Phytochemistry Profile, Antimicrobial and Antitumor Potential of the Methanolic Extract of Tabernaemontana catharinensis A DC and Eragrostis plana NEES

Natural compounds that have the potential to act as antimicrobials and antitumors are a constant search in the field of pharmacotherapy. Eragrostis plana NEES (Poaceae) is a grass with high allelopathic potential. Allelopathy is associated with compounds generated in the primary and secondary metabolism of the plant, which act to protect it from phytopathogens. Tabernaemontana catharinensis A DC (Apocynaceae), a tree in which its leaves and bark are used for the preparation of extracts and infusions that have anti-inflammatory and antinociceptive effects, is attributed to its phytochemical constitution. The objective of this study was to elucidate the phytochemical constitution, the antibacterial potential, the toxicity against immune system cells, hemolytic potential, and antitumor effect of methanolic extracts of E. plana and T. catharinensis. The phytochemical investigation was carried out using the UHPLC-QTOF MS equipment. The antibacterial activity was tested using the broth microdilution plate assay, against Gram-negative and Gram-positive strains, and cytotoxicity assays were performed on human peripheral blood mononuclear cells (PBMC) and in vitro hemolysis. Antitumor activity was performed against the colon cancer cell line (CT26). Results were expressed as mean and standard deviation and analyzed by ANOVA. p < 0.05 was considered significant. More than 19 possible phytochemical constituents were identified for each plant, with emphasis on phenolic compounds (acids: vanillic, caffeic, and quinic) and alkaloids (alstovenine, rhyncophylline, amezepine, voacangine, and coronaridine). Both extracts showed antibacterial activity at concentrations below 500 µg/mL and were able to decrease the viability of CT26 at concentrations below 2000 µg/mL, without showing cytotoxic effect on PBMCs and in vitro hemolysis at the highest concentration tested. This is the first report of the activity of E. plana and T. catharinensis extracts against colon cancer cell line (CT26). Studies should be carried out to verify possible molecular targets involved in the antitumor effect in vivo.


Introduction
Te signifcant increase in drug-resistant pathogens is associated with the inappropriate and indiscriminate prescription of drugs used in clinical practice, treatment time, among other factors, and although there is a wide range of antimicrobial drugs on the market, resistance is a limiting factor, considered a major public health problem [1,2].
Another problem that has a great impact on public health is the increasing number of cancer cases.It is estimated that more than 18 million cases are diagnosed each year [3].Colon cancer, or colorectal cancer (CRC), appears among the three main types of cancer diagnosed in the population, afecting both sexes, in diferent life cycles; however, data from the literature support that the highest incidence occurs from 50 years old [3,4].
Treatment for CRC has evolved tremendously in recent decades, with the removal of tumors being the frst form of treatment, hence the importance of obtaining an early diagnosis.Te surgical procedure can be curative, depending on the stage of the tumor [5].When the removal procedure is not the best option, chemotherapy and/or radiotherapy treatment is necessary as adjuvants which, although they have evolved with the advancement of new drugs being developed, is still a high-cost treatment that culminates in the deterioration of physiological functions of the individual [6,7].Tere is a constant search for new therapeutic alternatives for both antimicrobial and anticancer agents, and herbal medicine is among their agents.
Te use of plants as an alternative treatment for diseases is an ancient practice, and it is estimated that more than 80% of the world's population has already made use of some type of plant.With a healing purpose, Brazil has one of the greatest plant diversity in its fora in the world, and a large part is used for therapeutic purposes, many with scientifc evidence of their efectiveness and a wide range still being studied [8].Te advance in the discovery of active compounds in products resulting from the secondary metabolism of plants brought a new perspective for the treatment of numerous pathologies, as well as clarifcation on the toxic potential of species used indiscriminately [9,10].
Eragrostis plana NESS (Lovegrass) popularly known as "annoni" (Poaceae) is a grass originally from South Africa, brought to Brazil by accident in the 1950s, mixed with pasture seeds [11].Initially seen as a promising pasture due to its great resistance to climate change, the annoni grass quickly spread through the felds of southern Brazil, due to its easy propagation.However, the predominance in soil compared to native species of the Gaucho pampa and the low nutritional value made it a problem for the agricultural sector [11], having its sale was banned in 1979.Since the prohibition of the sale of lovegrass seeds, studies involving growing this type of plant are mostly focused on the area of agrarian sciences, with the aim of trying to control its dissemination.Lovegrass has high resistance even to desiccant products, and its small seeds spread easily, making it practically impossible to control the pest around the soil.
Te allelopathic profle of this dominant plant is due to its chemical constituents and secondary metabolites, which have diferent functions in the plant, such as nutrition and protection against insects [12].Isolated fractions, containing diterpenes, obtained through the plant extract (Eragrostis plana) show action against phytofungi, justifying the studies aimed at the development of natural defensive agriculture taking advantage of the allelochemical potential of the plant [13].
Tabernaemontana catharinensis A DC, popularly known as "cobrina," is a bushy plant, belonging to the Apocynaceae family, widely found in the southern region of Brazil, as well as in neighboring countries such as Uruguay, Argentina, and Paraguay [14].
Te medicinal use of infusions and macerations of diferent parts of the plant has been reported for a long time, commonly used by indigenous civilizations as an antiophidic, and this property gives rise to its popular name ("snake � cobra in Portuguese").Te antinociceptive efect of the plant made it popularly used to relieve pain.Many activities attributed to T. catharinensis have already been scientifcally proven.Some of its healing properties are antiinfammatory, analgesic, antifungal, antioxidant, and even antineoplastic activity [15][16][17][18].
Te therapeutic properties are related to its rich phytochemical constitution, mainly to the numerous indole alkaloids, which are reported in leaves, fowers, bark, and roots of the plant [19][20][21][22].
Te therapeutic efect of plant extracts is related to their phytochemical constituents, either raw or isolated.Both plants, T. catharinensis [16,[26][27][28][29][30][31] and E. plana [12,32], have therapeutic efects already described and attributed to their phytochemical composition; however, there are few research using crude extract of T. catharinensis, and no reports for E. plana use against the CT26 tumor lineage, justifying the present study.
In this sense, the aim of this research was to evaluate the activity of crude methanolic extract of Eragrostis plana NEES and Tabernaemontana catharinensis A DC against diferent bacterial strains, test its cytotoxic efectiveness against colon cancer strain (CT26), and in contrast to assessing whether the plant exerts a toxic efect on cell culture of the peripheral blood mononuclear cell (PBMCs), and identify possible phytochemical constituents.

2
Evidence-Based Complementary and Alternative Medicine ® , the column was maintained at 35 °C, and the QTOF-MS mass analyzer (Impact II, Bruker Daltonics).Te QTOF-MS system is equipped with an electrospray ionization (ESI) source, operating in both positive and negative modes.Te separation method was in gradient mode A: acetonitrile (0.1% formic acid) and B: aqueous phase (0.1% formic acid), the elution gradient started with 5% A maintained for 2 min, increasing to 95% up to 10 min, and maintained for 3 min, after which it was decreased to 5% in 2 min, and maintained for 5 min.Te fow rate was 0.3 mL•min −1 , and the injection volume was 10 μL.

Materials and Methods
Te operating parameters of the ESI were capillary voltage, 2500 V; end plate ofset, 500 V; nebulizer pressure, 3 bar (N 2 ); drying gas, 9 L•min −1 (N 2 ); and drying temperature, 200 °C, for negative mode, and capillary voltage, 4000 V; end plate ofset, 500 V; nebulizer pressure, 3 bar (N 2 ); drying gas, 9 L•min −1 (N 2 ); drying temperature, 200 °C, for positive mode.Te QTOF-MS system operated in broadband collision-induced dissociation (bbCID) acquisition mode in a range of m/z 60−1600 with an acquisition rate of 2 Hz, and bbCID mode provides MS and MS/MS information at the same time.Calibration of the QTOF-MS system was performed with a sodium formate solution.Te data were processed using Data Analysis 4.2 software.Te mass accuracy tolerance was less than 5 ppm.

Antibacterial Activity Assay.
To assess the minimal inhibitory concentration (MIC), the 96-well plate microdilution was used, containing Mueller Hinton broth [33].Ten diferent concentrations of extracts were evaluated against diferent microorganisms to determine the lowest concentration capable of inhibiting bacterial growth.
Extracts were serial diluted in decreasing concentration from 500 to 0.97 μg/mL against 13 MO in a 96-well plate containing Mueller Hinton broth (Sigma-Aldrich ® ) in 100 μL.
Five microliters of inocula in Mueller Hinton broth were added.After 24 h of incubation in a bacteriological incubator (35 °C), 20 µL of 70% ethanol solution of 2,3,5-triphenyl-tetrazolium chloride (CTT) at 0.5% was added in each well.Ten, the plates were incubated again for 120 minutes, at 35 °C.Te CTT has the ability to react with MO which has some metabolic activity, changing its color in the medium to red when the microorganism is viable, allowing visual diferentiation.

In Vitro Hemolysis Assay.
For this test, erythrocytes were obtained from 3 mL of human venous blood, collected in a heparinized Vacutainer ® (approved by the Research Ethics Committee of the Universidade Federal do Pampa, no 27045614.0.0000.5323),separated by centrifugation (1000 × g for 5 minutes), washed, centrifuged three times with saline solution, and then suspended in saline solution forming a hematocrit of 1%.Te lyophilized extracts were resuspended in 0.9% saline solution, at a concentration of 2000 μg/mL.Tis solution was fltrate; 2 mL of the fltrate was mixed with 1 mL of the erythrocyte suspension, with subsequent incubation under continuous homogenization for 20 minutes at room temperature [34].Saline solution was used as negative control and distilled water as positive control.
Te occurrence of erythrocyte lysis was observed after centrifugation (1000 × g for 5 minutes).Te results obtained were determined through reading in a spectrophotometer at 530 nm, being considered as capable or not of promoting hemolytic action in vitro.Tests were performed in triplicate.Te average of the optical density (OD) readings was used in the following equation, by which it is possible to measure the % of in vitro hemolysis.

Hemolysis(%):
OD Cell plating was performed with 1.2 × 10 4 cells/well with cell viability greater than 95% and treated with plant extracts at concentrations of 500, 1000, and 2000 μg/mL.After 24 hours of treatment, the culture medium with extract was removed and washed with PBS, preserving the treated cells on the plate, where 90 μL of RPMI 1640 without phenol red and 10 μL of [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium] solution (MTT solution) were added to each well and incubated for 4 hours, at 37 °C, in a controlled atmosphere of 5% of CO 2 .After the incubation period, 75 μL of the medium from each well was removed, and then, DMSO reagent (50 μL/well) was added to solubilize the formazan crystals generated in the assay.Te plate was reincubated for 10 minutes under the same conditions previously described, and then, the reading was performed on a spectrophotometer (Victor ® ) at a wavelength of 540 nm.Te MTT method consists of a colorimetric assay to evaluate cell viability, being highly sensitive to cytotoxic agents [36].All tests were performed in triplicate.Results were expressed as % cell viability.

Statistical Analysis.
Te results were expressed as mean and standard deviation of the mean.Analysis of variance was performed using one-way ANOVA, followed by Tukey's post hoc test, and was considered signifcant when p < 0.05, in statistical software GraphPad Prism 9.

Results and Discussion
3.1.Phytochemical Analysis.Te assay carried out by UHPLC-QTOF MS made it possible to fnd 30 main peaks for the methanolic extract of E. plana (Table 1) and 20 for the methanolic extract of T. catharinensis (Table 2).From the mass (m/z) obtained from UHPLC-QTOF MS data analysis and structure through the software Data Analysis 4.2, we suggested the identifcation of the compound and obtained through data available in these databases (PubChem ® , massbank.eu).Te mass spectra obtained in the assay are presented in Figure S1 (E.plana) and Figure S2 (T.catharinensis), in supplementary materials, and follow the order presented in the respective tables.
Cafeic acid is a phenolic compound of great relevance present in a wide range of plant species.Tests using the compound in isolation reveal antioxidant, anti-infammatory, antimicrobial, and antitumor properties, with a mechanism of action of the molecular pathway of oncogenesis [37,38].Tis compound is also described in phytochemical investigation assays of extracts obtained from diferent parts of the same plant (E.plana), using other extraction methods, and employing methodological analysis such as thin-layer chromatography and high-performance liquid chromatography [32].Cafeic acid and its derivatives are reported to have efects against several types of cancer (breast, hepatocellular carcinoma, skin, lung, oral, and cervical) [37].
With the exception of the previously described phenolic compounds [12,38], our study suggests the presence of more than 20 compounds that had not yet been related to E. plana.Te phytochemical constituent number 6 (m/z 371.2406, presented in table 1) and peak 6 in Figure S1, was identifed as the compound Tamoxifen, already used as a chemotherapy drug for the treatment of breast cancer [40].
Interestingly, peak number 7 (m/z 375.1841), in Figure S1, indicates the presence of a compound identifed as benzylmorphine, a morphine derivative, which, in turn, is known worldwide for having a sedative and potent analgesic efect, one of the frst efective drugs to treat pain in people sufering from cancer, for example [41].Tis is the frst report of the presence of this molecule in E. plana extract.
Also, peak 1 (m/z 163.0389), which can be observed both in the E. plana extract and in the T. catharinensis extract, indicates the presence of a heterocyclic azole compound, and the class is widely used in the treatment of infections fungicides and also as a microbial and antitumor agent [42].
Among the compounds suggested for the T catharinensis extract, we highlight the following: compounds of the phenolic class (vanillic acid and quinic acid), where these compounds are present in a wide variety of fruits, vegetables, and herbs and are often studied due to their potential pharmacological use [43].4 Evidence-Based Complementary and Alternative Medicine Alkaloids suggested for the T catharinensis, among which alstovenine, rhynchophylline, amezepine, voacangine, and coronaridine already have biological activity described in the literature [44].Rhynchophylline, identifed in Figure S2, in peak 8, has a cytotoxic activity for tumor cells, being able to reduce resistance to chemotherapy by a mechanism of decreased efux of drugs via inhibition of the ATP-binding cassette (ABCB1) transporter, reducing multidrug resistance to chemotherapy [45].
Other phytochemical studies involving Tabernaemontana also indicate the presence of some alkaloids suggested in our study.Te present voacangine and coronaridine are described in diferent plants of the Tabernaemontana genus.Tese alkaloids are related to the strong antimicrobial activity of the plant, in addition to in vitro antitumor and anticholinesterase activity, whether isolated or synergistically with the other constituents [46][47][48].

Antibacterial Activity.
Te methanolic extract had bactericidal activity against all tested microorganisms, in all tested concentrations.Te extract of E. plana showed the lowest MIC (15.3 μg/mL) found for Staphylococcus aureus subsp.aureus ATCC ® 25923, an important Gram-positive pathogen that is involved in many infectious processes [49].Te extract of Tabernaemontana catharinensis A DC has the lowest inhibitory concentration (3.13 μg/mL) as it was observed against MO Staphylococcus aureus subsp.aureus ATCC ® 29213, and a Gram-positive, methicillin-resistant pathogenic microorganism is associated with a wide number of infections, including necrotizing pneumonia [50].Te results for both extracts are shown in Table 3.
Other study tested the methanolic extract of roots and aerial parts of grass annoni against Staphylococcus aureus (ATCC 27213), Staphylococcus epidermidis (ATCC 12228), Pseudomonas aeruginosa (ATCC 27853), and Salmonella  typhi (ATCC 19196), fnding an MIC of 100 mg/mL only for MO treated with the extract prepared with the aerial part of the plant, a concentration much higher than that presented in our study.Te authors report that there was no inhibition of bacterial growth compared to extracts from the plant's roots, suggesting that most of the metabolites are present in the aerial parts of the plant [13].
Gindri et al. [51] evaluated the activity of crude ethanolic extract and three fractions of T. catharinensis against the following MO: Micrococcus sp., Staphylococcus aureus and Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Aeromonas sp.(all with MIC >1000 mg/mL), Enterococcus faecalis, and Proteus mirabilis (MIC: 62.50 mg/mL), where only the n-butanol fraction showed some antimicrobial activity.Te authors suggest that this result is related to polar compounds (phenolic compounds, tannins, and favonoids) present in the extract fraction and that these, in turn, have greater microbiological activity.Our results, using crude extract, present antimicrobial activity at lower concentrations than that indicated in the cited study, being less than 500 µg/mL for both extracts, emphasizing the potential use of plants as an antimicrobial agent.Te solvent chosen for preparing and obtaining the crude extract seems to be closely related to the increase or decrease in antimicrobial activity [52,53].

In Vitro Hemolysis Assay.
Both extracts tested did not cause hemolysis in erythrocytes (Figure 1) when tested at a concentration of 2000 µg/mL, and the concentration chosen for the test contemplates the concentrations used in the other tests of the present study.Te hemolysis assay is used to assess the toxicity of compounds in vitro, in a stage prior to in vivo assays [34].Te range of protocols to assess hemolysis is a bias that hinders the comparison between results.A recent study proposes a standardization for the in vitro hemolysis assay to assess cytotoxicity, and this standardized protocol will facilitate the comparison between studies [61].

Cell Culture and Viability.
Te extracts showed no cytotoxic efect on the PBMC culture in vitro, at any of the concentrations tested (Figures 2(a) and 2(b)), and this is one of the main objectives when looking for a new therapeutic agent, which is efcient without afecting healthy cells, enabling the expansion of the range of the assay.
On the other hand, both extracts decreased the cell viability of the CT26 cell line in vitro at all concentrations tested, as shown in Figures 3(a) and 3(b).Furthermore, a morphological change in the cells (Figure 4 and 5) was observed at concentrations of 1000 (b) and 2000 (c) μg/mL.Te negative control group (a), untreated cells, maintained high viability and did not have its morphology altered, which allows us to state that the result is in fact related to the treatment with the extracts, being this the frst report of in vitro assays of the extract E. plana in front of a tumor lineage.
Artico et al. [62] evaluated the cytotoxic, genotoxic, and mutagenic potential of two extracts of E. plana in the experimental model of Allium cepa.Te extract used as a positive control (PC) was obtained from E. plana collected in a region of charcoal-contaminated soil, and the other (negative control) obtained from E. plana was collected in a region with a low probability of contamination.Te E. plana extract obtained from plants in contaminated (PC) soil showed a greater mutagenic and genotoxic potential, although both had an antiproliferative efect.Te authors attribute the fact that the extract had this efect due to the large number of secondary metabolites produced by the plant, as evidenced by Favaretto and collaborators in their study on the plant's phytochemistry [32].Evidence-Based Complementary and Alternative Medicine Te toxic efect of fractions of the extract obtained from the bark and roots of T. catharinensis was tested against tumor cell lines (HeLa; B-16; Hep-2) and a nontumor cell line of 3T3 fbroblasts.Te authors found a cytotoxic efect against tumor cell lines; however, the same result was identifed in normal cells [23].Rosales et al. [22] identifed the cytotoxic activity of an alkaloid isolated from the ethanol extract of T. catharinensis bark against human melanoma cell lines (A375, WM1366, and SK-MEL-28).Similarly, others report the antiproliferative efect of the alkaloid fraction also isolated from the bark of the plant, against a human colorectal cancer strain (HT-29), with a low toxic efect in nontumor strains [24].Te antitumor efects found by the action of the E. plana and T. catharinensis extracts may be related to the compounds, suggested by the phytochemical analysis.For E. plana extracts, we suggest cafeic acid [37,38], benzothiazole derivatives [42], and tamoxifen [40] which is already used in clinical medicine as chemotherapy.Quinoline alkaloids and derivatives have anticancer activity [60,63].For T. catharinensis extracts, the presence of some alkaloids reinforces these actions, such as Evidence-Based Complementary and Alternative Medicine voacangine, coronaridine [47,48], and rhynchophylla, and exhibits cytotoxicity towards tumor cell lines [45].Vanillic acid has activity against colon cancer cells (HCT 116) [64] and quinic acid in nanoparticles against solid tumors [65,66].
Considering that we used the crude extract of T. catharinensis, our results corroborate the fndings in the literature, evidencing the antitumor potential of the genus Tabernaemontana.Nevertheless, it was possible to observe that the extract caused morphological changes in the CT26 strain.We suggest that these changes were related to the decrease in cell viability.

Conclusion
Te methanolic extracts of E. plana and T. catharines showed antiproliferative efect against tumor lineage (CT26), as well as antibacterial efect against diferent pathogenic strains (MIC <500 µg/mL), without showing cytotoxic efect in PBMCs and hemolysis in erythrocytes.Te antibacterial and antineoplastic activities of the extracts showed a cytosafe concentration.Te phytochemical constitution suggested for both extracts, with compounds already known to exert antitumor and antibacterial activity, and possibly the synergism between them, can be involved in the results shown in this study.Our study is the frst to show that the crude extract of E. plana is efective against human pathogens, as well as the CT26 tumor lineage, without causing toxic efects in the immune system cells.Studies should be carried out to verify possible molecular targets involved in the antitumor efect in vivo.

Figure 3 :Figure 4 :Figure 5 :
Figure 3: Percentage of viable cells (CT26) in vitro exposed to treatment with methanolic extract of Eragrostis plana NEES (a) and Tabernaemontana catharinensis A DC (b) at diferent concentrations.NC: negative control; results were expressed as mean and standard deviation.Diferent letters mean a statistically signifcant diference (p < 0.05).03 assays in triplicate were performed.

Table 1 :
Mass/charge (m/z), suggested structural formula of possible phytochemical constituents in E. plana extract obtained through UHPLC-QTOF MS analysis.

Table 2 :
Mass/charge (m/z), suggested structural formula of possible phytochemical constituents in T. catharinensis extract obtained through UHPLC-QTOF MS analysis.

Table 3 :
Minimum inhibitory concentration (MIC) of the extract of Eragrostis plana NEES and Tabernaemontana catharinensis A DC, against diferent microorganisms.
Percentage of hemolysis of erythrocytes exposed to treatment with Eragrostis plana NEES and Tabernaemontana catharinensis A DC extracts, at a concentration of 2000 µg/mL.NC: negative control; PC: positive control.Results were expressed as mean and standard deviation.Diferent letters mean statistically signifcant diference (p < 0.05).Assays were performed in triplicate.