Unraveling the Metabolomic Profile and Bioactivities of the Paratoid Gland Secretion from Rhinella granulosa

Toads of the Rhinella genus have a pair of paratoid glands that store biological secretions of high toxicity and varied chemical composition, rich in biologically active compounds. The present work aimed to carry out the investigation of the metabolomic profile and evaluation of the biological potential of the secretion paratoid glands (PGS) from Rhinella granulosa. The paratoid secretion was collected in the Piauí state (Brazil), extracted with methanol and the extract was analyzed by ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry. Fifty chemical constituents were identified. The extract showed cytotoxicity against tumor cell lines of the central nervous system (half maximal inhibitory concentration (IC50) = 1.9 µg mL-1) and prostate (IC50 = 1.6 µg mL-1), unsatisfactory antimicrobial potential (minimal inhibitory concentration (MIC) > 312 µg mL-1) and inhibited the enzyme acetylcholinesterase (IC50 = 5.119 mg mL-1). The results presented relevant information about the PGS and contributed to the understanding of the metabolomic and biological potential of R. granulosa.


Introduction
Natural products have been widely used by humanity since ancient times, mainly in the prevention, treatment, and cure of various diseases. 1The use of animal body parts and products of their metabolism (biological secretions and excrements) for the treatment of diseases, as well as for hunting, defense, and execution of prisoners, was a common practice among peoples of the Ancient Age. 2,3razil has a rich herpetofauna, with a large number of amphibian species. 4A total of 1026 species of amphibians have already been recorded in the country, of which 988 belong to the order Anura, representing the largest known anurofauna in the world, distributed in all Brazilian biomes. 5,6he Bufonidae family has a cosmopolitan geographic distribution, being present on all continents except Antarctica. 7,8In Brazil, the Bufonidae family is represented by eight genera, totaling 85 species, with the Rhinella genus being the most common. 9Toads of the Rhinella genus have a worldwide distribution, totaling about 200 species, most frequently in humid and tropical regions. 10,11nurans of the Bufonidae family have a pair of paratoid glands, present in the dorsolateral region of the body, which store biological secretions of high toxicity and varied chemical composition, having the function of defense against infections, microorganisms, and predators. 12A wide variety of compounds such as steroids (bufadienolides and bufotoxins), arginine diacids, alkaloids, peptides and proteins can be found in the paratoid secretions of amphibians. 13Bufadienolides have proven cardiotonic activity, while peptides and proteins are biomolecules, commonly referred to as toxins, which generally act on the central nervous system. 14About 2000 peptides have already been identified in amphibian glandular secretions.Among them are angiotensins, neuropeptides, myotropic peptides, antimicrobials and several others with a wide variety of biological activities. 15he biological secretions produced through glands found in the skin of anurans are rich in biologically active components and have great biotechnological potential. 16In ancient civilizations, secretion paratoid glands (PGS) of toads was already used as a diuretic, cardiac stimulant, expectorant, analgesic, and anti-inflammatory. 17,18In vitro studies showed compounds in paratoid secretions of these animals with several biological activities, such as antimalarial, antifungal, antitrypanosomal, antiviral, antileishmanial, antibacterial, insecticidal, anesthetic, and cytotoxic. 4,11,19hinella granulosa (Figure 1) is a toad found in different ecosystems with a wide distribution in the Brazilian Northeast and some southeastern Brazilian states, occurring mainly in the Caatinga biome, being more easily evidenced in the vicinity of streams, ponds, and water puddles. 20,21It is a small animal (48 to 53 mm) that has a nocturnal habit, explosive reproduction, and a diet consisting of arthropods, ants, and coleopterans. 22These toads have a back covered by small irregular granules (wart-like), varied coloration (from burnt yellow to brown), a whitish belly (cream), small dark spots scattered along the body, and inconspicuous paratoid glands, located just behind the eyes. 23onsidering the significant occurrence of R. granulosa in the southern region of the state of Piauí (Northeast Brazil) and the lack of scientific research on the PGS of this species, the present work aimed to carry out the investigation of the metabolomic profile and evaluation of the cytotoxicity, antimicrobial and anticholinesterase activities of the paratoid gland secretion from R. granulosa.

Obtaining the PGS
The toads of the species Rhinella granulosa were identified by biologists of the Federal University of Piauí, Picos campus, under the supervision of herpetologist, Prof Dr Mariluce Gonçalves Fonseca (IBAMA/SISBIO No. 22508-2).Through manual compression of the animal's paratoid glands in their natural habitat, the biological secretion of interest in this study was obtained.PGS was collected from toads distributed in the city of Picos (7°04'48"S, 41º26'10"W), located in the southern region of the Piauí state (Northeast Brazil), during the month of February 2022.After collection, the animals were returned to their natural habitat without injuries and/or bruises.
The collection of PGS from the animals was carried out after authorization from the Ethics Committee on the Use of Animals of the Federal University of Piauí (CEUA/UFPI No. 52107-2), supported by the research registration (SisGen No. AE58A09) and the permanent license for collection of zoological material (IBAMA/ SISBIO No. 55970-1).

Preparation of extract from paratoid secretion
During collection, the biological secretion was deposited in disposable plastic bottles and stored in a desiccator with silica for 72 h at room temperature (under vacuum).After this period, the dry PGS was transferred to a glass bottle and placed in a freezer at 4 °C.The extract was prepared by adding 50 mL of methanol (MeOH, Synth, Diadema, Brazil) to 1 g of PGS powder.The mixture was subjected to sonication in an ultrasonic (Ultronic, Indaiatuba, Brazil) bath for 15 min (four times), followed by simple filtration.The MeOH extract of PGS (yield of 55%) was obtained after rotoevaporation (Heidolph, Schwabach, Germany) of the solvent.
UPLC-QToF-MS/MS analysis U l t r a -p e r f o r m a n c e l i q u i d c h r o m a t o g r a p hy with quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS/MS) analysis was performed on a Waters Acquity UPLC Xevo G2-XS Q-TOF instrument (Waters, Milford, USA) with an electrospray ionization interface (ESI).The chromatographic separation was performed using an Acquity UPLC ® HSS T3 column (2.1 × 100 mm, 1.8 µm), with a mobile phase composed of ultrapure H 2 O + 0.1% formic acid (Merck, Darmstadt, Germany) (A) and MeCN (Sigma-Aldrich, Saint Louis, USA) + 0.1% formic acid (B).The elution gradient used was: 10 to 100% B in 8 min, maintaining the condition of 100% B for 0.2 min, returning to 10% B at t = 8.20 min, and maintaining this gradient until the time of 10 min.0.1 µL aliquots of the samples were injected at a flow rate of 0.5 mL min -1 , and the samples were solubilized (0.5 mg mL 1 ) in H 2 O/MeCN (3:7, v/v).
UPLC-MS data were obtained in positive ion detection mode.The parameters defined for MS were mass range m/z between 100-1500 Da, source temperature of 100 °C, capillary voltage of 2500 V, desolvation temperature of 250 °C, 40 V cone voltage, desolvation gas flow of 550 L h -1 , cone gas flow of 50 L h -1 , centroid mode, and 0.2 s -1 scan time.The MS/MS mode analysis was performed based on the application of a collision-induced dissociation energy gradient ranging from 10 to 30 eV.

Cytotoxic assay
The tumor lines used, HCT-116 (human colon), SNB-19 (glioblastoma), and PC-3 (prostate), were provided by the National Cancer Institute (USA), cultivated in RPMI-1640 medium (Thermo Fisher Scientific, Paisley, UK), and the non-tumor lineage L929 (murine fibroblast) was cultured in Dulbecco's Modified Eagle Medium (DMEM) medium (Thermo Fisher Scientific, Paisley, UK), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Paisley, UK) and 1% antibiotics (Penicillin-Streptomycin, Thermo Fisher Scientific, New York, USA), and kept in an oven at 37 ºC and an atmosphere containing 5% CO 2 .The sample was diluted in pure sterile dimethyl sulfoxide (DMSO, Tedia, Fairfield, USA), obtaining a stock solution with a concentration of 50 mg mL -1 , which was further diluted in working solutions to concentrations between 250 and 1.95 µg mL -1 .
The absorbances obtained in the test were used to calculate the concentration capable of inhibiting 50% of cell growth (IC 50 ) of the sample through non-linear regression using the GraphPad Prism program (version 8.0). 25 The MeOH extract from R. granulosa was tested in triplicate in three independent experiments.Doxorubicin (Sigma-Aldrich, Saint Louis, USA) was used as a positive control.

Antimicrobial assay
Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Candida albicans (ATCC 90028), and Candida krusei (ATCC 6258) were kindly provided by the Biology Laboratory Microorganism Collection of the Federal Institute of Maranhão, Monte Castelo Campus.Bacteria were cultured on Mueller-Hinton agar (MH, Merck, Darmstadt, Germany) at 37 ºC for 24 h, and the yeasts were cultured onto Sabouraud dextrose agar (SDA, Merck, Darmstadt, Germany) at 37 ºC for 48 h before tests.During the experiments, each culture medium was kept at 4°C.
The minimal inhibitory concentration (MIC) of the MeOH extract from R. granulosa was determined using the broth dilution method, as recommended by the Clinical Laboratory Standards Institute (CLSI). 26For this method, 190 μL per well of MH broth or 200 μL per well RPMI-1640 (Sigma-Aldrich, Poole, UK) buffered with 0.165 mol L -1 morpholinepropanesulfonic acid (MOPS, Sigma-Aldrich, Saint Louis, USA) for bacteria or yeasts, respectively, were added to 96-well microplates.
Before experiments, extract powder dissolved in dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany) was diluted in RPMI-1640 or MH broth medium, depending on whether the tests were against yeast or bacteria, respectively.Then, an aliquot of the MeOH extract (100 µL per well) was added to the first well of 96-well microplates, and serial dilutions were carried out in subsequent wells.Tested concentrations of MeOH extract were 2500-4.88µg mL -1 .Fluconazole (FLZ, Sigma-Aldrich, Poole, UK) and ciprofloxacin (CPR, Sigma-Aldrich, Poole, UK) were used as positive controls.
Following that, 100 µL of RPMI-diluted Candida inoculum (1 × 10 3 colony forming units (CFU) mL -1 ) or 10 µL of saline-diluted bacteria inoculum (1.5 × 10 8 CFU mL -1 ) was added to each well and incubated at 37 ºC for 24 h in RPMI-1640 medium.After the incubation period, the MIC was defined as the lowest concentration that visibly inhibited fungal growth.MIC values were confirmed after adding 10 µL of resazurin 0.03% (Thermo Fisher Scientific, Waltham, USA) to each well and incubating for 4 h in the dark at 37 ºC.RPMI-1640 or MH (100 µL) plus standardized inoculum was used as a negative control.Sterile DMSO (1% in saline) was also used as a negative control.The results were obtained from three independent assays performed in triplicate.
The quantitative test for AchE enzyme inhibition was performed, in triplicate, by dissolving 10 mg of MeOH extract in buffer A and 10% ethanol (Synth, Diadema, Brazil) (stock solution).From this solution, working solutions were prepared (dilution in buffer A) in concentrations of 10, 5, 2.5, 1.25, and 0.625 mg mL -1 .25 µL of MeOH extract solution, 50 µL of buffer B, and 25 µL of AchE 0.22 U mL -1 were added to the microplate wells.The blank was prepared with 25 µL of buffer A with 10% ethanol in 50 µL of buffer B with 25 µL of AchE 0.22 U mL -1 .The microplate with the extract solutions was kept in an oven for 15 min at 37 °C.Subsequently, 125 µL of 3 mmol L -1 DTNB and 25 µL of 15 mmol L -1 ATCI were added to each investigated extract solution, and the plate was measured at λ = 405 nm, at 0 and 5 min, using a microplate reader model Polaris (Celer, Belo Horizonte, Brazil).Rivastigmine (Exelon, Basel, Switzerland) was used as the positive control.

Identification of chemical constituents of PGS
The investigation of the chemical profile of the methanolic extract of PGS from Rhinella granulosa, collected in the city of Picos (Caatinga biome) in the southern region of the state of Piauí (Brazil), allowed the identification of 50 constituents (Figure 2), distributed in six classes of compounds: one amino acid, one carboxylic acid, three indole alkaloids, five arginine derivatives, 16 bufadienolides, and 24 bufotoxins.The compounds were identified by comparison with data reported in the literature, considering the relative error, the elution order of the chromatographic column (retention time), and the main fragment ions of the protonated molecules (Table 1).9][30] However, this is the first time that the amino acid arginine (1) and martinelic carboxylic acid (50) are identified in the PGS of toads of the genus Rhinella, especially from the South American continent.
Compound 1 was identified as the amino acid L-arginine, being reported for the first time in the secretion of the paratoid glands of toads of the Rhinella genus, Cao et al. 28 identified this amino acid in Chansu, a commercial product from the glandular secretions of Bufo gargarizans Cantor,   used in traditional Chinese medicine for the treatment of various diseases, including cancer.Compounds 3, 5, and 6 belong to the class of alkaloids.Although they occur mostly in plants, they can be found in animals, especially amphibians, which use these substances for their defense and protection against pathogens and predators. 39,40Studies 41,42 have shown that the concentration of alkaloids in anurans can vary depending on the species, geographic location, diet, and other associated complex characteristics.
Compound 3 was identified as indole-3-acetaldehyde and was evidenced in the chemical composition of of anurans of the Bufonidae family. 44These steroids are primarily responsible for the biological activities identified in amphibian glandular secretions. 45More than one hundred bufadienolides identified in biological secretions of toads are described in the literature. 31The bufadienolides identified in this work showed a similar fragmentation pattern, evidenced by the successive losses of water molecules

and the carbon monoxide group [M + H
The bufadienolides identified in the MeOH extract of R. granulosa can be divided into two groups.The compounds cinobufagin (10), resibufaginol (35), bufotalinin (36), marinobufagin (43), resibufagin (47), and resibufogenin (48) have an epoxy group at the C-14 and C-15 carbons, while the compounds bufarenogin (15), Ψ-bufarenogin (16), arenobufagin (23), hellebrigenin (29), gamabufotalin (33), argentinogenin (38), desacetylbufotalin (40), telocinobufagin (42), bufalin (46), and scillaridin A (49) have a hydroxyl group at the C-14 carbon.The presence of cyclic ether in the compounds alters the fragmentation pattern, generating distinct fragment ions between the two groups of bufadienolides. 37ufotoxins constitute the largest number of compounds identified in this study, totaling 24 metabolites.The number of bufotoxins identified in the glandular secretions of anurans is increasing, since there are numerous possibilities of combination between diacids, bufadienolides, and amino acids. 46Bufotoxins are widely described in glandular secretions of toads of the Bufo, Rhinella, and Rhaebo genera distributed around the world. 36A general fragmentation pattern for bufotoxins is the loss of the arginine-derived diacid [M + H − arginine diacid] + , followed by the loss of an amine group [M + H − NH 3 ] + and one or two water molecules by the generated diacid. 43A reduction of 28 Da in the mass of the compound was also observed, referring to the loss of the carbonyl group [M + H − CO] + and the loss of one or more water molecules by the bufadienolide portion. 37,38imilarly to the bufadienolides reported in this study, bufotoxins can also be organized into two distinct groups, characterized by the presence or absence of the epoxy group in the steroidal ring of the bufadienolide portion of the bufotoxin.The compounds 3-(N-suberoyl argininyl) hydroxybufotalinin ( 18 44), this structure is not observed.However, the observed structural difference does not change the fragmentation pattern of this class of compounds. 37,38ompound 50 was identified as martinelic acid, being evidenced for the first time in acetic extract from the skin of the Australian cane toad (Bufo marinus). 34here are no reports in the literature of the identification of this carboxylic acid in glandular secretions of toads in the Rhinella genus, distributed in the South American continent.It is the first time that martinelic acid has been detected in PGS from toads occurring in Brazil.

Cytotoxicity
9][30] This animal matrix has attracted the attention of the scientific community, mainly due to the richness of compounds and the amount of biologically active metabolites, making it a promising alternative in the development of new therapeutic resources for the treatment of the most varied types of cancer. 47he use of the MTT dye reduction method showed that the MeOH extract from the secretion of the paratoid glands of R. granulosa produced a considerable cytotoxic effect in central nervous system (SNB-19) and prostate (PC-3) tumor cells, with no similar behavior being observed in the colorectal tumor cell line (HCT-116).Against SNB-19 tumor cells (glioblastoma), the MeOH extract showed similar cytotoxicity (IC 50 = 1.9 µg mL -1 ) to the positive control used (doxorubicin, IC 50 = 2.0 µg mL -1 ).
The cytotoxicity of the R. granulosa MeOH extract was also evaluated in the non-tumor cell line L929 (murine fibroblast) and did not present effect on growth inhibition, even when tested at the highest extract concentration, being more selective for tumoral cells.The IC 50 value was above 125 µg mL -1 .Due to the low cytotoxicity of the extract on the L929 cell line, at the tested concentrations (1-125 µg mL -1 ), it was not possible to obtain a doseresponse curve to determine the exact IC 50 value.The IC 50 values, with a confidence interval of 95%, of the methanolic extract and doxorubicin against the tumor and non-tumor cell lines tested in this assay are presented in Table 2.

Antimicrobial activity
The glandular secretions from anurans are complex mixtures of bioactive compounds; however, the biological activities of many of these biomolecules are still unknown, especially regarding the antimicrobial activities of the compounds present in the secretions of the paratoid glands of toads. 48able 2 presents the values of the MIC detected for the MeOH extract from the secretion of the paratoid glands of R. granulosa against the standard strains of S. aureus (ATCC 29213), E. coli (ATCC 25922), C. albicans (ATCC 90028), and C. krusei (ATCC 6258).
The MeOH extract of R. granulosa showed low antibacterial potential against strains of S. aureus (MIC = 312 µg mL -1 ) and E. coli (MIC = 625 µg mL -1 ), not showing relevance for practical uses (MIC of interest < 1.0 µg mL -1 ).Similarly, the investigated extract did not show a satisfactory antifungal effect (MIC = 1250 μg mL -1 ) against yeasts of the genus Candida (MIC of interest < 8 μg mL -1 ).There are reports in the scientific literature 48 on the antimicrobial activity of PGS from other species of the genus Rhinella; however, this work is pioneer in showing the antibacterial potential of R. granulosa.

Potential to inhibit the enzyme acetylcholinesterase
The in vitro study carried out with the MeOH extract of PGS from R. granulosa at concentrations of 0.625, 1.25,  2.50, 5.00, and 10.00 mg mL -1 produced inhibitions of 17.45, 18.78, 27.83, 55.35, and 63.69% in the activity of the enzyme AChE, respectively.From this data set, the IC 50 of the MeOH extract was calculated, which corresponded to 5.119 mg mL -1 , ranging from 4.411 to 6.059 mg mL -1 , with a confidence interval of 95%.These data are reported in Table 2.
Although this scientific investigation represents a preliminary study of bioprospecting of the paratoid secretion of R. granulosa from Northeast Brazil, future research may lead to the isolation of identified metabolites and investigation of the biological potential of these substances, like the studies developed by Tempone et al., 19 Machado et al. 11 and Monção Filho et al. 37,40

Conclusions
The investigation of the chemical profile of the methanolic extract of PGS from Rhinella granulosa allowed the identification of 50 constituents, distributed in six classes of compounds: one amino acid, one carboxylic acid, three indole alkaloids, five arginine-derived diacids, 16 bufadienolides, and 24 bufotoxins.The compounds evidenced in the matrix studied are reported in glandular secretions of other anuran species of the genera Bufo, Rhinella and Rhaebo.However, this is the first time that the compounds arginine (1) and martinelic acid (50) are identified in the PGS of toads of the genus Rhinella, mainly from the South American continent.The extract from the of the paratoid glands of R. granulosa showed promising cytotoxicity, mainly against tumor cell lines of the central nervous system (SNB-19) and prostate (PC-3), unsatisfactory antimicrobial potential and considerable potential to inhibit the enzyme acetylcholinesterase.This study presents relevant information about the PGS analyzed and contributes to the understanding of the chemical and biological potential of R. granulosa, expanding the knowledge about the anurofauna of the state of Piauí (Brazil).

Figure 2 .
Figure 2. Total ion chromatogram of methanolic extract of the paratoid secretion from R. granulosa.

Figure 3 .
Figure 3.Chemical constituents identified in the methanolic extract of PGS from R. granulosa.

Table 1 .
Identification of compounds of the MeOH extract of paratoid secretion from R. granulosa by UPLC-QToF-MS/MS fragmentation pattern in positive ion mode + : protonated molecule; ppm: parts per million; Ψ: Psi.

Table 1 .
Identification of compounds of the MeOH extract of paratoid secretion from R. granulosa by UPLC-QToF-MS/MS fragmentation pattern in positive ion mode (cont.)

Table 2 .
Bioactivities of the MeOH extract of paratoid gland secretion from Rhinella granulosa