Chemical and biological profile of the species Batis maritima Linnaeus extracts

Perfil químico e biológico dos extratos da espécie Batis maritima Linnaeus

Abstract

Medicinal plants have been used empirically since ancient civilizations to alleviate and cure diseases. Secondary metabolites of these plants, such as phenolics and flavonoids, have important anti-inflammatory, antimicrobial, and antioxidant properties. Bacterial resistance to antibiotics poses a public health challenge. The use of plant products presents an alternative in the treatment of resistant microbial infections, provided that these plants are previously evaluated in toxicity tests, ensuring their safe use. In this study, we evaluated the composition and antioxidant, antimicrobial, and cytotoxic activities of aqueous and ethanolic extracts obtained from the leaves, stems, and roots of Batis maritima Linnaeus. The six extracts obtained were subjected to phytochemical screening, minimum inhibitory concentration (MIC), 1,1-diphenyl-2 picrylhydrazine (DPPH) reduction, and erythrocyte hemolytic activity assays. During the investigation, all extracts presented flavonoids in qualitative analyses, with emphasis on the ethanolic extract of the leaves, which also presented tannins, saponins, and coumarins. In the quantitative evaluation, this same extract stood out for the presence of phenols and flavonoids. The same effect was also observed in the antioxidant evaluation, where the same extract showed greater scavenging activity compared to the other extracts at all concentrations tested. All extracts inhibited bacterial growth of Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli. However, the ethanolic extract of the leaves was the only one that inhibited Pseudomonas aeruginosa and displayed bactericidal activity against S. aureus. This extract excelled in the cytotoxic assay, showing lower toxicity at lower concentrations (31.25 to 0.97 μg/mL). Chromatographic analyses revealed a flavonoid as the major compound in the ethanolic leaf extract, suggesting it may be responsible for the promising biological activities demonstrated by this extract in our findings. Overall, the evaluation of the biological potential of Batis maritima extracts yielded promising results in the search for natural antimicrobial compounds, with the ethanolic leaf extract standing out as a potential catalyst for future scientific investigations.

Keywords:
antimicrobial activity; toxicity; antioxidant; phytochemical; Batis maritima

Resumo

As plantas medicinais são usadas, desde antigas civilizações, de forma empírica, para aliviar e curar doenças. Metabólitos secundários dessas plantas, como fenólicos e flavonoides apresentam propriedades importantes como: anti-inflamatória, antimicrobiana e antioxidante. A resistência bacteriana aos antibióticos configura-se como um desafio à saúde pública. A utilização de produtos vegetais apresenta-se como alternativa no tratamento de infecções microbianas resistentes, desde que tais vegetais sejam previamente avaliados em testes de toxicidade, tornando o uso seguro. Neste trabalho, avaliamos a composição e as atividades antioxidante, antimicrobiana e citotóxica de extratos aquosos e etanólicos obtidos de folhas, caule e raízes de Batis marítima Linnaeus. Os seis extratos obtidos foram submetidos aos ensaios de triagem fitoquímica, concentração inibitória mínima (CIM), redução de 1,1-difenil-2-picrilhidrazina (DPPH) e atividade hemolítica de eritrócitos. Durante a investigação, todos os extratos apresentaram flavonoides nas análises qualitativas, com destaque para o extrato etanólico das folhas, que apresentou também taninos, saponinas e cumarinas. Na avaliação quantitativa, esse mesmo extrato se sobressaiu quanto a presença de fenóis e flavonoides. Assim também na avaliação antioxidante, onde o mesmo extrato apresentou maior atividade sequestradora comparado aos demais extratos, para todas as concentrações testadas. Todos os extratos inibiram o crescimento bacteriano de Staphylococcus aureus, Staphylococcus epidermidis e Escherichia coli. Entretanto, o extrato etanólico das folhas foi o único a inibir Pseudomonas aeruginosa e apresentar atividade bactericida contra S. aureus. O destaque desse extrato foi observado no ensaio citotóxico que apresentou menor toxicidade nas menores concentrações (31,25 a 0,97 μg/mL). As análises cromatográficas mostraram, como composto majoritário do extrato etanólico das folhas, um flavonoide, evidenciando ser o possível responsável pelas atividades biológicas promissoras que esse extrato apresentou nos nossos achados. No geral, a avaliação do potencial biológico dos extratos de Batis maritima mostrou dados promissores, na busca de compostos antimicrobianos naturais, onde o extrato etanólico das folhas destaca-se para subsidiar futuras investigações científicas.

Palavras-chave:
atividade antimicrobiana; citotoxicidade; antioxidante; fitoquímica; Batis maritima

1. Introduction

Medicinal plants have been used, since ancient civilizations, in an empirical manner through the ingestion of herbs and leaves to relieve and cure diseases. This knowledge was refined with the implementation of policies such as the National Policy on Medicinal Plants and Herbal Medicines, which aims to guarantee the population access to and safe use of medicinal plants and herbal medicines (Figueredo et al., 2014). Herbal medicines, being obtained from plant raw material, may play palliative, prophylactic, or curative roles, showing activity in the cosmetic field, such as extracts, oils, essential oils, and plant-derived phytocosmetics (Simão et al., 2019).

Through secondary plant metabolism, plants produce substances with biological and pharmacological activities, such as defense, growth regulation, anti-inflammatory, antioxidant, antimicrobial, and healing effects (Pereira and Cardoso, 2012). Phenolic compounds, characterized by the structure of an aromatic ring with a hydroxyl group forming the phenol group, exert several physiological effects such as: anti-inflammatory, antimicrobial, antithrombotic, cardioprotective, vasodilatory, and antioxidant actions (Panche et al., 2016). Among these are coumarins, tannins, flavonoids, and lignins (Borges and Amorim, 2020). Flavonoids stand out for their antioxidant, anti-inflammatory, and cell cycle inhibition activities (Machado et al., 2018).

Bacterial resistance to antibiotics is one of the greatest challenges to public health, generating clinical and economic consequences, increasing treatment costs and mortality rates (Silva and Nogueira, 2021). Some clinically relevant bacteria are highly resistant to available treatments, such as Pseudomonas aeruginosa, which produces extended-spectrum β-lactamases and carbapenemases (ESBL), Escherichia coli resistant to beta-lactams, and methicillin-resistant Staphylococcus aureus (Zou et al., 2022; Guo et al., 2020).

In this context, it is relevant to encourage research that seeks antibacterial activity from natural sources that are easily accessible to the population and that circumvent the current resistance of these pathogens to conventional antibiotics (Souza et al., 2022; Clementino et al., 2015). Thus, the use of plant-based products presents itself as a promising alternative in the treatment of microbial infections, especially those caused by bacteria (Silva and Nogueira, 2021; Ferreira et al., 2020).

Although plant extracts are constantly tested for possible biological activities, such as antimicrobial and antioxidant ones, depending on the substance class or its dose, they may alter the individual’s homeostasis, triggering toxicity (Vieira and Fernandes, 2021). In this scenario, there is a need for further research to clarify and confirm information from the popular use of medicinal plants, reducing possible toxicological and side effects to ensure safe use (Firmo et al., 2014; Bhattacharya, 2017).

Halophytic plants contain in their composition a range of chemical constituents, such as vitamins, minerals, and bioactive compounds, which ensure their physiological complexity (Ventura et al., 2011). Moreover, they have contributed to the advancement of nutraceutical, industrial, and pharmacological approaches (Hameed and Khan, 2011).

The perennial subshrub Batis maritima Linnaeus stands out among halophytes (Marcone, 2003). It belongs to the Bataceae family, found on the northeastern and southeastern Brazilian coast, along the edges of water bodies in artisanal salt pans (Souza and Lorenzi, 2008), in Central and North America, and in the Caribbean Islands (Lonard et al., 2011). Regarding its empirical use, indigenous tribes in Puerto Rico use its roots as a sweetener in coffee, its leaves in salads, and it also provides nutrition and cover for wild animals. In folk medicine, it is used in the treatment of gout, eczema, psoriasis, rheumatism, thyroid disorders, and for anti-inflammatory and antioxidant purposes (Lonard et al., 2013; Marcone, 2003).

Given the scarce reports in the literature regarding B. maritima, even though it is a plant present in Brazilian biomes, it is necessary to investigate the antioxidant and antimicrobial activity of this plant, as well as to evaluate its cytotoxic potential, since promising results ensure the future use of B. maritima in the development of herbal medicines. The objective of this study is to qualitatively and quantitatively characterize the aqueous and ethanolic extracts of B. maritima, as well as to elucidate the secondary metabolites present, and to analyze the antioxidant, antimicrobial, and cytotoxic activities of these extracts.

2. Material and Methods

2.1. Collection and preparation of Batis maritima extracts

The collection was carried out in the municipality of Grossos, Rio Grande do Norte, Brazil (4°59’06.6”S 37°08’43.3”W). A voucher specimen was prepared and archived at the Dárdano de Andrade Lima Herbarium – MOSS (UFERSA), under voucher number 15836. Leaves, stems, and roots were collected and taken to the Plant Physiology and Biochemistry Laboratory at the State University of Rio Grande do Norte (UERN) for the preparation of aqueous and ethanolic extracts. The collected plant material was dried in an oven at 60 °C for approximately 72 hours and subsequently ground in a knife mill to obtain a homogeneous powder. Next, for each extract, aliquots of the powder were individually weighed and then mixed with their respective solvents: distilled water or absolute ethanol (C₂H₅OH), both in a 1:5 ratio, in amber bottles to prevent interference from ambient light.

The extraction lasted 10 days with daily mechanical stirring, at room temperature around 25 °C. After this period, the mixtures were filtered with the aid of a vacuum pump coupled to a Büchner flask. Subsequently, to obtain the dry extracts of B. maritima, the solutions were concentrated in a rotary evaporator at a temperature below 60 °C until complete removal of the solvent. The extracts were abbreviated as follows: aqueous extract of leaves (AEL), aqueous extract of stems (AES), aqueous extract of roots (AER), ethanolic extract of leaves (EEL), ethanolic extract of stems (EES), and ethanolic extract of roots (EER).

2.2. Qualitative phytochemical screening

The preliminary qualitative phytochemical screening of the extracts aimed to detect the main classes of secondary metabolites through chemical reactions that result in color changes and/or precipitates characteristic of each class of substances. The dry extracts were previously weighed and diluted in a 2.5% DMSO solution at a concentration of 1000 µg/mL. This solution was used to detect the presence or absence of molecules in the qualitative phytochemical screening, among which were: alkaloids, flavonoids, tannins, coumarins, and saponins (Simões et al., 2004).

The presence of alkaloids, flavonoids, tannins, coumarins, and saponins was confirmed as previously described in the literature (Kloss et al., 2016). Alkaloids were detected by precipitation using 10% hydrochloric acid (HCl) with the addition of Dragendorff's reagent. Flavonoids were detected by visualization of the precipitate using 10% lead acetate. Tannins were detected using 10% ferric chloride, in which a blue color indicates hydrolyzable tannins, while a green color indicates condensed tannins. Coumarins were detected on filter papers impregnated with 10% sodium hydroxide (NaOH) solution and examined under ultraviolet light; yellow or green fluorescence indicates the presence of coumarins. Saponins were detected by mixing the extracts with boiling distilled water and subjecting them to mechanical agitation. Foam formation indicates the presence of this metabolite (Kloss et al., 2016).

2.3. Quantification of phenolics and flavonoids

For the quantification of phenolic compounds, the Folin-Ciocalteu spectrophotometric method was used (Folin and Ciocalteu, 1927). Readings were taken in a spectrophotometer at a wavelength of 700 nm, under low light conditions. The calibration curve used was gallic acid, similar to the extracts. The total phenolic content was expressed in µg GAE/mL (micrograms of gallic acid equivalents per mL of sample). The quantification of flavonoids was based on the methodology of Bag et al. (2015), with modifications. Readings were taken in a spectrophotometer at a wavelength of 415 nm, under low light conditions. The calibration curve used was quercetin, similar to the extracts. The flavonoid content was expressed in µg QE/mL (micrograms of quercetin equivalents per mL of sample). All analyses of phenolic and flavonoid content were performed in triplicate.

2.4. Antioxidant activity

The quantitative evaluation of the free radical scavenging capacity of the extracts was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical method (Aldrich Co®), based on the methodologies of Brand-Williams et al. (1995) and Boroski et al. (2011), with modifications. A DPPH solution of 0.1192 mmol/L was prepared and stored protected from light. Samples of each extract were prepared at the following concentrations: 1000, 500, 250, 125, and 62.5 µg/mL, where the extracts were dissolved in methanol. The assay was performed in triplicate, and absorbance readings were taken at 515 nm, with pure methanol as the blank. The absorbance of the DPPH solution was also measured. To determine the IC50 values of each extract, the percentage inhibition of DPPH (%DPPH inhibition) was first calculated using Equation 1:

$\%DPPHinhibition=\left(\frac{AbsDPPH-Abssample}{AbsDPPH}\right)*100$ (1)

where: Abs DPPH corresponds to the absorbance of the methanolic DPPH radical solution. Abs Sample corresponds to the absorbance of the sample 30 minutes after reaction with DPPH.

Next, a curve of DPPH inhibition percentage versus extract concentration was plotted, obtaining the linear regression equation to determine the IC50 value, i.e., the extract concentration capable of inhibiting 50% of the DPPH radical. The lower the IC50 value, the greater the antioxidant efficiency of the extract.

2.5. Toxicity test

The hemolytic activity was determined by the ability of the tested extracts to lyse human erythrocytes. The methodology used was based on the study of Ribeiro et al. (2020). Blood samples of type O- anticoagulated with EDTA were used and stored under constant refrigeration. A 10% O- erythrocyte concentrate was prepared from these samples. In the assay, 1.5 mL of B. maritima extracts, dissolved in 2.5% DMSO, were added to test tubes at the following concentrations: 500, 250, 125, 62.5, and 31.25 µg/mL. The following controls were used: positive control (PC), 1.5 mL of 1% Triton X; negative control (NC), 1.5 mL of 0.85% saline solution; and solvent control (SC), 1.5 mL of 2.5% DMSO.

Subsequently, 200 µL of the 10% erythrocyte suspension was added, and the tubes were incubated at 37 °C for 1 hour, then left at room temperature for 15 minutes. After cooling, the tubes were centrifuged at 4000 rpm for 5 minutes, and 4 mL of the resulting supernatants were transferred to cuvettes and read in a spectrophotometer at 540 nm to obtain absorbance values. The percentage of hemolysis (H%) was determined by Equation 2:

$H\%=\left(\frac{AbsA-AbsNC}{AbsPC}\right)*100$ (2)

where: AbsA corresponds to the absorbance of the sample. AbsNC corresponds to the absorbance of the negative control. AbsPC corresponds to the absorbance of the positive control.

The absorbance of the solvent control (AbsS), although not applied in the formula for obtaining the percentage of hemolysis, was only useful to verify whether the solvent of the extracts (2.5% DMSO) was interfering with the hemolysis results. The hemolysis percentage values (H%) of the different concentrations of each extract were plotted on the Y-axis of a graph to obtain the HC50 values, i.e., the concentrations of B. maritima extracts capable of causing 50% hemolysis of erythrocytes. The assay was performed in triplicate.

2.6. Antibacterial activity

The microorganisms tested were maintained at 8 °C and included Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853). The inocula were prepared from growth on BHI agar (Brain Heart Infusion) at 37 °C/24 h. Colonies were suspended in 0.9% sterile saline solution and adjusted to 0.5 McFarland, equivalent to 10⁸ colony-forming units per mL (10⁸ CFU/mL).

The antibacterial activity of the extracts was quantitatively evaluated by the broth microdilution technique in 96-well microplates (Schuetz et al., 2025). Test samples (T) of each extract were serially diluted at the following concentrations: 1000, 500, 250, 125, 62.5, 31.25, 15.62, and 7.81 µg/mL. The positive control (PC) used serial dilutions of gentamicin (Gentamisan – SANTISA®), starting from a concentration of 16 μg/mL. As a negative control (NC), an aqueous solution of 2.5% DMSO (solvent of the extracts) was used. The growth control (GC) consisted only of culture medium and the respective bacterial strain to assess its viability. Then, 20 μL of the inoculum solution was added to each well. The microplates were incubated without agitation at 37 °C/24 h to determine the minimum inhibitory concentration (MIC). All tests were performed in triplicate.

To determine the MIC, 20 μL of 0.5% 2,3,5-triphenyltetrazolium chloride (TTC) solution was applied to each well to reveal bacterial growth. MIC readings were taken after incubation at 37 °C for an additional 2 h and defined as the lowest concentrations of extracts that visually inhibited microbial growth (Valgas et al., 2007). The bactericidal activity of the extracts was measured after MIC reading by seeding the contents of the microplate wells on BHI agar. The most representative wells of the T, PC, NC, and GC groups were selected. From each selected well, 8 μL were inoculated into specific numbered regions on the back of Petri dishes, which were then incubated at 37 °C. After 24 h of incubation, readings were taken to determine the bactericidal action of the extracts. The MBC (Minimum Bactericidal Concentration) was considered the lowest concentration that completely prevented microbial growth on BHI agar.

2.7. Chromatographic analysis

Chromatography was performed on the extract that showed the most promising results in the analyses described above, aiming to separate the major component and compare it with the standards of the equipment. This made it possible to determine the class of secondary metabolite to which the compound belongs. One gram of the ethanolic leaf extract of B. maritima, in its crude form obtained from extraction, was solubilized in hexane and applied to a separation column to fractionate by gravity the bands corresponding to the metabolite classes present in the extract. During fractionation, solvents were added in increasing order of polarity: hexane, ethyl acetate, and methanol. The bands were collected throughout the column elution into identified test tubes. The volume of the most intense band was collected, and this fraction obtained from partitioning was injected into the equipment used.

The compounds present in this sample were identified by Ultra Fast Liquid Chromatography (UFLC) Nexera XR of the UFLC-DAD type coupled to a diode array detector (Shimadzu). The chromatographic conditions were defined with a degassing system (DGU-20A-3R), ultra-high pressure pump LC-20AD XR (Nexera XR, Shimadzu), auto-injector (SIL 20A XR), oven for temperature control (CTO-20A), DAD detector (SPD-M20A), a controller (CBM-20A), and Shimadzu Labsolution® software. The mobile phase consisted of 0.1% formic acid (solvent A) and methanol (solvent B), with a flow rate of 0.6 mL/min, oven temperature of 33 °C, C18 Phenomenex pre-column (4.0 × 3.0 mm, 5 μm), and a C18 Phenomenex Kinetex column (250 × 4.6 mm; 5 μm). The diode array detector operated at wavelengths of 245, 260, 280, 320, and 355 nm. Analysis time was 60 min with an injection volume of 2 μL. The standards used in the UFLC assay and their respective wavelengths in nm, as shown inTable 1.

Table 1
Standards used in UFLC and their wavelengths.

Standards	Wavelenghts (λ) in nm
Acacetin	269/335
Amentoflavone	268/366
Apigenin	267/339
Chrysin	267/312
Coumarin	276/309
Galangin	266/358
Isoliquitrigenin	240/366
Kaempferol	266/366
Liquitrigenin	275/312
Luteolin	253/348
Myricitrin	259/351
Orientin	256/348
Pinobanksina	291/330
Pinocembrine	290/335
Pinostrombin	289/331
Quercetin	255/366
Routin	256/353
Vitexin	268/338

In the UFLC, it was possible to obtain the chromatogram of the sample equivalent to the fraction of the ethanolic leaf extract of B. maritima. This allowed analyzing the peak of the major compound and comparing the retention time and wavelength with the standards used, in order to facilitate the identification of the possible class of secondary metabolite present.

3. Results

3.1. Qualitative phytochemical screening

In the results of the qualitative phytochemical screening, it was observed that flavonoids are the predominant secondary metabolites, followed by tannins, which were absent only in the roots. The presence of coumarins and saponins was observed only in the ethanolic leaf extract, as shown in Table 2.

Table 2
Phytochemical screening of B. maritimaextracts.

Extract	Alkaloids	Flavonoids	Taninns	Coumarins	Saponins
AEL	-	+	+	-	-
AES	-	+	+	-	-
AER	-	+	-	-	-
EEL	-	+	+	+	+
EES	-	+	+	-	-
EER	-	+	-	-	-

• (+) Presence of Chemical compund; (-) Absence of chemical compund.

3.2. Quantification of total phenolics and flavonoids

From the equations of the calibration curves obtained with gallic acid (y = 0.0594x – 0.0024; R² = 0.9968) and quercetin (y = 0.0327x – 0.0043; R² = 0.9983), the absorbance values (triplicate) of each sample were substituted into Y to obtain, respectively, the values of total phenolic compounds (µg GAE/mL) (microgramas de equivalentes de ácido gálico por mL de amostra) and flavonoids (µg QE/mL) (microgramas de equivalentes de quercetina por mL de amostra), as shown in Table 3.

Table 3
Total phenolic compounds and flavonoid content of B. maritimaextracts.

Extracts	Total phenolics (µg GAE/mL)	Flavonoids (µg QE/mL)
AEL	16.45 ± 0.08	22.35 ± 0.70
AES	13.81 ± 0.41	19.52 ± 0.59
AER	10.61 ± 0.42	27.43 ± 0.25
EEL	40.54 ± 0.36	267.03 ± 0.67
EES	33.39 ± 1.49	30.68 ± 0.28
EER	24.67 ± 1.34	11.46 ± 0.81

3.3. Antioxidant activity

Based on the absorbance values obtained, the percentage inhibition of the DPPH radical at 515 nm and IC50 were calculated for each extract at different concentrations (1000, 500, 250, 125, and 62.5 µg/mL) (as shown in Table 4).

Table 4
Percentage of DPPH radical inhibition and IC50 by B. maritimaextracts.

Concentration (µg/mL)	DPPH scavenging
	AEL	AES	AER	EEL	EES	EER
1000	92.60 ± 0.47	91.59 ± 1.59	87.25 ± 1.78	96.41 ± 1.56	93.88 ± 1.61	92.64 ± 1.90
500	88.70 ± 1.08	87.64 ± 0.71	78.57 ± 1.67	93.77 ± 1.52	84.79 ± 0.98	84.12 ± 1.77
250	81.09 ± 0.45	72.37 ± 1.48	65.68 ± 1.78	92.65 ± 1.34	76.12 ± 1.51	72.23 ± 1.69
125	65.71 ± 1.87	63.30 ± 1.83	54.72 ± 1.35	91.60 ± 1.63	62.59 ± 1.50	58.17 ± 1.27
62.50	48.83 ± 1.84	43.22 ± 1.93	30.22 ± 1.43	88.64 ± 1.64	48.32 ± 0.85	37.22 ± 1.94
IC50	0.70	1.26	2.04	-21.06	0.96	1.62

3.4. Toxicity assay on erythrocytes

Based on the hemolysis percentage values obtained at different concentrations of B. maritima extracts, were obtained HC50 values (as shown in Table 5).

Table 5
Percentage of hemolysis (H%) and HC50 of B. maritimaextracts.

Concentration (µg/mL)	Hemolysis (%)
	AEL	AES	AER	EEL	EES	EER
500	48.72 ± 1.44	56.21 ± 5.03	94.76 ± 1.06	16.02 ± 0.76	17.02 ± 0.81	69.01 ± 2.53
250	31.20 ± 3.13	52.15 ± 2.48	82.84 ± 1.06	9.94 ± 0.21	13.87 ± 0.51	55.00 ± 0.69
125	24.92 ± 0.56	45.33 ± 0.66	72.33 ± 1.33	7.49 ± 0.71	11.65 ± 0.93	44.02 ± 1.54
62.50	17.66 ± 1.45	32.67 ± 0.82	61.35 ± 2.34	5.81 ± 0.31	10.44 ± 0.51	31.63 ± 0.55
31.25	8.70 ± 0.80	28.98 ± 1.34	53.59 ± 2.15	4.40 ± 0.35	8.93 ± 1.11	23.64 ± 0.61
HC50	550.55	185.70	18.24	993.41	676.89	173.15

3.5. Antibacterial activity

The Minimum Inhibitory Concentration (MIC) of the extracts obtained from Batis maritima against the strains of S. aureus, S. epidermidis, E. coli and P. aeruginosa was defined as the lowest concentration capable of inhibiting bacterial growth in triplicate, without the visual detection of TTC activity. The Minimum Bactericidal Concentration (MBC) was determined as the lowest concentration of each plant sample that resulted in complete inhibition of bacterial growth on BHI agar medium. The results of MICs and MBCs, as shown in Table 6.

Table 6
Results of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for the positive control (gentamicin) and B. maritima extracts (µg/mL).

Bacteria	S. aureus		S. epidermidis		E. coli		P. aeruginosa
Extracts	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
AEL	1000	BT	1000	BT	1000	BT	-	-
AES	1000	BT	1000	BT	500	BT	-	-
AER	1000	BT	1000	BT	500	BT	-	-
EEL	500	1000	500	BT	1000	BT	1000	BT
EES	250	BT	1000	BT	500	BT	-	-
EER	500	BT	1000	BT	1000	BT	-	-

• BT = Bacteriostatic; - = no inhibition observed.

In the antimicrobial assay, the controls efficiently validated the tests performed according to CLSI (2025). The positive control (PC) showed no bacterial growth at the highest concentrations, confirming the effectiveness of gentamicin as a standard, since the antibiotic demonstrated activity against all bacteria tested down to the concentration of 0.004 mg/mL (4 µg/mL), with equivalence between MIC and MBC values.In contrast, in the negative control (NC) and the growth control (GC), bacterial growth was observed in all assays, confirming respectively the absence of bacterial inhibition by 2.5% DMSO and the viability of the strains used. These findings demonstrate that the antibacterial effect observed was exclusively due to the constituents of the B. maritima extracts.

3.6. Chromatographic analysis

The most intense band of the ethanolic leaf extract (EEL) of B. maritima corresponded to the 1:1 fraction (ethyl acetate/methanol), whose major component peak presented a retention time of 30.447 minutes. This compound was the most concentrated in the sample and is possibly responsible for the biological activities described here. The chromatogram obtained is shown in Figure 1.

Figure 1
Chromatogram of the 1:1 (ethyl acetate/methanol) fraction of the ethanolic leaf extract ofB. marítima.

4. Discussion

The search for biological activities of different types of plants has aroused great scientific interest. In this context, the extracts of Batis maritima and their composition showed important biological activities, including against microorganisms.

Studies with different halophytes, such as Acanthus ilicifolius and Heliotropium curassavicum, demonstrated antimicrobial activity against medically important bacteria such as Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Streptococcus pneumoniae (Pothiraj et al., 2021). In addition to the antimicrobial potential reported in these halophytes, our findings with B. maritima showed inhibition of the growth of Gram-negative bacteria (E. coli and P. aeruginosa) and Gram-positive bacteria (S. aureus and S. epidermidis), demonstrating a broad antimicrobial spectrum.

Among the extracts tested here, the ethanolic leaf extract of B. maritima (EEL) was the only one that exhibited bactericidal activity, and only against the Gram-positive S. aureus. It is important to note that, according to Bodade et al. (2008), the antibiotics most commonly used in therapeutic treatments are active against Gram-positive bacteria, which highlights the importance of this study, since Gram-negative bacteria are more difficult to inhibit due to their cell structure. This was the case with P. aeruginosa, evaluated here as the most resistant, being inhibited only bacteriostatically by the EEL, at a concentration of 1000 μg/mL. Nevertheless, studies reveal that activity may be considered strong for MIC values between 50-500 μg/mL, moderate for MIC values between 600-1500 μg/mL, and weak above 1500 μg/mL (Sartoratto et al., 2004).

On the other hand, against S. epidermidis, the EEL did not show bactericidal action, possibly due to the fact that this bacterium produces more biofilm than S. aureus (Schilcher and Horswill, 2020), making it more difficult for antibacterial compounds present in the extract to penetrate. It is known that biofilm is one of the main resistance mechanisms of Staphylococcus species (Burke et al., 2024).

Thus, in agreement with the literature, the data obtained in this research indicate that B. maritima extracts show growth inhibition activity against all bacterial strains tested, except P. aeruginosa, which was inhibited only by the EEL. This extract stands out as the most promising in terms of antimicrobial activity, since in addition to inhibiting all strains, it was the only one to exert bactericidal activity against S. aureus. There are already reports in the literature of the antimicrobial activity of this plant’s leaves, specifically antifungal activity (Srikishen and Subramanian, 2022). Therefore, considering this potential and our findings, further research on the antibacterial properties of the ethanolic leaf extract of B. maritima is encouraged.

Complementing the promising results of antimicrobial activity, in the erythrocyte toxicity test, the ethanolic extracts were less toxic, indicated by lower hemolysis values ​​(%), up to the lowest concentration (31.25 µg/mL): 4.40 ± 0.35 (EEL); 8.93 ± 1.11 (EES); 23.64 ± 0.61 (EER), compared to the aqueous extracts, respectively: 8.70 ± 0.80 (AEL); 28.98 ± 1.34 (AES); 53.59 ± 2.15 (AER), as shown in Table 5. Notably, the ethanolic leaf extract (EEL) showed the lowest toxicity against erythrocytes among all extracts, as it had the highest HC50 value (993.41). It is of utmost importance that any molecule with effective antibacterial action does not show significant toxicity or adverse effects (Wijnakker et al., 2023). Therefore, the EEL, in addition to being an effective inhibitor of bacterial growth, also stands out as the least toxic, making B. maritima a promising antibacterial plant.

To elucidate the class of major compounds in the B. maritima extracts responsible for the biological activities, the qualitative phytochemical screening results (as shown in Table 2) showed that the extracts obtained from the leaves and stems contained higher levels of secondary metabolites compared to the root extracts. Among these metabolites, flavonoids predominated in the composition, being present in all extracts, followed by tannins, which were only present in the aerial parts of the plant. The study conducted by Srikishen and Subramanian (2022), which also qualitatively evaluated the phytochemistry of alcoholic extracts from B. maritima leaves, corroborates these results, showing the presence of the same metabolites cited here.

The predominance of secondary metabolites in the aerial parts of the plant is due to the fact that molecules such as flavonoids and tannins are polyphenols commonly responsible for pigmentation of plant structures such as leaves and stems (Panche et al., 2016). Furthermore, the presence of these constituents in polar extracts is explained by the solubility of such phytochemicals in polar solvents, such as water and ethanol (Wolff et al., 2019).

Flavonoids, which were found to be the predominant phytoconstituent here, make B. maritima a promising plant, as these metabolites are extremely beneficial. They are among the most important phytochemicals present in many plants, fruits, and vegetables, offering various health benefits, including anticancer, antioxidant, anti-inflammatory, antimicrobial, neuroprotective, and cardioprotective effects (Ullah et al., 2020). This occurs thanks to their specific chemical structure, which contains hydroxyl groups that increase their bioavailability and biological activity (Šamec et al., 2021).

Regarding the solvent used for extraction, it was observed that detection of secondary metabolites was more effective in ethanolic extracts than in aqueous ones. This is likely due to ethanol being more efficient in extracting antioxidant compounds compared to water (Costa et al., 2021). This was particularly evident in the leaves of B. maritima, where the ethanolic leaf extract (EEL), compared to the aqueous leaf extract (AEL), stood out for containing not only flavonoids and tannins but also other metabolites such as coumarins and saponins (as shown in Table 2). Thus, the EEL becomes even more promising, as coumarins in plants are pharmacologically known for their antioxidant, anticancer, anti-neurodegenerative, anticoagulant, anti-inflammatory, antidiabetic, and antimicrobial properties (Franco et al., 2021). Saponins have antidiabetic, anticoagulant, anticancer, cholesterol-reducing, anti-hypercalciuric, and kidney stone-preventive properties (Fernandes et al., 2019).

The quantification of total phenolics confirmed the results of the qualitative phytochemical screening, since the ethanolic extracts presented higher levels of phenolic compounds compared to aqueous extracts (Costa et al., 2021), especially the EEL. In the quantification of flavonoids, which belong to the phenolic class, it was also observed that the ethanolic extracts contained higher flavonoid levels, with the EEL again standing out. This confirms that this extract is the most promising among all tested and makes clear that flavonoids are the molecules that phytochemically characterize B. maritima in this study.

In the study by Scopel et al. (2021), which analyzed the aqueous leaf extract of B. maritima, a significant amount of phenolics and flavonoids was observed (0.99 ± 0.19 µg QE/mL), proving that flavonoids are the phenolic component in highest concentration in this plant. However, compared to the ethanolic extract analyzed here, the latter showed a much higher flavonoid concentration (267.03 ± 0.67 µg QE/mL), as shown in Table 3, once again highlighting the efficacy of the EEL in the quantification of this metabolite.

In the antioxidant activity assay using DPPH, the ethanolic leaf extract (EEL) was the most efficient in inhibiting free radicals. This result aligns with the flavonoid and phenolic compound quantifications (as shown in Table 3), as this extract had the highest levels of these metabolites. Flavonoids are possibly the main compounds responsible for the antioxidant activity observed. Their antioxidant action is highly significant among their pharmacological properties, as they act in the elimination of free radicals, enzyme receptor systems, enzymatic inhibition, inhibition of cyclooxygenase, and inhibition of lipoxygenase (Panche et al., 2016).

The chromatographic result corroborates the findings of the previous analyses, as the ethanolic leaf extract of B. maritima proved to be highly promising, with high levels of flavonoids, antioxidant molecules. The fraction equivalent to the major compound presented a retention time of 30.447 and absorption wavelengths of 254/355 nm, parameters characteristic of flavonoids (Bonta, 2017), suggesting that the major compound responsible for the biological activities described here belongs to this class of molecules (Figure 1).

These wavelengths, when compared to the standards used here (as shown in Table 1), most closely matched three compounds: quercetin, myricitrin, and rutin. Two other standards also showed some numerical similarity with the major compound’s wavelengths: luteolin and orientin. All these standards are flavonoids, confirming that the compound in question belongs to this class of phenolics. Moreover, the major compound may have a structure similar to the standard molecules mentioned and may even be a derivative of them, since they share strong structural and biosynthetic similarities (Liu et al., 2021). Reports in the literature regarding these flavonoids support this hypothesis concerning their various pharmacological properties, especially antimicrobial and antioxidant activities. Such activities have been observed for quercetin (Xu et al., 2019; Nguyen and Bhattacharya, 2022; Qi et al., 2022), routin (Ganeshpurkar and Saluja, 2017; Xia et al., 2022; Zielińska et al., 2025), myricitrin (Motlhatlego et al., 2020; Neves et al., 2024), luteolin (Adamczak et al., 2019; Caporali et al., 2022; Zhu et al., 2024) and orientin (Adamczak et al., 2019; Sowa et al., 2023; Fahmy et al., 2025). These findings highlight that the major compound in the ethanolic leaf extract of B. maritima may be derived from these molecules and could be responsible for the biological activities analyzed here.

5. Conclusion

Overall, our investigation explored for the first time aspects of the bioactive properties of Batis maritima. Through phytochemical analyses, we demonstrated the presence of important classes of biomolecules such as phenolics, tannins, saponins, coumarins, and flavonoids, with the latter being the most prominent. The ethanolic leaf extract of B. maritima proved to be the most promising, presenting the highest flavonoid content and standing out in all the activities analyzed. Furthermore, through high-performance liquid chromatography, we provided consistent results indicating that the antimicrobial, antioxidant, and lower toxicity potential of this extract is due to the presence of flavonoids, which may support future studies in this field.

Acknowledgements

We would like to thank CNPq, the Multicenter Program of Biochemistry and Molecular Biology, and the team of the Plant Physiology and Biochemistry Laboratory of the State University of Rio Grande do Norte (UERN).

• Data Availability Statement
  The entire set of data that supports the results of this study were published in the article itself.

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Edited by

• Editor:
  Jairo Lizandro Schmitt

Data availability

The entire set of data that supports the results of this study were published in the article itself.

Publication Dates

• Publication in this collection
  06 Mar 2026
• Date of issue
  2026

History

• Received
  02 Sept 2025
• Accepted
  05 Jan 2026