Chemical characterization, cytotoxicity, antimicrobial and antioxidant potential of Justicia pectoralis Jacq and Croton jacobinensis Baill extracts

Abstract In this research, aqueous and ethanolic extracts from Justicia pectoralis Jacq and Croton Jacobinensis Baill were characterized. The UPLC-QTOF-MSE analysis was performed on the extracts identified, predominantly, flavonoids, tannins and acids. The extracts did not indicate toxicity in human epithelial cells. C. jacobinensis presented a concentration of phenolics 60.5% higher than J. pectoralis in all scenarios evaluated and, for both samples, the hydroalcoholic extract at 70% exhibited the best efficiency in the extraction (14501.3 and 32521.5 mg GAE 100 g-1 for J. pectoralis and C. jacobinensis, respectively). The antioxidant activity presented a positive correlation with the concentration of phenolics, being 1.186,1 and 1.507,9 μM of Trolox for J. pectoralis and C. jacobinensis at 70% of ethanol; however, it was not verified statistical difference between the ethanolic solutions (p < 0.05). The antimicrobial activity of J. pectoralis extracts was highlighted once was the most effective against gram-positive bacteria. The results suggest that both J. pectoralis and C. jacobinensis extracts present the potential to be applied as natural additives due to their antioxidant and antimicrobial activity and safety. Thus, it is suggesting the development of studies that could investigate the interaction of these plant extracts with food matrices is required.


INTRODUCTION
Natural plant extracts have been studied as food preservation additives, with the aim of using their activities against pathogenic microorganisms in food.The relevance of the use of these metabolites is related to the desire of food consumers for high-quality natural additives, without synthetic components (Calo et al., 2015;Mak et al., 2013).Plant extracts can be incorporated into the formulations used in the food industry, such as edible coatings (Lv et al., 2011).In Brazil, some plant species have great potential for use as extracts given their simple cultivation and inexpensive production.
J. pectoralis is a plant popularly known in Brazil as chambá, chambabá, anador, or clover-cumaru (Kalyne et al., 2017).Chambá was previouly reported as an alternative source of natural antimicrobial compounds in the function of the presence of bioactive compounds as coumarin (1,2 benzopyran) and umbelliferon.It was previously reported Page 2/13 Braz.J. Pharm.Sci.2023;59: e21224 that aqueous extract of J. pectoralis was able to inhibit Staphylococcus aureus and Escherichia coli growth (Guimarães et al., 2020).Phenolics compounds such as the flavonoids and tannins are markedly present in chambá extracts and may contribute to its antioxidant and anti-inflammatory activities (Chanfrau et al., 2013).In addition, several species of the genus Justicia are used in popular medicine for the treatment of respiratory issues (Agra, Freitas, Barbosa-Filho, 2007), gastrointestinal issues (Corrêa, Alcântara, 2012), diabetes, and prostate diseases (Lizcano et al., 2010).
There is no reliable information regarding the use J. pectoralis and C. jacobinensis themselves in food preparations or processing.However, is undeniable the biological potential of both plants, which requires more studies to prospect their use in different research fields.Therefore, the purpose of this research was to investigate the antioxidant and antimicrobial activities of aqueous and hydroalcoholic extracts from leaves of J. pectoralis and C. jacobinensis to evaluate the potential of these for future application as natural food preservatives.

Plant material
The J. pectoralis and C. jacobinensis leaves were manually collected by Dra.Leilanne Marcia in the Prisco Bezerra Herbarium (EAC) belonged to Federal University of Ceará (UFC) localized in Fortaleza (State of Ceará, Brazil).The identity of both plants was determined by comparison with a number of exsiccates (dried samples) deposited in EAC, which were 61.197 and 61.198 for J. pectoralis and C. jacobinensis, respectively.The plant material (fresh leaves) was collected with scissors in the morning from 8 to 10 am under the conditions of maximum (32°C) and minimum temperature (24°C) and 65% relative humidity, during the period from January to March.The leaves were washed with distilled water and dried in an air circulation oven (35 ± 3°C for 24 h for J. pectoralis and 48 h for C. jacobinensis).The dried material was stored using light protection at 25 ± 1°C until the extracts were prepared, which preparation was performed in triplicate.

Preparation of extracts
The extracts were made from infusions of dried leaves (with 0.84 mm of granulometry).The water extracts were obtained using distilled water at 90°C at the proportion 1:20 (dried leaves: water).The hydroalcoholic extracts (prepared in the concentration of 50 and 70% -v/v) were obtained with slight heating (70°C) in the same proportion used for water extract preparation.The extractions were sustained for 30 min and subsequently filtered.The material was freeze-dried using a Beta 1-8 LD plus (CHRIST, Germany), at -40°C and the pressure of 0.025 mbar.After freeze-drying process, the extracts were stored in polyethylene vessels, protected from light, and kept in a freezer (-18°C) until analysis.

Chemical characterization
For chemical characterization, 10 mg of dried samples were solubilized in 10 mL of ultra-pure water.The solutions were homogenized in a USC 1400 bath ultrasound (Unique Indaiatuba, Brazil) for 5 minutes.After, the solutions were filtered in a 0.22µm Millipore filter, collected in vials e sent for analysis.
The chemical profile of samples was performed by coupling the Waters ACQUITY UPLC system to the QTOF mass spectrometer (Waters, Milford, MA, USA), with the electrospray ionization interface in negative ionization mode (ESI -).The (ESI -) mode was acquired in the rangeof 110-1180 Da, fixed source temperature at 120 °C, desolvation temperature 350 °C, desolvation gas flow of 500 L h -1 , 0.5 V extraction cone, 3 kV capillary voltage.Leucine enkephalin was used as lock mass.The MS mode used Xevo G2-XS QTOF.The spectrometer operated with MS and centroid programming, using a tension ramp from 20 to 40 V.The instrument was controlled by Masslynx 4.1 software (Waters Corporation, USA).

Total polyphenols content (TPC)
For the quantification of polyphenosls, solutions with a concentration equal to 1 mg/mL were prepared from the solubilization of dried samples in distilled water.After complete homogenization, the solutions were stored under refrigeration until the analysis.The TPC was determined by the method described by Larrauri, Rupérez and Saura-Calixto (1997), which uses Folin-Ciocalteu reagent and gallic acid as the standard.Aliquots of 0.1 mL of the extracts, 0.5 mL of distilled water, 0.5 mL of the Folin-Ciocalteau reagent (1:3), 1.0 mL of 20% NaCO 3 solution, and 1.0 mL of distilled water were used for this analysis.After homogenization, the product was protected from light for 30 min and was read at 700 nm using a Shimadzu spectrophotometer (model UV-1800).The results were expressed as milligrams of gallic acid equivalent (GAE)/100 g of extract.

Total Antioxidant Activity
The same solutions prepared for polyphenols quantification were used to determine the total antioxidant activity of J. pectoralis and C. jacobinensis.The method was based on the reduction of the 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS •+ ), according to the technique reported by Re et al. (1999) and modified by Rufino et al. (2010).In the dark, an aliquot of 30 μL of each extract was mixed with 3 mL of the ABTS •+ radical and homogenized.After 6 min, the reads were made at 734 nm using the Shimadzu spectrophotometer.The results were expressed as TEAC -Antioxidant Capacity Equivalent to Trolox (2-carboxylic acid-6-hydroxy-2,5,7,8-tetramethylchroman) in micrometers per gram of extract.
Microorganisms and antimicrobial activity E. coli (ATCC 25922), S. enteritidis (IAL 1132), S. aureus (ATCC 27664), and L. monocytogenes (ATCC 19115) were used to evaluate the antibacterial activity of the extracts of J. pectoralis and C. jacobinensis.This study was based on the plate microdilution method suggested by (MIC) and minimum bactericidal concentration (MBC) values.Water and ethanolic Branen and Davidson (2004) and Brandt et al. (2010), with adaptations.A method using 96-well microplates (MicrotestTM, Becton Dickinson and Co.) was used to determine the minimum inhibitory concentration extracts (50 and 70% -v/v) at 250 mg/ mL were prepared similarly to previsouly described.The extracts were diluted in water to obtain different concentrations (0.5-100 mg/mL) and sterilized using 0.22 μm membrane filters.In each well, TSB broth, bacterial suspension (approximately 10 5 CFU/mL) and an aliquot of each dilution of the extracts studied were incubated with shaking at 35°C for 24 h.The test was also carried out in wells containing culture medium, distilled water, and bacterial suspension, as a negative control.A commercial antimicrobial agent (amikacin) and inoculum was used as a positive control.Optical density (OD) was read at 630 nm using the ELx 808 absorbance reader (BioTek instruments, Inc.Winooski, VT, USA).The final results were obtained by variation of the absorbance at different times (0 and 24 hours after incubation).Considering the MICs, the concentrations that presented differences between the readings were ≤ 0.05 nm.From the wells that indicated inhibitory activity (≤ 0.05 nm), 100 μL was inoculated into culture media and specific differential media were provided for each species: Mac Conkey Agar (Oxoid) for E. coli, Listeria Oxford Agar (Hi Media) for L. monocytogenes, Baird-Parker Agar (BD) for S. aureus, and deoxycholate-lysine-xylose (XLD) agar for S. eteritidis.The plates were incubated at 35°C for 48 h, and MBC was considered the lowest test concentration at which a plate showed the absence of microbial growth.The results were expressed in milligrams per milliliter of dry extract.

Cytotoxicity Activity
Cytotoxicity activity tests were done with the extracts that presented the best antimicrobial activity, according to the method described by Mosmann (1983) using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).Cytotoxicity was measured by exposing intestinal epithelial cells (approximately 20.000 cells of the IEC 6 line previously cultured) to 100 μL of the extract solutions with concentraton varying from 0.25 to 20 mg/mL.The fractions obtained were compared to positive (cell culture only in culture medium) and negative (Clostridium difficile cytotoxin A) controls, being the positive control considered 100% cell viability.The results of the absorbances obtained were submitted to Bonferroni's multiple comparison test and the results expressed as mean and standard deviation.

Statistical analysis
The results of total extractable polyphenols and total antioxidant activity were analyzed statistically by comparing the means by Variance Analysis (ANOVA) and Tukey's test, at the 5% level of significance, using the software Statistica 10.0.The data obtained for the antimicrobial activity was expressed through the minimum inhibitory and bactericidal concentrations.

UPLC characterization
The UPLC results of the aqueous extract of J. pectoralis Jacq.showed the presence of twelve peaks (Figure 1A and Table I).For C. jacobinences extract, fourteen peaks were found (Figure 1B and Table II).The identification of the compounds present in each extract was made by comparison with results present in the literature database.Thus, for a higher confidence level, it was considered the maximum similarity with the mass calculated, fragments and empirical formula and, it was considered the minor error possible between the substances evaluated.Regarding to J. pectoralis Jacq., the ethanolic solution at 70% (v/v) (equal to 14.501,3 GAE.100g -1) presented an extraction efficience 16 and 42% higher than that prepared with 50% of ethanol (12.111,9 mg GAE.100g -1 ) and water (8.436,1 mg GAE.100g -1 ), respectivetly.In the same situation, for C. jacobinensis Baill., the values were 3.5 and 23.4%.Considering the average of the concentrations obtained for each solvent the amount of phenolics present in C. jacobinensis Baill.was 60.5% superior to that present in J. pectoralis Jacq (p>0.05).
Current research involves an evaluation of different solvents and methods of extracting bioactive compounds from plants (Feng et al., 2020).Artega et al. (2011) compared the effects of the use of water, ethanol, and ethyl acetate as Regarding the compounds found, derivatives of caffeic acid (caffeic acid dihexoside, caffeic acid hexoside and caffeic acid glucuronide) are reported in the literature associated with antioxidant and anti-inflammatory potential.In addition, they have antimicrobial activity (Moosavi et al. 2017).The substances p-Coumaroil glucoside and p-Coumaroil galactoside are derived from phenolic compounds with antioxidant activity as described by Tang et al. (2001).
The literature shows several classes of diterpenes associated with the molecular formulas of the compounds referring to peaks 7 to 14 (Xu, Liu, Liang, 2018).However, due to the large number of isomers, it was not possible to establish the identification of each substance and they were tentatively identified as diterpenes.Citric acid is known in the literature as a natural antioxidant (Ryan et al., 2019) and has an antimicrobial effect by modifying the pH of the medium.Diterpenes are reported with various biological activities, with larvicide (Geris et al., 2008), insecticide (Viegas Júnior, 2003) and antimicrobial (Sá Firmino et al., 2019).Isoquecitrin has a positive effect on osteogenesis (Li et al., 2019), antibacterial activity (Yun, Woo, Lee, 2018) and antioxidant (Silva et al., 2009).Kaempferol-O-rutinoside is reported in the literature with a hepatoprotective effect (Wang, Tang, Zhang, 2015), in addition to other phenolic compounds have antioxidant and anti-inflammatory activity (Ma et al., 2019).

Polyphenol content and antioxidant activity
It is possible to verify in Table III that, compared to water, the hydroalcoholic solutions presented more efficiency on the recovery of phenolics and, in all scenarios evaluated, C. jacobinensis Baill.presented the highest concentration of these compounds.Generally, the incrase of the medium polarity favors the solubization of different phytochemicals, such as the phenolics Between the hydroalcoholic.Thus, as was expected, the hydroalcoholic solution of C. jacobinensis prepared at 70% (v/v) was the most effective on the extraction of phenolics (32.521,5 mg GAE.100g -1 ), being statistically different from those prepared with 50% (v/v) (31.384,7 mg GAE.100g -1 ) and with water (24.908,9GAE.100g -1 ) with a significance of 5%.
solvents on the extraction of phenolics from J. pectoralis.The authors pointed out ethanol as the best extractant on the recovery of the polyphenols, being 42 and 66% more efficient than water and ethyl acetate, respectivetely.Agra, Freitas and Barbosa-Filho (2007) found higher levels of phenolics in the ethanolic extract of J. pectoralis with an average value of 23.795 mg AGE/100 g.This value was higher than the one reported in this research for that plant, and this may be function of the differences of the cultivar characteristics, as well as in the process extraction itself.
Previously, it was highlighted the considerable amount of phenolics presented by C. jacobinensis Baill; however, there is a lack of information regarding its phytochemical characterization in the literature.Thus, the amount of phenolic found in other species of Croton was compared to the data found in this research.da Silva et al. (2019) evaluated different phytochemicals in hydroalcoholic extracts of C. floribundus and C. urucurana and found a phenolic concentration equal to 9.141 and 7.997 mg AGE/100g, respectively.An average of 2.602 mg AGE/100g for the aqueous extract of C. cajucara Bentda was reported by da Silva et al. (2013).For comparision, the aqueous extracted produced in this research presented a concentration of phenolics 89.55% higher than that point out by the authors.Dall'Acqua et al. (2021) evaluated the use of methanol, water, dichloromethane, ethyl acetate and water in the extraction of phenolics present in the C. hirtus L'Hér.The author found concentrations equal to 1.796, 2.424, 2.251 and 2.238 997 mg AGE/100g, which were expressively lower than the average obtained in this research for C. jacobinensis Baill (29.604,3 mg AGE/100g).According to Dutta, Dey and Chaudhuru ( 2013), the leaves of Croton ssp are rich in phytochemicals (e.g., phenolic compounds) that possess several bioactive properties, meaning that the plant has the potential to be used as a therapeutic agent.According to the authors, the phenolic compounds present anti-inflammatory, vasodilator, and antioxidant actions and can prevent several diseases, like cancer and heart complications.The total phenolic content and the antioxidant activity could depend on the solvent used for extraction.
It is possible to verify in Table III that the average antioxidant activity was 900 and 1.320,7 μM of Trolox g -1 for J. pectoralis Jacq and C. jacobinensis Baill, respectively.It was found a positive correlation between the phenolics concentration and the antioxidant activity; thus, the hydroalcoholic extracts presented a higher antioxidant activity compared to aqueous extract, for both species evaluated.Between the species, the C. jacobinensis Baill.exhibited the higher antioxidant activity in all scenarios evaluated, however the extracts prepared with 50 and 70% of ethanol did not present a statistical difference (P>0.05).
The information regarding the antioxidant characterization of J. pectoralis species is limited.For the aqueous infusions prepared with the leaf of J. pectoralis, Lizcano et al. (2010) found an antioxidant activity of 18.8 and 89.7 μM of Trolox g-1 and employing ABTS •+ and the oxygen radical absorbance capacity (ORAC) methods, respectively.These results were 95 and 75.6%, respectively, lower than that presented by the aqueous extract evaluated in this research.Croton spp are considered a source of antioxidant compounds, being reported in the literature several studies demonstrating their free radical scavenging capacity in a dosedependent way (da Silva et al., 2019;Suresh Reddy et al., 2015;Baqueiro-Peña, Guerrero-Beltrán, 2017).For C. jacobinensis, Keerthana, Kalaivani and Sumathy (2013) reported that the ethanolic extract of C. bonplandianum leaves has good antioxidant capacity after using the DPPH radical capture method.
The research on natural plant antioxidants has been applied in tests for pharmaceutical and food grade additives, due to its therapeutic and nutritional properties.Regarding the food industry, these substances from plants are considered alternatives to the substitution of synthetic antioxidants, such as butylhydroxyanisole and butylhydroxytoluene, which can cause adverse health effects (Rameshkumar, Sivasudha, 2012).Thus, the preliminary results indicate both J. pectoralis and C. jacobinensis Baill as potential natural antioxidants.

Antimicrobial activity
It is possible to evaluate in Table IV that all plant materials showed activity against the bacterial strains tested.The aqueous and 50% ethanolic extracts from J.
The antimicrobial action is associated with the specific characteristics of the extract to be used as an antimicrobial agent: its composition will determine the performance of this product as a preservation additive in food.The particular aspects of the antimicrobial agent to be applied in the food may limit its action as a preservative.Some metabolites with antimicrobial activity have hydrophobic properties that can prevent dissolution of the extract in water, limiting their use as food additives.The concentrations required for inhibition/inactivation of microorganisms are intrinsically linked to the specific targets of the antimicrobial agent because they depend on the structure of the target microbial cell and the specific genetic machinery of each species of microorganism (Furtado et al., 2015).
The aqueous extracts provided greater antimicrobial activity (1 and 10 mg/L MIC, respectively) against S. aureus and L. monocytogenes.These bacteria are considered to be more sensitive to antimicrobial agents due to the constitution of their cell walls.These are mostly of peptideoglycan, which facilitates the permeability of these substances (Singh et al., 2011).Higher extract concentrations were required for cell growth inhibition and death of gram-negative bacteria.The same concentration for MIC and MBC (25 mg/ mL) was observed for the aqueous extracts and for 50% ethanolic extract for S. enteritidis (Table IV).
The aqueous extract was more efficient than the ethanolic one (at 50% v/v) for E. coli.The gram-negative bacteria are more resistant to antimicrobials than grampositive ones, because the cell wall of gram-negative microorganisms has an outer membrane made of lipopolysaccharides and lipoproteins.In addition, there are flow pumps that actively remove harmful molecules from the cells, thereby further increasing tolerance to antimicrobial treatment.However, some authors have found different behavior when J. pectoralis extract is jacobinensis leaf extracts showed action against the Gram-positive bacteria, S. aureus and L. monocytogenes (Table IV).Only the hydroalcoholic extract (70% v/v) presented inhibitory and bactericidal action against the two gram-negative bacteria tested.
Similar to this research, Nunes et al. (2018) did not found cytotoxic effects in normal peripheral blood mononuclear cells treated with J. pectoralis extract usinf a concetration of 100 μg/mL.On the other hand, the same extract reduced the viability of neoplastic cell lines, demonstrating its potential on cancer treatment.According to Brighenti et al. (2014) used against gram-negative bacteria.Furtado et al. (2015) Justicia pectoralis, Cymbopogon citratus frente a cepas de bactérias gram-positivas e gram-negativas.Trata-se de uma pesquisa de caráter experimental, quantitativo e observacional.O material botânico (folhas studied the antimicrobial activity of an aqueous extract of J. pectoralis made by agar diffusion and verified that the extract was not efficient against E. coli and Klebsiella pneumoniae, both Gram-negative.This is justified by the fact that different techniques were used to determine the antimicrobial activity, different methods were used to obtain the extracts, and the plants originated in different countries.The hydroalcoholic extract (50% ethanol v/v) was only effective for S. enteritidis.Singh et al. (2011) evaluated the antimicrobial potential of aqueous, methanolic, and petroleum ether extracts of C. bonplandianum at concentrations of 50-125 mg/mL.These were obtained using the agar diffusion method and were used against Bacillus (now Paenibacillus) macerans, S. aureus, Pseudomonas aeruginosa, and Pseudomonas striata.The authors verified that the aqueous extract had action only against the bacterium P. aeruginosa (37.5% inhibition at 100 mg/mL).The methanolic extract presented the best antimicrobial potential, being ineffective only for S. aureus.

Citotoxicity test
For the cytotoxicity test, it was evaluated the aqueous extract of J. pectoralis and the hydroalcoholic extract at 70% (v/v) of C. jacobinensis.According to Figure 2, it is possible to state that both extracts did not present cytotoxic effects for the IEC 6 culture from direct comparison with negative control (cytotoxin A), which, as expect, presented a notable reduction in the cell population.It was observed a proportional increase in the cell viability with the increase of the concentrations.At higher concentration (20 mg/mL) it was verified an increase of 40 and 10% in the cell culture growth, for the J. pectoralis and C. jacobinensis, respectively.This result means that the extracts stimulate the growth of the cells and implies the safety of both extracts.extract of each plant may reduce the cytotoxicity potential of the concentrations studied.The authors also emphasize that the use of MTT has methodological limitations, requiring more accurate studies with animal models.In this case, studying the toxicity of J. pectoralis microcapsules using the Zebrafish method, Guimarães et al. (2020) did not report any sedative effect or impairment in the locomotion in the animals tested with the sample in different concentrations.Also employing the Zebrafish method, Ramos et al. (2009) evaluated the cytotoxicity of different plant extracts used in Northeastern medicide, between them several species of Croton.Regarding C. jacobinensis, the authors pointed the extracts prepared with the leaves using ethanol and hexane did not present toxicity for the animals.On the other hand, the ethanolic extract prepared with stem presented elevated toxicity.In general, even being different methods to evaluate the toxicity of natural samples, the results reported by the authors may collaborate to state the safety of the extracts produced with both studied plants.

CONCLUSIONS
The 70% ethanolic extract proved to be more effective for the presentation of higher amounts of bioactive compounds, as polyphenol compounds and presented higher antioxidant activity.The organic extracts studied showed antimicrobial activity against S. aureus, L. monocytogenes, S. enteritidis, and E. coli.The J. pectoralis and C. jacobinensis extracts showed potential for use in food products, justified by the indicated antioxidant and antimicrobial properties and by the non-toxicity to human cells of the extracts from both plants.

TABLE I -
Compounds identified in the hydroalcoholic extract of Justicia pectoralis Jacq.

TABLE I -
Compounds identified in the hydroalcoholic extract of Justicia pectoralis Jacq.

TABLE II -
Compounds identified in the hydroalcoholic extract of Croton jacobinences Baill.

TABLE III -
Total polyphenols content (TPC) and antioxidant activity of J. Pectoralis Jacq and C. jacobinensis Baill.extracts.

TABLE IV -
Antimicrobial activity of extracts of J. pectoralis Jacq and C. jacobinensis Baill leaves showed antimicrobial activity against S. aureus, L. monocytogenes, S. enteritidis, and E. coli.The ethanolic extract (70% v/v) did not show antibacterial activity for E. coli.The extracts of J. pectoralis were more effective against gram-positive bacteria.The C. pectoralis