 2014) |
|
2 |
377.0854 |
377.0873 |
1 |
C18H18O9 |
Caffeic Acid |
(TOTH; TOTH; KERY, 2014) |
|
3 |
364.3480 |
364.34800 |
6 |
C21H16O6 |
Gerberinol |
(LENTA et al., 2015) |
|
4 |
325.1128 |
325.1128 |
0.6 |
C5H11NO2 |
L-Valine |
(ALOLGA; MAIS; ONOJA, 2017) |
|
5 |
381.0803 |
381.0803 |
4 |
C17H16O10 |
8-Hydroxy-5-O-beta-D-glucopyranosylpsoralen |
(XIAO; LI; MASAHIKO, 2001) |
|
6 |
277.1661 |
277.1661 |
3.6 |
C13H25O6 |
15α-Butoxy-14,15-dihydronorsecurinine |
(GAN; YUE, 2006) |
|
7 |
307.1767 |
307.1767 |
2.9 |
C17H25NO4 |
Alkaloid (sessilifoliamide A) |
(KAKUTA; HITOTSUYANAGI; MATSUURA; FUKAYA;TAKEYA, 2003) |
|
8 |
191.0205 |
191.0192 |
6.8 |
C6H8O7 |
Citric acid |
(WILLIAM et al., 2018) |
|
9 |
381.0803 |
381.822 |
3 |
C17H16O10 |
6-O-β-D-Glucopyranosyl-5-hydroxyangelicin |
(CHANG et al., 2005) |
|
10 |
577.1373 |
577.1405 |
5.5 |
C23H30O17 |
Procyanidins |
(WILLIAN et al., 2018) |
Source: Authors (2021)
In where: *m/z: ratio mass (m) over charge (z).
The compound 8-Hydroxy-5-O-beta-D-glucopyranosylpsoralen belongs to glycoside molecules and is widely distributed in the plant kingdom. These compounds are known for their anti-cancer and anti-inflammatory properties due to their affinity for proteins and their antioxidant properties (BIESAGA; PYRZYNSKA, 2009). Lima, Andrade, and Silva (2020) reported this glycoside in the extracts of Inga edulis leaves. The compounds 15α-Butoxy-14,15-dihydronorsecurinine and sessilifoliamide were also identified; they are considered alkaloid compounds. The pharmacological benefits of alkaloids are well known, such as activity in the circulatory system, metabolism against obesity, and cardiac dysfunction (e.g., antioxidant, anticancer, and neuroprotective properties), and often found in seeds and plant extracts (ROMEO; FABRONI; BALLISTRERI; MUCCILLI; SPINA; RAPISARDA, 2018).
Citric acid was also identified, and it is a known natural preservative and antioxidant (WILLIAM et al., 2018). This compound can be found in acidic fruits, especially in their residues (AYDOGAN; KARAKOÇ; DENIZLI, 2015). The compound 6-O-β-D-Glucopyranosyl-5-hydroxyangelicin is also present in the extract, and Chang et al., (2005) reported this compound in the leaf extracts of Ficus ruficaulis Merr, a Chinese medicinal plant, and reported antioxidant and anti-inflammatory action.
Thus, the characterization by ESI-ToF-MS allowed important phytochemicals of industrial interest to be identified. Most of the compounds identified are antioxidant compounds. Therefore, it can be said that the antibacterial and antifungal effects (Tables 2 and 3) observed in the Inga extract are directly associated with their phytochemical composition. Synergism and molecular antagonism may exist in the composition of fruit or extract, although further research is required to explore the endointeractions within a specific food or extract (FREEMAN; EGGETT; PARKER, 2010). In these cases, some compounds or only a major compound can be extracted from the fruit or vegetable parts (GORGANI; MOHAMMADI; NAJAFPOUR; NIKZAD, 2016).
3.4 Inga seed cytotoxicity
Cytotoxicity analysis has the main objective of detecting the potential of a sample to produce lethal or sublethal effects in biological systems at the cellular level. This assay is characterized as the intrinsic ability of any material or compound to promote metabolic changes in cultured cells. Cytotoxicity assays are among the most common in vitro methods used to predict the potential toxicity of a substance in cell cultures (ROGERO; LUGÃO; IKEDA; CRUZ, 2003). Therefore, we evaluated the cytotoxicity of the lyophilized sample of Inga seed at different concentrations by the MTT and NRU tests in the period of 24 h using 3T3 cell viability. The results were expressed as a percentage of viability in relation to control the cells, which have 100% viability (Figure 2).
Figure 2 – Evaluation of the cytotoxic activity of Inga seeds by the MTT and NRU viability tests for 24 h at different concentrations of the sample
Source: Authors (2021)
In where: Viability plots in 3T3 cell line exposed to different concentrations of sample by MTT and NRU tests. Control (untreated) cells at 100% viability considering there is significant difference when a p<0.05 level is reached (*) p<0.05/(****) p<0.0001. Samples with standard deviation.
Our findings showed that the Inga seed has cytotoxic activity only at the concentration of 250 µg mL-1 for both the cell viability tests by 2,5-diphenyl-3-(4,5-dimethyl-2-thiazolyl) tetrazolium bromide (MTT) and Neutral Red Uptake (NRU), differing statistically compared to the control 250 and 500 µg mL-1 (p<0.0001). In the MTT test and at low concentrations (15.6, 31.25, 62.5, and 125 µg mL-1), cell viability of 94, 97, 89, and 90% were obtained, respectively, with no statistically significant difference at these concentrations compared to the control, being considered non-cytotoxic because cell viability was greater than 90% (LOPES; FASCIO, 2004). For the NRU test, the sample was also non-cytotoxic at the concentrations of 15.6, 31.25, 62.5, and 125 µg mL-1, obtaining cell viability of 100, 99, 92, and 90%, respectively, with no statistically significant difference at these concentrations compared to the control. Fruit seeds can contain toxic substances, including tannins, acids, glycosides, and cyanogenic phenolics with high cyanide content. Hence, when the seeds undergo breakage or maceration, the content of the compounds change, and toxic substances are produced, which can be explained by the fact that plant-defense substances are released (SENICA; STAMPAR; VEBERIC; MIKULIC-PETKOVSEK, 2017).
4 CONCLUSIOn
This study brought evidence that the plant material under study, the Inga marginata Willd seed extract, has a potent phytochemical action since 30 ºC was the best condition to extract phenolic compounds and for antioxidant activity by DPPH and inhibitory capacity (IC50), obtaining better results at this temperature than at 60 ºC. The Inga seed extract obtained at 30 o C also inhibited microorganisms and acted as a bactericide and fungicide at low concentrations. In addition, it was possible to identify ten phytochemical compounds, most of which have antioxidant and antimicrobial properties. The cytotoxicity tests showed that the Inga seed does not present toxicity at low concentrations, maintaining cell viability above 90%. Given all these bioactive benefits, the use of Inga marginata Willd seeds as an extract is of great importance, as it can be considered a natural source of compounds that have antioxidant and antimicrobial effects.
ACKNOWLEDGMENTS
The authors would like to thank the Department of Food Science and Technology (Federal University of Santa Maria, RS, Brazil). This study was partially funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Financial Code 001. The authors would also like to thank Atlas Assessoria Linguística for providing support with the English version of this manuscript.
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Authorship contributions
1 – Déborah Cristina Barcelos Flores
Contribution: Conceptualization, Data Curation, Formal Analysis, Investigation, Writing – Original Draft, Writing – Review & Editing
2 - Caroline Pagnossim Boeira
Contribution: Formal Analysis, Methodology, Software, Visualization
3 - Clarice Madalena Bueno Rolim
Contribution: Methodology, Validation, Data Curation
4 - Daniele Rubert Nogueira Librelotto
Contribution: Methodology, Validation, Data Curation
5 - Frederico Luiz Reis
Contribution: Conceptualization, Funding Acquisition, Methodology
6 - Liziane Maria Barassuol Morandini
Contribution: Conceptualization, Funding Acquisition, Methodology
7 - Ademir Farias Morel
Contribution: Conceptualization, Funding Acquisition, Methodology
8 - Claudia Severo da Rosa
Contribution: Conceptualization, Formal Analysis, Project Administration, Supervision