Thiocarbamates from Moringa oleifera Seeds Bioactive against Virulent and Multidrug-Resistant Vibrio Species

Prospect of antibacterial agents may provide an alternative therapy for diseases caused by multidrug-resistant bacteria. This study aimed to evaluate the in vitro bioactivity of Moringa oleifera seed extracts against 100 vibrios isolated from the marine shrimp Litopenaeus vannamei. Ethanol extracts at low (MOS-E) and hot (MOS-ES) temperature are shown to be bioactive against 92% and 90% of the strains, respectively. The most efficient Minimum Inhibitory Concentration (MIC) levels of MOS-E and MOS-ES against a high percentage of strains were 32 µg mL−1. Bioguided screening of bioactive compounds showed that the ethyl acetate fraction from both extracts was the only one that showed antibacterial activity. Vibriocidal substances, niazirine and niazimicine, were isolated from the aforementioned fraction through chromatographic fractionation.


Introduction
Bacteria of Vibrio genus are ubiquitous in the marine environment and are part of the indigenous microbiota of marine invertebrates. Some species are recognized as human pathogens, often associated with diseases such as cholera and acute gastroenteritis [1,2]. Vibrios are also seen as opportunistic pathogens of cultured aquatic organisms, which is one of the reasons for observing the use of antibiotics in shrimp cultivation.
Furthermore, inappropriate use of antimicrobial drugs in aquaculture has been associated with negative environmental impacts: selection of bacterial populations resistant to drugs [3,4] and contamination of adjacent ecosystems to culture ponds.
Thus, detection of antibacterial activity in higher plants against vibrios with virulent, antimicrobial-resistant profiles is of utmost importance. In the present research, due to its high medicinal activity [5], the vibriocidal capacity of the angiosperm Moringa oleifera was investigated. The antimicrobial effect of Moringa has been researched since the 1980s and seems to be related to some specific components including pterygospermin and Moringa glycosides, as well as 4-( -L-rhamnosyloxy)-benzyl isothiocyanate and 4-( -L-rhamnosyloxy)-phenyl-acetonitrile, which act especially against Bacillus subtilis, Mycobacterium phlei, Serratia marcescens, Escherichia coli, Pseudomonas aeruginosa, Shigella sp., and Streptococcus sp. [6].
Despite the extensive scientific evidence of the bioactivity of Moringa against bacteria [7][8][9][10], studies on its effects against vibrios are still incipient. Thus, this research aimed to evaluate the bioactive potential from extracts of Moringa seeds against vibrios with virulent and multidrug antimicrobial profile.

Botanical
Material. Moringa oleifera seeds were collected from two specimens grown in a campus of the Federal University of Ceará (Pici, Fortaleza, Ceará). Separation from the fruit (pod), removal of husks, and posterior packing in plastic polyethylene bags followed the material collection.

Moringa oleifera Extracts.
All extraction procedures were performed in the Department of Organic and Inorganic Chemistry at Federal University of Ceará (UFC). Part of the crushed seeds of M. oleifera (110 g) was subjected to three extractions with 300 mL cold hexane (PA) at 24 h intervals. After filtration and evaporation of the solvent under reduced pressure in a rotary evaporator, 15.36 g of an extract of fluid and yellowish appearance called MOS-H was obtained. The resulting cake was subjected to three cold extractions with 300 mL ethanol (PA) in 24 h intervals. After filtration and evaporation of the solvent under reduced pressure in a rotary evaporator, 11.64 g of an extract of fluid and dark appearance called MOS-E was obtained. 139 g of crushed M. oleifera seeds was used for hot extraction in a Soxhlet apparatus with 800 ml of hexane (PA) for 48 h. After filtration and evaporation of the solvent under reduced pressure in a rotary evaporator, 28.29 g of an extract of fluid and yellowish aspect called MOS-HS was obtained. Another extraction was carried out with 800 ml of ethanol (PA) for 48 h. After filtration and evaporation of the solvent under reduced pressure in a rotary evaporator, 13.66 g of a pasty and dark-colored appearance called MOS-ES was obtained.

In Vitro Susceptibility Testing of Moringa oleifera Extracts.
Susceptibility of Vibrio sp. strains to the four extracts types (MOS-H, MOS-E, MOS-HS, and MOS-ES) was assessed using the disk diffusion method (DDM) and by Minimum Inhibitory Concentration (MIC) [13]. In order to proceed with the DDM, paper discs (6 mm) containing 100 L of each extract were applied in triplicate on Mueller-Hinton plates previously seeded with bacterial cultures (10 8 UFC mL −1 ). As negative and positive Gram control, strains of V. parahaemolyticus IOC and Staphylococcus aureus ATCC 25923, respectively, were used. For MIC determination, macrodilution technique in Mueller-Hinton broth containing 1% NaCl was used. Concentrations of 4, 8, 16, 32, and 64 g mL −1 were tested, using the MOS-E oils (cold extraction with ethanol) and MOS-ES (hot extraction with ethanol) in comparison to isolates susceptible to crude extracts in the DDM test. 07%. All fractions were subjected to antimicrobial activity test by disk diffusion method. The bioactive fraction was subjected to chromatographic fractionation by High-Performance Liquid Chromatography (HPLC) in order to isolate its active principles.

Chromatographic Fractionation of the Ethyl Acetate Fraction (MOS-ESA) by High-Performance Liquid Chromatography (HPLC) and Isolation of Active Substances.
Part of the MOE-ESA active fraction (285 mg) was analyzed by HPLC in a chromatograph Shimadzu5 (UFLC model) equipped with a UV-Vis detector with diode array (model SPD-M20A). Separation was performed in reverse phase conditions in semipreparative column (C-18.5 m), with isocratic elution using MeOH/H 2 O (1 : 1) with a 4.72 mL min −1 flow. Chromatographic fractionation of the ethyl acetate's fraction from the MOS-ES fixed oil resulted in the detection (at 284 nm) and isolation of three main substances (Figure 1), which were obtained as whitish, amorphous solids: the compound related to the peak 1 (23.3 m; = 4.99 min) was called MOS-ES-1, related to peak 2 (4.0 mg; = 7.06 min) was called MOS-ES-2, and related to peak 3 (65.1 mg, = 17.45 min) was called MOS-ES-3. Isolated substances (S1 and S3) had their structures determined by the analysis of Nuclear Magnetic Resonance (NMR) and Infrared (IR) spectral data and also by comparison to other findings described in the literature [14,15].

Disk Diffusion Test.
From all the 100 strains tested, only five were resistant to MOS-E fixed oil; on the other hand, 36 had its growth inhibited, with average inhibition zones ranging from 13 to 15 mm (Table 1). When comparing the oils, MOS-ES tests demonstrated inhibitory bacterial rate somewhat lower, since most strains ( = 37) had inhibitions indicated by halos in the range 10 to 12 mm (Table 2). In addition, seven strains were resistant to the MOS-ES. The larger inhibition halo (22 to 24 mm) was observed on MOS-E test against a V. navarrensis strain (Table 1). Both extracts showed antibacterial effect against the Grampositive (Staphylococcus aureus) and Gram-negative (Vibrio parahaemolyticus) controls (Tables 1 and 2). MOS-H and MOS-HS extracts showed no bioactivity against any of the isolates ( = 100).

Minimum Inhibitory Concentration (MIC)
. MIC levels of the MOS-E show that 83 (90.2%) of the strains were inhibited in the presence of a 32 g mL −1 concentration. Levels of 8, 16, and 64 g mL −1 were able to inhibit 1, 6, and 2 strains, respectively. Also, MIC levels of the MOS-ES able to inhibit the highest percentage of strains ( = 88; 97.8%) were that of 32 g mL −1 . On the other hand, only 2 (2.2%) of the strains were inhibited by a MOS-ES MIC of 16 g mL.

Bioactivity of the Fractions.
Study of the bioactivity of dichloromethane (CH 2 Cl 2 ), ethyl acetate (EtOAc), and methanol (MeOH) fractions of MOS-E and MOS-ES revealed that only EA fractions from both extracts showed antimicrobial activity (Table 3). Ethyl acetate fractions were selected and subjected to chromatographic fractionation by HPLC in order to isolate bioactive substances.

Identification of Individual Substances.
From the MOS-EA fraction, three substances were identified S1 (MOS-ES-1), S2 (MOS-ES-2), and S3 (MOS-ES-3). Due to the small amount obtained from MOS-ES-2 substance (S2), it was impossible to obtain spectroscopic data enough for a structural characterization. Those two substances derived from MOE-EA fraction were also obtained in small amounts and identified based only by Thin Layer Chromatography (TLC). Furthermore, the drain of this fraction yielded 23 mg of unidentified less polar substances. Analysis of spectral data and the comparison with information in the literature [15] were used to characterize S1 as the 4-[( -L-rhamnosyloxy)benzyl] nitrile or niazirine.

Structural Characterization of MOS-ES-1 (S1) and MOS-ES-3 (S3). S1 was isolated as a white amorphous solid
S3 was also isolated as a white amorphous solid and pre- Analysis of spectral data and the comparison with information in the literature [14] were used to characterize S3 as O-ethyl-4-[( -L-rhamnosyloxy)benzyl] thiocarbamate or niazimicine.

Evaluation of Antibacterial Activity of the Isolated Substances.
Bioactivity results of S1 and S3 substances against ten   Vibrio sp. strains are summarized in Table 4. It is possible to attest, in a comparison between substances 1 and 3 (S1 and S3) using the size of the inhibition zone as a criterion, a greater antibacterial efficiency of S3 against all strains.

Discussion
Studies on the bioactive properties of Moringa seeds highlight multiple uses of this phanerogam, for example, turbidity removal of contaminated water by coagulation [16], biosorption of heavy metals in effluents [17], anti-inflammatory [18], and antibacterial activity against S. aureus and E. coli [19]. However, bioactivity against vibrios has not been widely researched. Thus, the high inhibition level of both extracts (95% for MOS-E; 93% for MOS-ES) (Tables 1 and 2)  Vibriocidal activity of aerial parts of M. oleifera was reported by Peixoto et al. [20]. The authors tested the bioactivity of its extracts against standard V. parahaemolyticus strain and found average inhibition halos of 21.9 and 20.7 mm for ethanol and aqueous extracts, respectively.
Moringa seed extracts have also been used in tests against standard strain of V. cholerae. Atieno et al. [21] observed the bioactivity of hexane and methanolic extracts of M. oleifera and M. stenopetala seeds against Salmonella ser. Typhi, E. coli, and V. cholerae. For the species of Vibrio, the authors reported inhibition halo sizes of 22.2 and 13.8 mm for hexane and methanolic extracts of M. oleifera, respectively. The aforementioned data support the assertion that Moringa seeds have vibriocidal potential; however, it cannot be compared to the ones presented in the present study, since antibacterial activity in leaf extracts was not detected (MOS-H and MOS-HS).
Despite the occurrence of compounds with antibacterial activity in different parts of Moringa is being reported in the scientific literature since the early 1980s [22], their use for epizootic purposes has been little explored. The data obtained by the disk diffusion test and the results of the Minimum Inhibitory Concentration (MIC) serve as evidence of the high antibacterial potential of ethanol extracts of MOS-E and MOS-ES against vibrios.
Satisfactory results in disk diffusion tests and the definition of MIC levels were pivotal for the decision of carrying out complementary studies, starting with the bioguided screening of bioactive compounds. The selective antimicrobial effect of Moringa's crude extract fractions was also verified by Nantachit [23]. The author noted that the dichloromethane fraction was active against E. coli.
Since the 1990s, biological activities of pharmacological interest concerning nitriles isolated from Moringa have been described, and its antitumor [28] and antihypertensive [14] functions are noteworthy, along its use in the prevention of carcinogenesis [24].
The presence of niazimicine in Moringa seeds is often cited as a strong antitumor factor. It can be used as a prophylactic or therapeutic measure while treating HSV-1 infections [29].
Although this is not the first description of carbamate glycosides in constituent parts of the M. oleifera species, its unexploited vibriocidal potential must be highlighted.
In the present study, niazirine and niazimicine showed high antibacterial efficiency against vibrios with phenotypic profiles compatible to the presence of virulence factors (exoenzymes and -hemolysis producers), cross-resistance tolactams, and mono/multiresistance to antibiotics. These findings suggest a new class vibriocidal compounds. Moreover, the results are consistent with the demand for new alternatives to antibacterial drugs in order to mitigate the impact caused by the indiscriminate use of antimicrobials in aquaculture.

Conflicts of Interest
The authors declare that they have no conflicts of interest.