Metabolic profile, antimicrobial and toxicity evaluation of Azadirachta indica roots

ABSTRACT: The constituents of the hydroethanolic extract ofAzadiractaindicaroot were investigated using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-QTOOF-MSE). Acute toxicity was evaluated in an experimental animal model. We investigated the antibacterial activities ofA. indicaroots againstSalmonella typhimuriumandStaphylococcus aureusand the antifungal activities against strains ofTrichophyton rubrum, Candida albicansandCandida tropicalis. We identified nine secondary metabolites in the hydroethanolic extract by UPLC-QTOOF-MSE. The extract was highly effective in inhibiting the growth of T. rubrum strains, so it can be effective in combating the dermatophyte tested,but it had no inhibition potential on any bacterial strains orCandidaspecies evaluated. It was possible to infer that the extract had no acute toxicity in relation to the animal model Danio rerio. Therefore, since neem has a high bioactive potential and adapts well to the climate of semiarid regions, growing this species could become a source of income for farmers by its use to produce naturals fungicide and drug, as alternatives to conventional products, which can cause microbiological resistance and/or are toxic to the environment, besides being expensive.


IntROduCtIOn
Neem (A. indica) is a plant native to the region between India and Burma, belonging to the family Meliaceae (KUMAR & NAVARATNAM, 2013;ZHU et al., 2017).This species has gained global importance because it is an extremely versatile medicinal plant (DALLAQUA et al., 2013).In India, A. indica has been used in traditional medicine for over 2000 years because of its anti-inflammatory, anti-ulcer, anti-malarial, anti-bacterial and anti-oxidant activities (BHOWMIK et al., 2011;KURIMOTO et al., 2014).In countries where it is cultivated, studies have reported that its preparations are effective against ringworm and other fungal infections, as well as dermatitis, eczema and acne.Also, leaf cataplasms or decoctions are used against boils and the oil is used to cure skin diseases such as scrofula and indolent ulcers (BHOWMIK et al., 2011).
The azadiractins, preferentially reported in neem seeds, are complex limonoid tetranortriterpenoids well known to be the most active constituents contained in the species, presenting antifungal and antifeedant properties against insects (MORIELLO & NISBET, 2000;DAVID et al., 2017).Azadirachtin, who named the class, is the active principle of several neembased products and the most studied substance of the species.Despite the wide use of this active principle, due to its complexity it has not yet been synthesized, so all products available in the market are prepared by the extraction of compounds from the plant (COSTA, 2010).Besides azadirachtin, meliantriol, limonene, odoratone and other triterpenoids are biologically active among the more than 100 compounds isolated (DELEITO & BORJA, 2008).
The search for bioactive plant species with antimicrobial action has intensified in recent years due to the increased resistance of pathogenic microorganisms against synthetic products.The specialized literature has shown that the use of crude extracts or essential oils against pathogens and phytopathogens, including viruses, is promising, and they can have fungitoxic activity, through direct antimicrobial action as an eliciting action, activating defense mechanisms in plants.Some plant extracts and essential oils are as efficient against fungi and bacteria as conventional fungicides and antibiotics, with the advantage of not being toxic to humans and the environment (CUNICO et al., 2003;SHAAPAN et al., 2021).The literature contains studies that, in addition to evaluating the bioactive potential of neem, compare the results obtained with the efficiency of commercial antibiotics or antifungals, emphasizing its high efficiency for the control of S. typhimurium and S. aureus, and antifungal activity against strains of T. rubrum, C. albicans and C. tropicalis.S. typhimurium is a non-specific zoonotic bacterium responsible for causing self-limiting gastroenteritis.Furthermore, some strains of have been shown to be highly invasive, crossing the intestinal wall and reaching the systemic circulation, causing more severe infections (SANTOS et al., 2019).S. aureus is a Gram-positive, immobile and coagulase-positive bacterium with coccoid shape belonging to the phylum Firmicutes.Among the 52 species and 28 subspecies of the genus, S. aureus is the most clinically relevant, being found in the human commensal microbiota of the nasal mucosa in 20-40% of the general population (LEE et al., 2018).S. aureus is well adapted to its human host and the hospital environment, causing endocarditis, bacteremia, osteomyelitis and skin and soft tissue infections.S. aureus has emerged as of the main causes of infections associated with the hospital environment (TURNER et al., 2019).
Candida spp are commensal fungi of the skin, mouth and gastrointestinal tract.Despite being part of the human flora, they also have the ability to become pathogenic, causing a wide spectrum of conditions, ranging from superficial infections of the hair and nails to deadly systemic infections.The most common species are Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei.In recent decades, C. albicans has been responsible for 50% of all candidemia cases.Its growth and spread are controlled by the coexistence of microbial flora, intact epithelial barriers and innate immune system defenses (PFALLER et al., 1997;BERKOW & LOCKHART, 2017).
T. rubrum is an anthropophilic fungus commonly related to the dermatophytosis known as tinea pedis (athlete's foot).This species of dermatophyte is atypical in animals, but due to the close contact between dogs, cats and humans, there are reports of isolation of strains in these animals, even where the owners did not suffer from athlete's foot at the time of the study (KUSHIDA & WATANABE, 1975;BALDA et al., 2004;MORIELLO et al., 2017;REIS et al., 2020).Both in humans and animals, dermatophytosis causes superficial infections and lesions of keratinized tissues such as the stratum corneum, hair and nails (CECONI et al., 2018).This type of ringworm is important in canine and feline hosts, and due to its zoonotic nature, it is naturally transmissible between animals and humans, variously through direct contact, food, water and/or the environment (ANDRADE & ROSSI, 2019).
The literature mainly elucidates the metabolic profiles of the aerial organs of the species and the bioactivity of its constituents.To the best of our knowledge, this paper reports the first study of the metabolic profile bactericidal and fungicidal activities of the hydroethanolic extract of A. indica roots.The constituents of the hydroethanolic root extract were investigated using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-QTOOF-MS E ).This technique is currently considered the state of the art for compound separation and identification of substances.We investigated the antibacterial activities against S. typhimurium and S. aureus and the antifungal activities against strains of T. rubrum, C. albicans and C. tropicalis, along with the acute toxicity of the hydroethanolic extract (evaluated in an experimental animal model).

Identification and extraction of the plant material
The roots were collected from adult plants with approximate age of 5 years, at the campus of the State University of Ceará (Universidade Estadual do Ceará -UECE), Fortaleza, Brazil (03º 43 '02' 'S and 38º 32' 35'' W).The specimen was identified by Luiz Wilson Lima-Verde of the Prisco Bezerra Herbarium of Federal University of Ceará (Universidade Federal do Ceará -UFC), Fortaleza, Brazil, on March 13, 2018, where an exsicata was deposited under number 61196.
The hydroethanolic extract was obtained from 200 g of roots previously washed in running water and dried in an oven at 40 °C.The plant material was crushed and immersed in 1 L of EtOH-H 2 O (75:25, v/v) for seven days at room temperature, protected from light, with occasional stirring.The extract, passed through filter paper, was evaporated to dryness in a rotary evaporator, resulting in 1.2 g of crude extract.

Analysis by UPLC-QTOOF-MS E
The analyses were conducted with an Acquity UPLC chromatograph (Waters, USA) coupled to a Xevo quadrupole and time-of-flight mass spectrometer (QTOF, Waters).The chromatographic runs were performed with a Waters Acquity BEH UPLC column (150 mm x 2.1 mm I.D., 1.7 µm) at 40 °C.The mobile phases were water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B), eluted with 2%-95% B (0-15 min); 100% B (15.1 to 17 min) and equilibrating with 2% B (17.1 to 19.1 min) at a flow rate of 0.4 mL min -1 and an injection volume of 5.0 µL.Ionization was performed with electrospray ionization source (negative mode -ESI), acquired in the range of 110-1180 Da, source temperature set at 120 °C, desolvation temperature of 350 °C, desolvation gas flow rate of 500 L h -1 , extraction cone of 0.5 V, and capillary voltage of 3.2 kV.Leucine enkephalin was used as lock mass.The mode of acquisition was MS E .The instrument was controlled by the Masslynx 4.1 software (Waters Corporation).

Acute toxicity evaluation with Danio rerio
For each drug and concentration evaluated, groups of six wild adult specimens of D. rerio (zebrafish), aged between 60 and 90 days, were used, to which 20 μL of the drug/sample was given orally at concentrations 0.2, 0.5, 1.0, 1.5 and 2.0 mg mL -1 , solubilized in distilled water/Tween 80 (2% v/v).As a negative control, distilled water/Tween 80 (2% v/v) was administered, and one group received only water.After 24, 48 and 72 h, the numbers of dead animals were counted.Statistical analysis was performed by the trimmed Spearman-Karber method with 95% confidence intervals, to estimate the median lethal dose (the dose that promotes the death of 50% -LD 50 ) of the zebrafish.

Antifungal activity
The antifungal activity was evaluated by the broth microdilution technique in 96-well plates, carried out in duplicate using clinical isolates of C. albicans, C. tropicalis (2.5 -5 x 10 4 CFU mL -1 and n=4) and Trichophyton rubrum (5 x 10 4 CFU mL -1 and n=3).Concentration ranges from 2 to 2500 μg mL -1 were evaluated.In the control test, the standard drugs amphotericin B and ketoconazole were used at concentrations of 0.125 to 64 μg mL -1 .
Initially, 100 μL of RPMI medium was added to all wells and 10 mg mL -1 of extracts diluted in DMSO was then added to all wells of the first column to form serial dilutions.Finally, 100 μL of the inoculum was added to the wells.The plates were covered with parafilm and incubated at 37 °C.The readings were carried out by measuring the diameters of the fungal growth inhibition zones in millimeters, after incubation of two days for the Candida strains and four days for the T. rubrum strains.The minimum inhibitory concentration (MIC), defined as the lowest concentration of extract capable of inhibiting 100% of the visible growth of the fungus, was determined by visualization as recommended by the guidelines M27-A3 from the Clinical and Laboratory Standards Institute (CLSI) and FONTENELLE et al., (2007).

Romão et al.
The minimum fungicidal concentration (MFC) for the species was determined by sub-culturing 100 μL of the turbidity-free well solution in potato agar at 28 °C (FONTENELLE et al., 2007).

Antibacterial activity
The antibacterial activity was investigated with the strains of S. typhimurium ATCC 14028 and S. aureus ATCC 27664.The tests were performed by the disc diffusion method as described in CLSI (CLSI, 2008).The extract was evaluated at concentrations of 1.4, 5.0 and 10 mg mL -1 .Laborclin chloramphenicol discs (30 μg mL -1 ) were used as positive control.Three discs for each dose of extract were prepared for each bacterium.In addition, an empty disc used to prepare the extract discs was used for control.The MIC and minimal bactericidal concentration (MBC) of each extract was determined by inhibition zones caused by the extract, measured in millimeters.The bacteria were inoculated in Petri dishes containing Mueller-Hinton agar (MH) and incubated inverted in a bacteriological oven for 24 h at 35 °C.According to this technique, a growth inhibition halo of any degree around the paper discs is considered a positive result.The experiment was performed in triplicate.

Identification by UPLC-QTOOF-MS E
Table 1 presents the compounds tentatively identified in positive mode by their retention time, fragmentation pattern, molecular formula and error (in ppm) in studies reported previously for the Azadirachta genus.Figure 1 presents the chromatogram of hydroethanolic extract of the A. indica roots by UPLC-QToF-MS E in according of elution order.Figure 2 showed the compounds tentatively identified.
The analysis of the spectra, through their specific fragments in MS E , along with mechanistic analysis of literature data and the molecular formula, allowed us to provisionally identify nine molecules present in the hydroethanolic extract of neem roots: azadirachtin H, margosinolide, 6-deacetylnimbin, deacetylsalanine, nimbanal, nimbinene, salanol acetate, salanin and nimbin (Table 1 and 2).

Acute toxicity to D. rerio
After 48 h of toxicity tests of the neem hydroethanolic extract, mortality of test organisms was found to be less than 50%, even at the concentration of 2 mg mL -1 .These data indicated that the extract was not toxic to the evaluated organisms.
In the literature, we did not found studies evaluating the toxicity of the hydroethanolic extract of neem roots in animal models.
However, ASHAFA et al., 2012, observed that the oral administration in rats of the ethanol extract from steamed bark of A. indica produced alteration in the biochemical parameters of the animals' organs.Acute and subacute toxicity studies performed by DORABABU et al., (2006), using aqueous extract from neem leaves indicate no mortality with a dose of 2.5 g kg -1 in mice and no significant changes in body weight or tissue appearance, or in the metabolism of this species.KANAGASANTHOSH et al., (2015), working with the ethanol extract from neem leaves, also identified no mortality in mice using doses up to 2 g kg -1 .

Antifungal activity
The tests revealed that the hydroalcoholic extract of neem has considerable antifungal activity against T. rubrum for both strains tested.However, it was inactive against Candida ssp for all strains evaluated (Table 2).In the literature, there are no reports of investigation of root activity in relation to the organisms evaluated in this study.However, in the discussion below, some studies carried out with neem that support this study are described.MAHMOUD et al. (2011), evaluated the neem leaf hydroalcoholic extract against the fungi Aspergillus flavus, A. fumigatus, A. niger, A. terreus, C. albicans and Microsporum gypseum and found that the concentration of 5% (mass/volume) caused 44% inhibition of A. flavus and 20% of C. albicans, while the concentration of 15% caused absence mycelial growth.NATARAJAN et al. (2003), reported that the ethanol extracts from neem leaves had equal MIC and MFC values of 250 µg mL -1 against T. rubrum.GOVINDACHARI et al.
(1998), investigating the antifungal action of neem seed oil and its isolated compounds against Drechslera oryzae, Fusarium oxysporum and Alternaria tenuis, identified a reduction of the antifungal activity caused by the majority of isolated compounds, higher for salannim and azadiradione, while nimbin had low activity against most of the strains evaluated.The epoxyazadiradione in the pure form did not present any activity.The authors stated it is possible the main triterpenoids isolated has little or no antifungal activity, while in combination they presented excellent activity against all three fungi, suggesting potentiating/additive/synergetic effects.
For T. rubrum, the activity reported in the present study is similar to literature reports.For Candida ssp, the results found are not similar.The inactivity of the hydroalcoholic root extract can be related to the age of the plant, the time of collection, the soil at the collection site (variables that influence the production of secondary metabolites of the plant), as well as differences in the concentrations of bioactive compounds or the extractant solvent used, or even the absence of active substances found in other organs of the plant.

Antibacterial activity
The extract was not effective at any of the concentrations evaluated in inhibiting the growth   Alves and collaborators, (2009), working with the hydroalcoholic extract of neem leaves against strains S. aureus, S. thiphymurium, Bacillus subtilis, Aspergillus niger and Escherichia coli, verified that the extract was effective only to inhibit the growth of S. aureus (MIC of 10 4 µg mL -1 ).MEHROTRA et al., 2010, observed that the ethanol extract of neem leaves had strong activity against Vibrio cholerae, Pseudomonas aeruginosa and S. aureus (MIC of 0.25 µg µL l-).MAHFUZUL et al., 2007, observed that the neem leaves extract presented higher antimicrobial activity against Gram-positive than Gram-negative bacteria.Among the microorganisms evaluated, the authors observed antibacterial activity against Listeria monocytogenes, Vibrio parahaemolyticus, Bacillus cereus and S. aureus (MIC of 4500 µg mL -1 ), but no activity against Escherichia coli and Salmonella enteritidis.
The data in the literature only pertain to the activities of ethanol extracts of leaves, indicating moderate to strong activities against the evaluated organisms (S. typhimurium and S. aureus).However, it is necessary to highlight that the strains used in this study are different from those evaluated in previous studies and the substances were obtained from different organs of the plant, so the metabolic profile of the extract and the concentrations of the constituents are different.Although, the extract of neem root has been found to contain limonoid tetranortriterpenoids, include azadirachtin (a recognized bactericidal compound), it is possible that its low concentration in the extracts influenced the bioactivity against the evaluated bacteria.
Despite the high bioactive potential of neem, some municipal governments in Brazil have banned its cultivation (BOM JESUS DA LAPA (BA), 2021).This ban has occurred because some studies have shown that it can affect the reproductive activity of humans and animals, by promoting a decline of seminal parameters (volume and concentration of the ejaculate, motility and morphological changes of sperm), as well as through spermicidal action, vaginal contraceptive effect and interference in the estrous cycle of females in early embryonic development, promoting stillbirth (SILVA, 2010;URIBE-CLAVIJO et al., 2012;BRASIL, 2013).
Even so, due to its high bioactive potential, the plant would be an excellent source of income for rural populations in the semiarid region of Northeast Brazil, where it could be used a source to extract various natural products, since the species can be easily cultivated in this region.According to literature data, the plant adapts well to tropical and subtropical climates, with ideal cultivation temperatures of 21 to 32 °C, and the ability to withstand temperatures above 44 °C for short periods.It resists long periods of drought and annual rainfall between 400 and 800 mm.Finally, it can be cultivated in dry soils and soils poor in nutrients without harming its flowering (NEVES et al., 2003).Strains ------Hydroethanolic extract of A. indica-------------------Drug (µg mL -1 )-----------MIC (µg mL -1 ) MCF (µg mL

Figure 1 -
Figure 1 -Chromatogram of hydroethanolic extract of the Azadirachta indica roots by UPLC-QToF-MSE in according of elution order.

Figure 2 -
Figure 2 -Compounds tentatively identified in hydroethanolic extract of the Azadirachta indica roots by by UPLC-QTOF-MS E .

Table 2 -
Antifungal activity of the hydroethanolic roots extract of A. indica against fungal test organism.