# Thiocarbamates fromMoringa oleifera Seeds Bioactive against Virulent and Multidrug-ResistantVibrio Species

Authors: Renata Albuquerque Costa; Oscarina Viana de Sousa; Ernesto Hofer; Jair Mafezoli; Francisco Geraldo Barbosa; Regine Helena Silva dos Fernandes Vieira
Journal: BioMed Research International
 (2017)

---

## Abstract

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 ofMoringa oleifera seed extracts against 100 vibrios isolated from the marine shrimpLitopenaeus 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.


---

## Body

## 1. Introduction

Bacteria ofVibrio 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 angiospermMoringa oleifera was investigated. The antimicrobial effect ofMoringa has been researched since the 1980s and seems to be related to some specific components including pterygospermin andMoringa glycosides, as well as 4-(α-L-rhamnosyloxy)-benzyl isothiocyanate and 4-(α-L-rhamnosyloxy)-phenyl-acetonitrile, which act especially againstBacillus subtilis,Mycobacterium phlei,Serratia marcescens,Escherichia coli,Pseudomonas aeruginosa,Shigella sp., andStreptococcus sp. [6].Despite the extensive scientific evidence of the bioactivity ofMoringa against bacteria [7–10], studies on its effects against vibrios are still incipient. Thus, this research aimed to evaluate the bioactive potential from extracts ofMoringa seeds against vibrios with virulent and multidrug antimicrobial profile.


## 2. Material and Methods

### 2.1. Origin of the Strains

One hundredVibrio sp. strains isolated from the hemolymph ofLitopenaeus vannamei shrimp were used, all of which belonged to the bacteriological collection of the Laboratory of Environmental and Fishery Microbiology at Sea Sciences Institute (LABOMAR-UFC-Brazil). The 100 strains were phenotypically identified asV. navarrensis (n=53),V. brasiliensis (n=15),V. parahaemolyticus (n=10),V. xuii (n=8),V. coralliilyticus (n=5),V. cholerae (n=4),V. neptunis (n=2),V. alginolyticus (n=1),V. diazotrophicus (n=1), andV. vulnificus B3 (n=1) [11]. The enzymatic profile and antimicrobial resistance were used as a criterion for selection [12].


### 2.2. Botanical Material

Moringa oleifera seeds were collected from two specimens grown in acampus 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.


### 2.3.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 ofM. 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 crushedM. 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.


### 2.4. In Vitro Susceptibility Testing ofMoringa oleifera Extracts

Susceptibility ofVibrio 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 µL of each extract were applied in triplicate on Mueller-Hinton plates previously seeded with bacterial cultures (108 UFC mL−1). As negative and positive Gram control, strains ofV. parahaemolyticus IOC andStaphylococcus 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 µg 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.


### 2.5. Chromatographic Fractionation of MOS-E and ES-MOS and Obtaining Active Fraction Ethyl Acetate (MOS-ESA)

Part of the MOS-E extract (3.1 g) was adsorbed onto 7.3 g of silica gel and chromatographed on 52.5 g of silica gel in open column (Ø 5,0 cm). Elution was done in order of increasing polarity with dichloromethane (1200 ml) (MOS-ED), ethyl acetate (800 mL) (MOS-EA), and methanol (600 mL) (MOS-EM). Solvents were evaporated under reduced pressure in a rotary evaporator, yielding the following mass and yields: MOS-ED 1,920.8 mg, 61.96%; MOS-EA 231.6 mg, 7.47%; MOS 660.8 mg, 21.31%. MOS-ES extract (2.6 g) was adsorbed onto 3.9 g of silica gel and chromatographed on 48.3 g of silica gel in open column (Ø 5,0 cm). Elution was done in order of increasing polarity with dichloromethane (500 ml) (MOS ESD), ethyl acetate (700 ml) (MOS-ESA), and methanol (600 ml) (MOS-ESM). Solvents were evaporated under reduced pressure in a rotary evaporator, yielding the following mass and yields: MOS-ED 34.8 mg, 1.33%; MOS-EA 335.3 mg, 12.89%; MOS 1,978 mg, 76.07%. 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.


### 2.6. 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 Shimadzu® (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-18.5μm), with isocratic elution using MeOH/H2O (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; tr = 4.99 min) was called MOS-ES-1, related to peak 2 (4.0 mg; tr = 7.06 min) was called MOS-ES-2, and related to peak 3 (65.1 mg, tr = 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].Figure 1
HPLC chromatogram analysis of an active fraction of MOS-ESA and isolation of MOS-ES-1, MOS-ES-2, and MOS-ES-3.


## 2.1. Origin of the Strains

One hundredVibrio sp. strains isolated from the hemolymph ofLitopenaeus vannamei shrimp were used, all of which belonged to the bacteriological collection of the Laboratory of Environmental and Fishery Microbiology at Sea Sciences Institute (LABOMAR-UFC-Brazil). The 100 strains were phenotypically identified asV. navarrensis (n=53),V. brasiliensis (n=15),V. parahaemolyticus (n=10),V. xuii (n=8),V. coralliilyticus (n=5),V. cholerae (n=4),V. neptunis (n=2),V. alginolyticus (n=1),V. diazotrophicus (n=1), andV. vulnificus B3 (n=1) [11]. The enzymatic profile and antimicrobial resistance were used as a criterion for selection [12].


## 2.2. Botanical Material

Moringa oleifera seeds were collected from two specimens grown in acampus 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.


## 2.3.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 ofM. 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 crushedM. 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.


## 2.4. In Vitro Susceptibility Testing ofMoringa oleifera Extracts

Susceptibility ofVibrio 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 µL of each extract were applied in triplicate on Mueller-Hinton plates previously seeded with bacterial cultures (108 UFC mL−1). As negative and positive Gram control, strains ofV. parahaemolyticus IOC andStaphylococcus 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 µg 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.


## 2.5. Chromatographic Fractionation of MOS-E and ES-MOS and Obtaining Active Fraction Ethyl Acetate (MOS-ESA)

Part of the MOS-E extract (3.1 g) was adsorbed onto 7.3 g of silica gel and chromatographed on 52.5 g of silica gel in open column (Ø 5,0 cm). Elution was done in order of increasing polarity with dichloromethane (1200 ml) (MOS-ED), ethyl acetate (800 mL) (MOS-EA), and methanol (600 mL) (MOS-EM). Solvents were evaporated under reduced pressure in a rotary evaporator, yielding the following mass and yields: MOS-ED 1,920.8 mg, 61.96%; MOS-EA 231.6 mg, 7.47%; MOS 660.8 mg, 21.31%. MOS-ES extract (2.6 g) was adsorbed onto 3.9 g of silica gel and chromatographed on 48.3 g of silica gel in open column (Ø 5,0 cm). Elution was done in order of increasing polarity with dichloromethane (500 ml) (MOS ESD), ethyl acetate (700 ml) (MOS-ESA), and methanol (600 ml) (MOS-ESM). Solvents were evaporated under reduced pressure in a rotary evaporator, yielding the following mass and yields: MOS-ED 34.8 mg, 1.33%; MOS-EA 335.3 mg, 12.89%; MOS 1,978 mg, 76.07%. 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.


## 2.6. 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 Shimadzu® (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-18.5μm), with isocratic elution using MeOH/H2O (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; tr = 4.99 min) was called MOS-ES-1, related to peak 2 (4.0 mg; tr = 7.06 min) was called MOS-ES-2, and related to peak 3 (65.1 mg, tr = 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].Figure 1
HPLC chromatogram analysis of an active fraction of MOS-ESA and isolation of MOS-ES-1, MOS-ES-2, and MOS-ES-3.


## 3. Results

### 3.1. 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 (Table1). When comparing the oils, MOS-ES tests demonstrated inhibitory bacterial rate somewhat lower, since most strains (n = 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 aV. navarrensis strain (Table 1). Both extracts showed antibacterial effect against the Gram-positive(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 (n = 100).Table 1
Distribution, according to the average inhibition zone (mm), of the number of strains inhibited by cold ethanol extract ofMoringa oleifera seeds (MOS-E).
Vibrio species
n
Average inhibition zone (mm)
22–24
19–21
16–18
13–15
10–12
7–9
0
V. navarrensis
53
1
2
5
27
15
3
—
V. brasiliensis
15
—
1
2
4
2
5
1
V. parahaemolyticus
10
—
—
—
—
—
9
1
V. xuii
8
—
—
—
1
—
5
2
V. coralliilyticus
5
—
—
2
1
1
1
—
V. cholerae
4
—
—
—
2
1
1
—
V. neptunis
2
—
—
1
—
1
—
—
V. alginolyticus
1
—
—
—
1
—
—
—
V. diazotrophicus
1
—
1
—
—
—
—
—
V. vulnificusB3
1
—
—
—
—
—
—
1
V. parahaemolyticus
I
O
C
a
1
—
—
1
—
—
—
—
S. aureus ATCC25923b
1
—
1
—
—
—
—
—
Total
102
1
5
11
36
20
24
5
n: number of isolates. a: standard strain used as Gram-negative control. b: standard strain used as Gram-positive control.Table 2
Distribution, according to the average inhibition zone (mm), of the number of strains inhibited by hot ethanol extract ofMoringa oleifera (MOS-ES).
Vibrio species
n
Average inhibition zone (mm)
19–21
16–18
13–15
10–12
7–9
0
V. navarrensis
53
1
6
14
26
5
1
V. brasiliensis
15
1
2
2
4
3
3
V. parahaemolyticus
10
—
—
—
—
9
1
V. xuii
8
—
—
—
1
7
—
V. coralliilyticus
5
—
—
1
3
1
—
V. cholerae
4
—
—
1
1
1
1
V. neptunis
2
—
—
—
2
—
—
V. alginolyticus
1
—
—
1
—
—
—
V. diazotrophicus
1
—
1
—
—
—
—
V. vulnificusB3
1
—
—
—
—
—
1
V. parahaemolyticus
I
O
C
a
1
—
1
—
—
—
—
S. aureusATCC25923b
1
—
1
—
—
—
—
Total
102
2
11
19
37
26
7
n: number of isolates. a: standard strain used as Gram-negative control. b: standard strain used as Gram-positive control.


### 3.2. 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 32µg mL−1 concentration. Levels of 8, 16, and 64 µg 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 (n = 88; 97.8%) were that of 32 µg mL−1. On the other hand, only 2 (2.2%) of the strains were inhibited by a MOS-ES MIC of 16 µg mL.


### 3.3. Bioactivity of the Fractions

Study of the bioactivity of dichloromethane (CH2Cl2), 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.Table 3
Average inhibition halos of ethyl acetate (EtOAc) fractions of ethanolic extracts ofMoringa oleifera seeds extracted cold (MOS-E) and hot (MOS-ES) with ethanol against ten Vibrio sp. strains isolated from the hemolymph of Litopenaeus vannamei.
Strain
Vibriospecies
EtOAc (MOS-E)
EtOAc (MOS-ES)
1
V. coralliilyticus
14.51
±
0.07
12.10
±
0.08
7
V. alginolyticus
12.65
±
0.06
10.52
±
0.45
13
V. navarrensis
15.71
±
0.21
14.03
±
0.07
35
V. diazotrophicus
17.14
±
0.04
16.58
±
0.42
40
V. navarrensis
15.75
±
0.35
11.82
±
0.31
42
V. xuii
12.39
±
0.24
11.80
±
0.25
46
V. parahaemolyticus
8.53
±
0.15
7.66
±
0.12
89
V. neptunis
18.54
±
0.42
11.18
±
0.09
97
V. cholerae
8.46
±
0.09
7.32
±
0.10
98
V. brasiliensis
15.01
±
0.05
14.61
±
0.15


### 3.4. 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.


### 3.5. Structural Characterization of MOS-ES-1 (S1) and MOS-ES-3 (S3)

S1 was isolated as a white amorphous solid and presented 129–132°C, [M + Na]+ = 302,1012. RMN 1H (500 MHz, CD3OD): δ 7,28 (2H, d,J = 8,6 Hz); 7,07 (2H, d,J = 8,6 Hz); 5,43 (1H, d,J = 1,4 Hz); 3,84 (1H, dd,J = 3,4 e 9,5 Hz); 3,82 (2H, s); 3,82 (2H, m); 3,62 (1H, m); 3,46 (1H, t,J = 9,5 Hz); 1,21 (3H, d,J = 6,2 Hz). RMN 13C (125 MHz, CD3OD): δ 157,4 (C-1); 118,1 (C-2 e C-6); 130,2 (C-3 e C-5); 125,8 (C-4); 22,7 (C-7); 119,8 (C-8); 99,9 (C-1′); 72,0 (C-2′); 72,0 (C-3′); 73,8 (C-4′); 70,7 (C-5′); 18,0 (C-6′). IV νmax/cm−1 (KBr) 3411 (νO-H), 2948 (ν-C-H), 2250 (νC≡N), 1611–1513 (νC=C), 1227 (νC-O).Analysis of spectral data and the comparison with information in the literature [15] were used to characterize S1 as the 4-[(α-L-rhamnosyloxy)benzyl] nitrile or niazirine.S3 was also isolated as a white amorphous solid and presented 130–133°C, [M + Cl]− = 392,0951, RMN 1H (500 MHz, CD3OD): δ 7,25 (2H, d,J = 8,7 Hz); 7,01 (2H, d,J = 8,7 Hz); 5,40 (1H, d,J = 1,8 Hz); 4,63 (2H, s); 4,46 (2H, q,J = 7,1 Hz); 3,99 (1H, dd,J = 1,8 e 3,4 Hz); 3,83 (1H, dd,J = 3,4 e 9,5 Hz); 3,62 (1H, m); 3,45 (1H, t,J = 9,5 Hz); 1,29 (3H, t,J = 7,1 Hz); 1,21 (3H, d,J = 6,2 Hz), RMN 13C (125 MHz, CD3OD): δ 157,1 (C-1); 117,6 (C-2 e C-6); 130,1 (C-3 e C-5); 133,1 (C-4); 49,0 (C-7); 192,3 (C-8); 66,9 (C-9); 14,7 (C-10); 100,2 (C-1′); 72,1 (C-2′); 72,3 (C-3′); 73,9 (C-4′); 70,6 (C-5′); 18,0 (C-6′). IV νmax/cm−1 (KBr) 3405 (νO-H), 2948 (ν-C-H), 1611–1513 (νC=C), 1233 (νC=S).Analysis of spectral data and the comparison with information in the literature [14] were used to characterize S3 asO-ethyl-4-[(α-L-rhamnosyloxy)benzyl] thiocarbamate or niazimicine.


### 3.6. Evaluation of Antibacterial Activity of the Isolated Substances

Bioactivity results of S1 and S3 substances against tenVibrio 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.Table 4
Average size of inhibition halos from substances 1 (S1) and 3 (S3) isolated from fractions of ethyl acetate (EtOAc) ofMoringa seed in cold extraction with ethanol (MOS-E) and in hot extraction with ethanol (MOS-ES), against ten Vibrio sp. strains isolated from the hemolymph of Litopenaeus vannamei.
Strains
Species
Fraction of EtOAc MOS-ES
S1
S2
S3
1
V. coralliilyticus
8.11
±
0.09
nt
14.04
±
0.02
7
V. alginolyticus
8.32
±
0.02
nt
15.98
±
0.09
13
V. navarrensis
9.76
±
0.43
nt
19.89
±
0.11
35
V. diazotrophicus
7.42
±
0.24
nt
21.21
±
0.24
40
V. navarrensis
9.77
±
0.15
nt
17.89
±
0.12
42
V. xuii
8.76
±
0.29
nt
20.33
±
0.06
46
V. parahaemolyticus
7.66
±
0.45
nt
9.90
±
0.07
89
V. neptunis
7.14
±
0.11
nt
20.11
±
0.03
97
V. cholerae
7.30
±
0.27
nt
10.15
±
0.18
98
V. brasiliensis
7.99
±
0.02
nt
18.75
±
0.06
n
t: not tested.


## 3.1. 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 (Table1). When comparing the oils, MOS-ES tests demonstrated inhibitory bacterial rate somewhat lower, since most strains (n = 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 aV. navarrensis strain (Table 1). Both extracts showed antibacterial effect against the Gram-positive(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 (n = 100).Table 1
Distribution, according to the average inhibition zone (mm), of the number of strains inhibited by cold ethanol extract ofMoringa oleifera seeds (MOS-E).
Vibrio species
n
Average inhibition zone (mm)
22–24
19–21
16–18
13–15
10–12
7–9
0
V. navarrensis
53
1
2
5
27
15
3
—
V. brasiliensis
15
—
1
2
4
2
5
1
V. parahaemolyticus
10
—
—
—
—
—
9
1
V. xuii
8
—
—
—
1
—
5
2
V. coralliilyticus
5
—
—
2
1
1
1
—
V. cholerae
4
—
—
—
2
1
1
—
V. neptunis
2
—
—
1
—
1
—
—
V. alginolyticus
1
—
—
—
1
—
—
—
V. diazotrophicus
1
—
1
—
—
—
—
—
V. vulnificusB3
1
—
—
—
—
—
—
1
V. parahaemolyticus
I
O
C
a
1
—
—
1
—
—
—
—
S. aureus ATCC25923b
1
—
1
—
—
—
—
—
Total
102
1
5
11
36
20
24
5
n: number of isolates. a: standard strain used as Gram-negative control. b: standard strain used as Gram-positive control.Table 2
Distribution, according to the average inhibition zone (mm), of the number of strains inhibited by hot ethanol extract ofMoringa oleifera (MOS-ES).
Vibrio species
n
Average inhibition zone (mm)
19–21
16–18
13–15
10–12
7–9
0
V. navarrensis
53
1
6
14
26
5
1
V. brasiliensis
15
1
2
2
4
3
3
V. parahaemolyticus
10
—
—
—
—
9
1
V. xuii
8
—
—
—
1
7
—
V. coralliilyticus
5
—
—
1
3
1
—
V. cholerae
4
—
—
1
1
1
1
V. neptunis
2
—
—
—
2
—
—
V. alginolyticus
1
—
—
1
—
—
—
V. diazotrophicus
1
—
1
—
—
—
—
V. vulnificusB3
1
—
—
—
—
—
1
V. parahaemolyticus
I
O
C
a
1
—
1
—
—
—
—
S. aureusATCC25923b
1
—
1
—
—
—
—
Total
102
2
11
19
37
26
7
n: number of isolates. a: standard strain used as Gram-negative control. b: standard strain used as Gram-positive control.


## 3.2. 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 32µg mL−1 concentration. Levels of 8, 16, and 64 µg 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 (n = 88; 97.8%) were that of 32 µg mL−1. On the other hand, only 2 (2.2%) of the strains were inhibited by a MOS-ES MIC of 16 µg mL.


## 3.3. Bioactivity of the Fractions

Study of the bioactivity of dichloromethane (CH2Cl2), 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.Table 3
Average inhibition halos of ethyl acetate (EtOAc) fractions of ethanolic extracts ofMoringa oleifera seeds extracted cold (MOS-E) and hot (MOS-ES) with ethanol against ten Vibrio sp. strains isolated from the hemolymph of Litopenaeus vannamei.
Strain
Vibriospecies
EtOAc (MOS-E)
EtOAc (MOS-ES)
1
V. coralliilyticus
14.51
±
0.07
12.10
±
0.08
7
V. alginolyticus
12.65
±
0.06
10.52
±
0.45
13
V. navarrensis
15.71
±
0.21
14.03
±
0.07
35
V. diazotrophicus
17.14
±
0.04
16.58
±
0.42
40
V. navarrensis
15.75
±
0.35
11.82
±
0.31
42
V. xuii
12.39
±
0.24
11.80
±
0.25
46
V. parahaemolyticus
8.53
±
0.15
7.66
±
0.12
89
V. neptunis
18.54
±
0.42
11.18
±
0.09
97
V. cholerae
8.46
±
0.09
7.32
±
0.10
98
V. brasiliensis
15.01
±
0.05
14.61
±
0.15


## 3.4. 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.


## 3.5. Structural Characterization of MOS-ES-1 (S1) and MOS-ES-3 (S3)

S1 was isolated as a white amorphous solid and presented 129–132°C, [M + Na]+ = 302,1012. RMN 1H (500 MHz, CD3OD): δ 7,28 (2H, d,J = 8,6 Hz); 7,07 (2H, d,J = 8,6 Hz); 5,43 (1H, d,J = 1,4 Hz); 3,84 (1H, dd,J = 3,4 e 9,5 Hz); 3,82 (2H, s); 3,82 (2H, m); 3,62 (1H, m); 3,46 (1H, t,J = 9,5 Hz); 1,21 (3H, d,J = 6,2 Hz). RMN 13C (125 MHz, CD3OD): δ 157,4 (C-1); 118,1 (C-2 e C-6); 130,2 (C-3 e C-5); 125,8 (C-4); 22,7 (C-7); 119,8 (C-8); 99,9 (C-1′); 72,0 (C-2′); 72,0 (C-3′); 73,8 (C-4′); 70,7 (C-5′); 18,0 (C-6′). IV νmax/cm−1 (KBr) 3411 (νO-H), 2948 (ν-C-H), 2250 (νC≡N), 1611–1513 (νC=C), 1227 (νC-O).Analysis of spectral data and the comparison with information in the literature [15] were used to characterize S1 as the 4-[(α-L-rhamnosyloxy)benzyl] nitrile or niazirine.S3 was also isolated as a white amorphous solid and presented 130–133°C, [M + Cl]− = 392,0951, RMN 1H (500 MHz, CD3OD): δ 7,25 (2H, d,J = 8,7 Hz); 7,01 (2H, d,J = 8,7 Hz); 5,40 (1H, d,J = 1,8 Hz); 4,63 (2H, s); 4,46 (2H, q,J = 7,1 Hz); 3,99 (1H, dd,J = 1,8 e 3,4 Hz); 3,83 (1H, dd,J = 3,4 e 9,5 Hz); 3,62 (1H, m); 3,45 (1H, t,J = 9,5 Hz); 1,29 (3H, t,J = 7,1 Hz); 1,21 (3H, d,J = 6,2 Hz), RMN 13C (125 MHz, CD3OD): δ 157,1 (C-1); 117,6 (C-2 e C-6); 130,1 (C-3 e C-5); 133,1 (C-4); 49,0 (C-7); 192,3 (C-8); 66,9 (C-9); 14,7 (C-10); 100,2 (C-1′); 72,1 (C-2′); 72,3 (C-3′); 73,9 (C-4′); 70,6 (C-5′); 18,0 (C-6′). IV νmax/cm−1 (KBr) 3405 (νO-H), 2948 (ν-C-H), 1611–1513 (νC=C), 1233 (νC=S).Analysis of spectral data and the comparison with information in the literature [14] were used to characterize S3 asO-ethyl-4-[(α-L-rhamnosyloxy)benzyl] thiocarbamate or niazimicine.


## 3.6. Evaluation of Antibacterial Activity of the Isolated Substances

Bioactivity results of S1 and S3 substances against tenVibrio 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.Table 4
Average size of inhibition halos from substances 1 (S1) and 3 (S3) isolated from fractions of ethyl acetate (EtOAc) ofMoringa seed in cold extraction with ethanol (MOS-E) and in hot extraction with ethanol (MOS-ES), against ten Vibrio sp. strains isolated from the hemolymph of Litopenaeus vannamei.
Strains
Species
Fraction of EtOAc MOS-ES
S1
S2
S3
1
V. coralliilyticus
8.11
±
0.09
nt
14.04
±
0.02
7
V. alginolyticus
8.32
±
0.02
nt
15.98
±
0.09
13
V. navarrensis
9.76
±
0.43
nt
19.89
±
0.11
35
V. diazotrophicus
7.42
±
0.24
nt
21.21
±
0.24
40
V. navarrensis
9.77
±
0.15
nt
17.89
±
0.12
42
V. xuii
8.76
±
0.29
nt
20.33
±
0.06
46
V. parahaemolyticus
7.66
±
0.45
nt
9.90
±
0.07
89
V. neptunis
7.14
±
0.11
nt
20.11
±
0.03
97
V. cholerae
7.30
±
0.27
nt
10.15
±
0.18
98
V. brasiliensis
7.99
±
0.02
nt
18.75
±
0.06
n
t: not tested.


## 4. Discussion

Studies on the bioactive properties ofMoringa 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 againstS. aureus andE. 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) against antimicrobial-resistant vibrios with virulence factors must be stressed. This vibriocidal activity is congruent with the findings of Vieira et al. (2010), who investigated antibacterial activity of ethanol extracts ofMoringa seeds and found inhibition zones ranging from 26 to 29.5 mm against classicalV. cholerae 569B.Vibriocidal activity of aerial parts ofM. oleifera was reported by Peixoto et al. [20]. The authors tested the bioactivity of its extracts against standardV. 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 ofV. cholerae. Atieno et al. [21] observed the bioactivity of hexane and methanolic extracts ofM. oleifera andM. stenopetala seeds againstSalmonella ser. Typhi,E. coli, andV. cholerae. For the species ofVibrio, the authors reported inhibition halo sizes of 22.2 and 13.8 mm for hexane and methanolic extracts ofM. oleifera, respectively. The aforementioned data support the assertion thatMoringa 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 ofMoringa 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 ofMoringa’s crude extract fractions was also verified by Nantachit [23]. The author noted that the dichloromethane fraction was active againstE. coli.Guevara et al. [24] reported similar structures described in this study, namely,O-ethyl-4-(α-L-rhamnosyloxy) benzyl carbamates, 4(α-L-rhamnosyloxy)-benzyl isothiocyanate, niazimicine, 3-O-(6′-O-oleoyl-β-D-glucopyranosyl)-β-sitosterol, β-sitosterol-3-O-β-D-glucopyranoside, niazirine, β-sitosterol, and glycerol-1-(9-octadecanoate) by studying the ethanol extracts ofMoringa seeds. The authors mentioned the bioactivity potential of niazimicine and niazirine. Furthermore, Gilani et al. [25] isolated four bioactive compounds out of the bioguided fractionation of ethanolic extracts ofM. oleifera leaves and also found compatible substances with those described in the present study: niazinine A, niazinine B niazinine, niazimicine, and niazinine A + B.In this context, Padla et al. [10] isolated 4-(alpha-L-Rhamnosyloxy)benzyl isothiocyanate and 4-(4′-O-acetyl-alpha-L-rhamnosyloxy)-benzyl isothiocyanate fromMoringa oleifera seeds and demonstrated that both substances were bioactive against Gram-positive bacteriaStaphylococcus aureus,Staphylococcus epidermidis, andBacillus subtilis. In the present study, we isolated fromMoringa seeds the substances (1) 4-[(α-L-rhamnosyloxy)benzyl]nitrile (niazirine) and (2)O-ethyl-4-[(α-L-rhamnosyloxy)benzyl] thiocarbamate (niazimicine).The compoundsO-ethyl-p-hydroxybenzene carbamate andO-methyl-4-[(2′,3′,4′-tri-O-acetyl-α-L-rhamnosyloxy)benzyl] thiocarbamate must also be added to the previously mentioned substances as isolates from leaf extracts ofM. oleifera [26, 27]. The addition of methanol or ethanol to isothiocyanate is considered a pathway to the synthesis of thiocarbamates glycosides inMoringa [26].Since the 1990s, biological activities of pharmacological interest concerning nitriles isolated fromMoringa 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 inMoringa 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 theM. 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 β-hemolysis producers), cross-resistance to β-lactams, 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.



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*Source: 7963747-2017-07-09.xml*