# Trigonella foenum-graceum (Seed) Extract Interferes with Quorum Sensing Regulated Traits and Biofilm Formation in the Strains of Pseudomonas aeruginosa and Aeromonas hydrophila

Authors: Fohad Mabood Husain; Iqbal Ahmad; Mohd Shahnawaz Khan; Nasser Abdulatif Al-Shabib
Journal: Evidence-Based Complementary and Alternative Medicine
 (2015)

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## Abstract

Trigonella foenum-graecum L. (Fenugreek) is an important plant of the Leguminosae family known to have medicinal properties. However, fraction based antiquorum sensing and antibiofilm activities have not been reported from this plant. In the present study T. foenum-graecum seed extract was sequentially fractionated and sub-MICs were tested for above activities. The methanol fraction of the extract demonstrated significant inhibition of AHL regulated virulence factors: protease, LasB elastase, pyocyanin production, chitinase, EPS, and swarming motility in Pseudomonas aeruginosa PAO1 and PAF79. Further, QS dependent virulence factor in the aquatic pathogen Aeromonas hydrophila WAF38 was also reduced. Application of T. foenum-graecum seed extract to PAO1, PAF79, and WAF38 decreased the biofilm forming abilities of the pathogens by significant levels. The extract also exhibited reduced AHL levels and subsequent downregulation of lasB gene. In vivo study showed an enhanced survival of PAO1-preinfected C. elegans after treatment with extract at 1 mg/mL. Further, the major compound detected by GC-MS, caffeine, reduced the production of QS regulated virulence factors and biofilm at 200 µg/mL concentration indicating its role in the activity of the methanol extract. The results of the present study reveal the potential anti-QS and antibiofilm property of T. foenum-graceum extract and caffeine.


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## Body

## 1. Introduction

Formation of biofilm by many pathogens is closely associated with density dependent cell-cell communication known as quorum sensing (QS), in which small diffusible signaling molecules called autoinducers regulate gene expression. Quorum sensing helps bacterial populations to switch from acting as individual cells to operating in a concerted, multicellular fashion [1]. In clinical settings, biofilms are major threat and challenge because bacteria living within the mode are more protected against host immune responses and are significantly more resistant to various antimicrobial drugs [2, 3].Pseudomonas aeruginosa is an opportunistic, nosocomial, and biofilm forming gram negative pathogen that has three main QS pathways. The rhlI/rhlR and lasI/lasR pathways are (acyl homoserine lactone) AHL based and PQS-MvfR pathway is regulated by 2-heptyl-3-hydroxy-4(1 H)-quinolone signal molecule [4–6].P. aeruginosa utilizes these signal molecules for the production of biofilms and virulence factors during pathogenesis. Several studies have also shown that QS deficientP. aeruginosa has reduced biofilm forming abilities [7, 8]. The above-mentioned observations imply that the quorum sensing inhibitors (QSIs) may have the potential to circumvent the challenge of combating multidrug resistance in bacteria [6]. Thus, it is envisaged that QS inhibitors will also be equally effective against biofilms formed by pathogenic bacteria.Plant-derived compounds have been used to treat microbial infections for centuries and are supposed to be safe for human consumption [9]. Screening of plant-derived compounds with improved strategy may facilitate the discovery of compounds that attenuate bacterial pathogenesis/biofilms. It is expected that antipathogenic drugs will generate less pressure for the evolution of resistance as compared to antibiotic therapy [10]. Plant-derived compounds such as ursolic acid, naringenin, cinnamaldehyde, salicylic acid, methyl eugenol, essential oils, and extracts from Indian medicinal plants, garlic, and edible fruits have shown various extents of antibiofilm and quorum sensing inhibitory properties against several pathogens [10, 11]. However the majority of Indian medicinal plants are yet to be screened and evaluated for such novel activities. In our previous study, interference in QS mediated violacein production by crude extracts ofT. foenum-graceumin CV12472 was observed [12]. Therefore, the present study was undertaken to investigate its anti-QS and anti-biofilm potential in search of effective antipathogenic drug principles against bacterial infections.Trigonella foenum-graecum L. (fenugreek) is an important annual medicinal plant of the Leguminosae family and its leaves and seeds have been used in various illnesses and as a health tonic for a very long time. Fenugreek is known to have hypoglycemic, hypocholesterolaemic, antioxidant potency, digestive stimulant action, and hepatoprotective effects [16]. Recent research demonstrated that fenugreek is a valuable medicinal plant of multipurpose uses and may be used for preparing various products such as steroidal hormones [17]. To the best of our knowledge, there is no report available on the antibiofilm activity ofT. foenum-graceum against PAO1. Therefore, we have selected this plant and elucidated the broad spectrum anti-QS and antibiofilm activity of methanolic extract against pathogenic bacteria.


## 2. Methods

### 2.1. Bacterial Strain and Growth Conditions

The strains used in this study are listed in Table1. Unless otherwise stated, all of the strains were grown in LB medium.Table 1
Bacterial strains used in the study.
Strains
Relevant genotype
Source
C. violaceum CVO26
Mini Tn5 mutant of 31532
McLean et al. [13]
P. aeruginosa PAO1
Wild type
McLean et al. [13]
P. aeruginosa PAO1 MW1
DlasI::Tet DrhlI::Tn501-2 strain PAO1 derivative
Schuster and Greenberg [14]
E. coli MG4/pKDT17
E. coli DH5α harboring plasmid pMG4/pKDT
Zhou et al. [15]
P. aeruginosa PAF79
AHL producing strain
Laboratory strain
A. hydrophila WAF38
AHL producing strain
Laboratory strain


### 2.2. Plant Material and Preparation of Extracts

T. foenum-graceum (L.) (Fenugreek) was purchased from a local market in Aligarh, India. The voucher specimen (MBD-34/09) was deposited in the Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh, India. Plant extract was prepared as described earlier [18]. Briefly, five hundred (500) grams of dry seed powder was soaked in 2.5 L of methanol for 5 days with intermittent shaking and was filtered through Whatman filter paper number 1 (Whatman Ltd., England). The filtered extract was concentrated to dryness under reduced pressure in a rotary evaporator at 40°C and stored at 4°C for future use.


### 2.3. Determination of Minimum Inhibitory Concentration (MIC)

Minimum inhibitory concentration (MIC) of plant extract against test strains was determined by the microbroth dilution method, using specific dye (p-iodonitrotetrazolium violet) as an indicator of growth as described by Eloff [19]. MIC is defined as the minimum concentration of plant extracts which inhibited the visible growth of test strains.


### 2.4. Effect of Methanolic Extract on Quorum Sensing Regulated Virulence Factors

Effect of sub-MICs of plant extract on virulence factors ofP. aeruginosa andA. hydrophila such as LasB elastase, protease, pyocyanin, chitinase, swarming motility, EPS extraction, and quantification was assessed as described previously [20].


### 2.5. Effect on Biofilm Formation

The effect ofT. foenum-graceum (seed) extract on biofilm formation was measured using the polyvinyl chloride biofilm formation assay [21]. Briefly, overnight (treated and untreated) cultures of PAO1, PAF79, and WAF38 were visualized for biofilm formation by staining with 0.1% crystal violet solution and measuring the absorbance at OD470.


### 2.6. Scanning Electron Microscopy

Biofilms were grown on glass coverslips, in the treated and untreated cultures of PAO1. After 24 h of incubation, the cover slips were rinsed with distilled water to remove planktonic cells and processed for scanning electron microscopy (SEM) examination [20].


### 2.7. Analysis of lasB Transcriptional Activity inE. coli

Measurement ofβ-galactosidase luminescence inE. coli MG4/pKDT17 was done by the method described previously [20]. Briefly, culture supernatant was extracted by ethyl acetate for quorum sensing signal molecules (AHLs) from overnight cultures of PAO1 grown in presence and absence of sub-MICs of seed extract. Then, 2 mL of reporterE. coli MG4 (pKDT17) strain and 0.5 mL of the ethyl acetate extracted supernatant were incubated at 30°C in a water bath for 5 h with rotation at 100 r.p.m. After centrifugation (3200 g for 15 min) of the reporter cell cultures, cell pellet was suspended in an equal volume of Z buffer (Na2HPO4·7H2O, 0.06 M; NaH2PO4·H2O, 0.04 M; KCl, 0.01 M; MgSO4·7H2O, 0.001 M; β-mercaptoethanol, 0.05 M; pH 7.0). To 1 mL of cell suspension 1 mL of Z buffer, 200 μL of chloroform, and 100 μL of 0.1% sodium dodecyl sulphate were added to lyse cells, and 0.4 mL of o-nitrophenol-β-D-galactopyranoside [4 mg/mL in phosphate buffered saline (PBS)] was also added. Reaction was stopped after the development of yellow colour by the addition of 1 mL of 1 M Na2CO3. OD of the reaction samples was measured at 420 and 550 nm. Units of β-galactosidase were calculated as 1000 × OD420nm-(
1.75
×
OD
550
nm
)
/
time
×
volume
×
OD
600
nm.


### 2.8. Determination of Effect of Extract onCaenorhabditis elegans Survival

The method described by Musthafa et al. [22] was adopted to study thein vivo efficiency ofT. foenum-graceum (seed) extract inC. elegans Nematode infection model. Briefly, the young adult Nematodes were infected with PAO1 in the 24-well microtiter plate and incubated at 25°C for 12 h. After incubation,C. elegans from the wells was washed thrice with M9 (KH2PO4 3 g, Na2HPO4 6 g, NaCl 5 g, 1 M MgSO4 1 mL, and distilled water 1000 mL) buffer to remove surface-bound bacteria. Around 10 infected worms were transferred to the wells of microtiter plate containing 10% LB broth in M9 buffer and incubated at 25°C with or without extract.


### 2.9. GC-MS Analysis of Plant Extracts

The composition of the extract was analysed by Perkin Elmer GC Autosystem XL and Turbomass with EI source using PE-Wax column (30 m × 0.25 mm i.d., film thickness 0.25 mm); carrier gas was helium with column head pressure 7 psi connected to data station. The linear temperature program of 60°C to 200°C was set at a rate of 4°C/min−1 with hold time at 200°C for 21 minutes. The components were identified by comparing their retention times to those of authentic samples, as well as by comparing their mass spectra with those of Wiley 8 and NIST 05 Libraries. Quantitative data were obtained by the peak normalization technique using integrated FID response.


### 2.10. HPTLC Analysis

Stock standard solutions containing 100μg/mL of caffeine in methanol were freshly prepared. A sample solution was prepared by dissolving 50 mg ofT. foenum-graceum (seed) extract in methanol and volume was adjusted to obtain a concentration of 10 mg/mL; the spots were applied on precoated TLC plates with the help of HPTLC applicator Linomat V (CAMAG). The stationary phase used was precoated silica gel 60F254 plates (20 cm × 20 cm) from E-Merck and the mobile phase composition was optimized 2-Propanol : Ethyl acetate (4 : 6). The densitometric analysis was performed on CAMAG TLC scanner at 254 nm.


### 2.11. Statistical Analysis

All experiments were performed in triplicate and the data obtained from the experiments were presented as mean values and the differences between control and test were analyzed using Student’st test.


## 2.1. Bacterial Strain and Growth Conditions

The strains used in this study are listed in Table1. Unless otherwise stated, all of the strains were grown in LB medium.Table 1
Bacterial strains used in the study.
Strains
Relevant genotype
Source
C. violaceum CVO26
Mini Tn5 mutant of 31532
McLean et al. [13]
P. aeruginosa PAO1
Wild type
McLean et al. [13]
P. aeruginosa PAO1 MW1
DlasI::Tet DrhlI::Tn501-2 strain PAO1 derivative
Schuster and Greenberg [14]
E. coli MG4/pKDT17
E. coli DH5α harboring plasmid pMG4/pKDT
Zhou et al. [15]
P. aeruginosa PAF79
AHL producing strain
Laboratory strain
A. hydrophila WAF38
AHL producing strain
Laboratory strain


## 2.2. Plant Material and Preparation of Extracts

T. foenum-graceum (L.) (Fenugreek) was purchased from a local market in Aligarh, India. The voucher specimen (MBD-34/09) was deposited in the Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh, India. Plant extract was prepared as described earlier [18]. Briefly, five hundred (500) grams of dry seed powder was soaked in 2.5 L of methanol for 5 days with intermittent shaking and was filtered through Whatman filter paper number 1 (Whatman Ltd., England). The filtered extract was concentrated to dryness under reduced pressure in a rotary evaporator at 40°C and stored at 4°C for future use.


## 2.3. Determination of Minimum Inhibitory Concentration (MIC)

Minimum inhibitory concentration (MIC) of plant extract against test strains was determined by the microbroth dilution method, using specific dye (p-iodonitrotetrazolium violet) as an indicator of growth as described by Eloff [19]. MIC is defined as the minimum concentration of plant extracts which inhibited the visible growth of test strains.


## 2.4. Effect of Methanolic Extract on Quorum Sensing Regulated Virulence Factors

Effect of sub-MICs of plant extract on virulence factors ofP. aeruginosa andA. hydrophila such as LasB elastase, protease, pyocyanin, chitinase, swarming motility, EPS extraction, and quantification was assessed as described previously [20].


## 2.5. Effect on Biofilm Formation

The effect ofT. foenum-graceum (seed) extract on biofilm formation was measured using the polyvinyl chloride biofilm formation assay [21]. Briefly, overnight (treated and untreated) cultures of PAO1, PAF79, and WAF38 were visualized for biofilm formation by staining with 0.1% crystal violet solution and measuring the absorbance at OD470.


## 2.6. Scanning Electron Microscopy

Biofilms were grown on glass coverslips, in the treated and untreated cultures of PAO1. After 24 h of incubation, the cover slips were rinsed with distilled water to remove planktonic cells and processed for scanning electron microscopy (SEM) examination [20].


## 2.7. Analysis of lasB Transcriptional Activity inE. coli

Measurement ofβ-galactosidase luminescence inE. coli MG4/pKDT17 was done by the method described previously [20]. Briefly, culture supernatant was extracted by ethyl acetate for quorum sensing signal molecules (AHLs) from overnight cultures of PAO1 grown in presence and absence of sub-MICs of seed extract. Then, 2 mL of reporterE. coli MG4 (pKDT17) strain and 0.5 mL of the ethyl acetate extracted supernatant were incubated at 30°C in a water bath for 5 h with rotation at 100 r.p.m. After centrifugation (3200 g for 15 min) of the reporter cell cultures, cell pellet was suspended in an equal volume of Z buffer (Na2HPO4·7H2O, 0.06 M; NaH2PO4·H2O, 0.04 M; KCl, 0.01 M; MgSO4·7H2O, 0.001 M; β-mercaptoethanol, 0.05 M; pH 7.0). To 1 mL of cell suspension 1 mL of Z buffer, 200 μL of chloroform, and 100 μL of 0.1% sodium dodecyl sulphate were added to lyse cells, and 0.4 mL of o-nitrophenol-β-D-galactopyranoside [4 mg/mL in phosphate buffered saline (PBS)] was also added. Reaction was stopped after the development of yellow colour by the addition of 1 mL of 1 M Na2CO3. OD of the reaction samples was measured at 420 and 550 nm. Units of β-galactosidase were calculated as 1000 × OD420nm-(
1.75
×
OD
550
nm
)
/
time
×
volume
×
OD
600
nm.


## 2.8. Determination of Effect of Extract onCaenorhabditis elegans Survival

The method described by Musthafa et al. [22] was adopted to study thein vivo efficiency ofT. foenum-graceum (seed) extract inC. elegans Nematode infection model. Briefly, the young adult Nematodes were infected with PAO1 in the 24-well microtiter plate and incubated at 25°C for 12 h. After incubation,C. elegans from the wells was washed thrice with M9 (KH2PO4 3 g, Na2HPO4 6 g, NaCl 5 g, 1 M MgSO4 1 mL, and distilled water 1000 mL) buffer to remove surface-bound bacteria. Around 10 infected worms were transferred to the wells of microtiter plate containing 10% LB broth in M9 buffer and incubated at 25°C with or without extract.


## 2.9. GC-MS Analysis of Plant Extracts

The composition of the extract was analysed by Perkin Elmer GC Autosystem XL and Turbomass with EI source using PE-Wax column (30 m × 0.25 mm i.d., film thickness 0.25 mm); carrier gas was helium with column head pressure 7 psi connected to data station. The linear temperature program of 60°C to 200°C was set at a rate of 4°C/min−1 with hold time at 200°C for 21 minutes. The components were identified by comparing their retention times to those of authentic samples, as well as by comparing their mass spectra with those of Wiley 8 and NIST 05 Libraries. Quantitative data were obtained by the peak normalization technique using integrated FID response.


## 2.10. HPTLC Analysis

Stock standard solutions containing 100μg/mL of caffeine in methanol were freshly prepared. A sample solution was prepared by dissolving 50 mg ofT. foenum-graceum (seed) extract in methanol and volume was adjusted to obtain a concentration of 10 mg/mL; the spots were applied on precoated TLC plates with the help of HPTLC applicator Linomat V (CAMAG). The stationary phase used was precoated silica gel 60F254 plates (20 cm × 20 cm) from E-Merck and the mobile phase composition was optimized 2-Propanol : Ethyl acetate (4 : 6). The densitometric analysis was performed on CAMAG TLC scanner at 254 nm.


## 2.11. Statistical Analysis

All experiments were performed in triplicate and the data obtained from the experiments were presented as mean values and the differences between control and test were analyzed using Student’st test.


## 3. Results

The methanol extract ofT. foenum-graceumexhibited MIC values of 1200, 2400, and 1200 μg/mL against PAO1, PAF79, and WAF38, respectively.The extract was evaluated against QS regulated virulence factors inP. aeruginosa PAO1 and clinical strain PAF79. At sub-MICs (125, 250, 500, and 1000 μg/mL), the extract showed a concentration dependent effect on virulence factors ofP. aeruginosa PAO1 (Table 2). Statistically significant reduction (p
≤
0.05) in elastase, total protease, chitinase activity, pyocyanin production, and swarming motility was recorded at the 500 μg/mL concentration. The extract at 1000 μg/mL concentration caused maximum percent decrease in virulence factors such as elastase (61.3%), protease (59.7%), chitinase (47.5%), pyocyanin (55.7%), and swarming motility (59.7%) inP. aeruginosa PAO1 over the untreated control (Table 2). However, exopolysaccharide (EPS) produced by the extract treated cultures of PAO1 exhibited a maximum decrease of 46.5% at 1000 μg/mL concentration. In the PAF79 strain, maximum inhibition in the activity of elastase (67.6%), protease (55%), chitinase (87%), pyocyanin production (82.1%), and swarming motility (62.5%) was observed at 1000 μg/mL concentration of extract. EPS production was reduced significantly at all tested concentrations and a maximum decrease of 77.5% was observed at the highest tested concentration of extract (Table 3). ThelasI rhlI mutant (PAO MW1) grown in the absence of acylated HSL was included as a negative control.Table 2
Effect of sub-MICs of methanolic extract ofTrigonella foenum-graceum (seed) on inhibition of quorum sensing regulated virulence factors in P. aeruginosaPAO1.
Extract concentration(μg/mL)
Elastase activitya
Total proteaseb
Pyocyanin productionc
Chitinase activityd
EPS productione
Swarming motilityf
PAO1-MW1
0.024 ± 0.005
0.061 ± 0.009
0.26 ± 0.012
0.021 ± 0.006
0.118 ± 0.01
9 ± 0.4
Control
0.181 ± 0.044
1.110 ± 0.027
5.2 ± 0.6
0.120 ± 0.009
1.075 ± 0.015
72 ± 1.5
125
0.138 ± 0.040(23.7)
0.770 ± 0.024(30.6)
4.3 ± 0.72(17.3)
0.094 ± 0.003(21.6)
1.039 ± 0.026(03.3)
57 ± 3.2(20.8)
250
0.124 ± 0.037(31.4)
0.715 ± 0.019(35.5)
4.0 ± 0.35(23.0)
0.080 ± 0.013(33.3)
0.916 ± 0.023(14.7)
42 ± 1.8(41.6)*
500
0.086 ± 0.020(52.4)**
0.480 ± 0.010(56.7)*
2.8 ± 0.39(46.1)*
0.069 ± 0.013(42.5)*
0.825 ± 0.023(23.2)
38 ± 1.1(47.2)*
1000
0.070 ± 0.025(61.3)**
0.447 ± 0.008(59.7)*
2.3 ± 0.11(55.6)*
0.063 ± 0.008(47.5)*
0.575 ± 0.016(46.5)*
29 ± 2.0(59.7)**
PAO1-MW1 grown in the absence of acylated HSL was included as a negative control.
aElastase activity is expressed as the absorbance at OD495.
bTotal protease activity is expressed as the absorbance at OD600.
cPyocyanin concentrations were expressed as micrograms of pyocyanin produced per microgram of total protein.
dChitinase activity is expressed as the absorbance at OD570.
eEPS production is expressed as absorbance at OD480.
fSwarming motility is expressed as diameter of swarm in mm.
The data represents mean values of three independent experiments.*Significance at p
≤
0.05, **significance at p
≤
0.005.
Values in the parentheses indicate percent reduction over control.Table 3
Effect of sub-MICs of methanolic extract ofTrigonella foenum-graceum(seed) on inhibition of quorum sensing regulated virulence factors in P. aeruginosaPAF-79.
Extract concentration(μg/mL)
Elastase activitya
Total proteaseb
Pyocyanin productionc
Chitinase activityd
EPS productione
Swarming motilityf
PAO1-MW1
0.029 ± 0.008
0.057 ± 0.004
0.29 ± 0.01
0.020 ± 0.005
0.121 ± 0.014
8 ± 0.7
Control
0.167 ± 0.025
1.039 ± 0.041
3.8 ± 0.25
0.139 ± 0.005
0.886 ± 0.036
48 ± 1.5
125
0.105 ± 0.022(37.1)
0.564 ± 0.024(45.7)*
2.6 ± 0.42(31.5)*
0.094 ± 0.016(32.3)
0.414 ± 0.025(53.2)*
39 ± 2(18.7)
250
0.085 ± 0.021(49.1)*
0.547 ± 0.033(47.3)*
1.2 ± 0.27(68.4)**
0.065 ± 0.023(53.3)*
0.357 ± 0.019(59.7)**
32 ± 2(33.3)*
500
0.057 ± 0.016(65.8)*
0.499 ± 0.003(51.9)*
0.94 ± 0.016(75.2)**
0.038 ± 0.006(72.6)**
0.309 ± 0.031(65.1)**
23 ± 0.5(52)*
1000
0.054 ± 0.003(67.6)*
0.468 ± 0.010(55)*
0.68 ± 0.028(82.1)***
0.018 ± 0.004(87)***
0.199 ± 0.014(77.5)**
18 ± 3(62.5)*
PAO1-MW1 grown in the absence of acylated HSL was included as a negative control.
aElastase activity is expressed as the absorbance at OD495.
bTotal protease activity is expressed as the absorbance at OD600.
cPyocyanin concentrations were expressed as micrograms of pyocyanin produced per microgram of total protein.
dChitinase activity is expressed as the absorbance at OD570.
eEPS production is expressed as absorbance at OD480.
fSwarming motility is expressed as diameter of swarm in mm.
The data represents mean values of three independent experiments.*Significance at p
≤
0.05, **significance at p
≤
0.005,  and ***significance at p
≤
0.001.
Values in the parentheses indicate percent reduction over control.The extract demonstrated a 24.1–68.7% decrease in the biofilm forming ability of PAO1 at sub-MICs tested (125–1000μg/mL) (Figures 1 and 2). Similarly, significant concentration dependent decrease (p
≤
0.005) in biofilm formation was also observed in PAF79 strain when grown in presence of sub-MICs of the extract. The extract demonstrated a maximum of 65.5% inhibition over untreated control at the highest sub-MIC tested as shown in Figure 1.Figure 1
Effect ofT. foenum-graceum (seed) extract on biofilm formation inP. aeruginosa PAO1 andP. aeruginosa PAF79 at respective sub-MICs.Figure 2
Light and scanning electron microscopic images ofP. aeruginosa PAO1 andA. hydrophila WAF38 biofilm in the presence and absence of sub-MICs ofT. foenum-graceum (seed) extract: (a, c) untreated control PAO1, (b, d) treated with 1000 μg/mL concentration ofT. foenum-graceum extract, (e, g) untreated control WAF38, and (f, h) treated with 800 µg/mL concentration ofT. foenum-graceum extract.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)The effect of the extract was also assessed against virulence factors ofA. hydrophila WAF38 at sub-MICs 100–800 μg/mL. Highest tested concentration (800 μg/mL) significantly reduced total protease activity (71.6%), EPS production (46.3%), and biofilm formation (76.9%) (Table 4, Figure 2).Table 4
Effect of sub-MICs of methanolic extract ofTrigonella foenum-graceum(seed) on inhibition of quorum sensing regulated virulence factorsin Aeromonas hydrophila WAF-38.
Concentration (μg/mL)
Total proteasea
EPS productionb
Biofilm formationc
Control
0.589 ± 0.051
0.748 ± 0.021
0.226 ± 0.006
100
0.520 ± 0.025(11.7)
0.721 ± 0.064(03.6)
0.126 ± 0.011(44.2)*
200
0.431 ± 0.049(26.8)
0.642 ± 0.035(14.1)
0.077 ± 0.018(65.9)**
400
0.321 ± 0.033(45.5)*
0.516 ± 0.013(31.0)
0.068 ± 0.004(69.9)**
800
0.167 ± 0.018(71.6)**
0.401 ± 0.021(46.3)*
0.052 ± 0.010(76.9)**
aTotal protease activity is expressed as the absorbance at OD600.
bEPS production is expressed as absorbance at OD480.
cBiofilm formation is expressed as OD470 after incubation with crystal violet.
The data represents mean values of three independent experiments.*Significance at p
≤
0.05, **significance at p
≤
0.005.
Values in the parentheses indicate percent reduction over control.


### 3.1. Effect of Methanolic Extract ofTrigonella foenum-graceum (Seed) on lasB Transcriptional Activity inE. coli

Impact of sub-MICs ofT.foenum-graceum (seed) extract on β-galactosidase activity ofE. coli MG4/pKDT17 exhibited a dose dependent decrease. Untreated control produced 768 miller units (MU) whereas 623, 557, 483, and 361 miller units of AHL production were recorded at 200, 400, 800, and 1000 μg/mL concentration (Figure 3). The reduction in the levels of AHL demonstrates that the inhibition oflasB promoter activity involves LasR controlled transcription.Figure 3
Effect ofT. foenum-graceum (seed) extract on β-galactosidase activity inE. coli MG4/pKDT17. All of the data are presented as mean ± SD. ∗Significance at p ≤ 0.05 and significance at p ≤ 0.005.


### 3.2. Anti-Infective Potential of Seed Extract inC. elegans Nematode Model

In the absence of plant extracts, complete mortality of the PAO1-preinfectedC. elegans was observed within 72 h, which shows the potent pathogenicity of PAO1 towards theC. elegans nematode. However,C. eleganspreinfected with PAO1 further treated withT. foenum-graceum extract (1000 μg/mL) displayed enhanced survival of 48% (Figure 4). However, the extract ofT. foenum-graceum alone demonstrated no significant effect on the mortality ofC. elegans at tested concentrations.Figure 4
Anti-infection potential of sub-MICs (1000μg/mL) ofT. foenum-graceum (seed) extract in increasing survival of preinfectedC. elegans Nematode model. Means values of triplicate independent experiments and SDs are shown.


### 3.3. GC-MS Analysis

A total of 18 chemical components were identified in methanol seed extract by GC-MS analysis. These numbers may be extended with the help of chemometric techniques. The major compounds identified were 1,3,7-trimethyl-3,7-dihydro-1h-purine-2,6-dione [caffeine] (40.82%) followed by methyl 14-methylpentadecanoate (8.22%), palmitic acid (6.41%), 1,2,3-benzenetriol (6.13%), linoleic acid, methyl ester (5.58%), and capric acid (4.2%). The remaining compounds were present in percentages from 2.01 to 0.1 as depicted in Table5.Table 5
Components ofTrigonella foenum-graceum (seed) extractas identified by GC-MS analysis.
Peak number
Components
Retention time
Area (%)
1
1,2,3-Benzenetriol
8.76
6.13
2
Capric acid
11.42
4.20
3
1,3,7-Trimethyl-3,7-dihydro-1h-purine-2, and 6-dione (Caffeine)
13.85
40.82
4
Methyl 14-methylpentadecanoate
14.29
8.22
5
Palmitic acid
14.70
6.41
6
Linoleic acid, methyl ester
15.93
5.58
7
9,12,15-Octadecatrienoic acid, methyl ester
16.00
9.17
8
Phytol
16.11
1.45
9
Methyl stearate
16.20
1.75
10
10,12-Hexadecadien-1-ol
16.34
2.56
11
cis-11-Eicosenoic acid, methyl ester
17.75
0.43
12
Palmidrol
17.89
0.10
13
Methyl 18-methylnonadecanoate
17.95
0.12
14
Hexacosane
18.54
0.11
15
1-Monopalmitin trimethylsilyl ether
19.37
0.13
16
Methyl 18-methylnonadecanoate
22.87
0.40
17
2-Hydroxy-3-(palmitoyloxy)propyl (9E)-9-octadecenoate
28.14
1.48
18
Stigmasterol
29.46
2.01
19
Stigmast-8(14)-en-3.beta.-ol
29.98
0.48


### 3.4. HPTLC Analysis

The mobile phase resolved caffeine efficiently from other components ofT. foenum-graceum(seed) extract. The Rf of caffeine was found to be 0.33 as shown in Figure 5. The findings of the HPTLC analysis clearly indicate the presence of caffeine in the extract.Figure 5
HPTLC chromatogram ofT. foenum-graceum (seed) extract: (a) methanol extract and (b) pure caffeine.


### 3.5. Evaluation of QS Inhibitory Activity of Caffeine

Caffeine (1,3,7-trimethyl-3,7-dihydro-1h-purine-2,6-dione) was tested for its possible role in contributing to the anti-QS activity of the plant extract againstChromobacterium violaceum CVO26 andP. aeruginosa PAO1. Caffeine, at a maximum concentration of 200 μg/mL, showed 87% violacein inhibition in CVO26 (Figure 6). Further, at the same concentration, the compound reduced the biofilm formation (64%), swarming motility (70%), total protease (78%), LasB elastase (68%), and pyocyanin (74%) inP. aeruginosa PAO1 (Figure 6). Moreover, caffeine showed no growth inhibitory effect on the test bacterial pathogen.Figure 6
Effect of caffeine on quorum sensing regulated functions and biofilm. All of the data are presented as mean ± SD.∗Significance at p ≤ 0.05, ∗
∗significance at p ≤ 0.005, and ∗
∗
∗significance at p ≤ 0.001.


## 3.1. Effect of Methanolic Extract ofTrigonella foenum-graceum (Seed) on lasB Transcriptional Activity inE. coli

Impact of sub-MICs ofT.foenum-graceum (seed) extract on β-galactosidase activity ofE. coli MG4/pKDT17 exhibited a dose dependent decrease. Untreated control produced 768 miller units (MU) whereas 623, 557, 483, and 361 miller units of AHL production were recorded at 200, 400, 800, and 1000 μg/mL concentration (Figure 3). The reduction in the levels of AHL demonstrates that the inhibition oflasB promoter activity involves LasR controlled transcription.Figure 3
Effect ofT. foenum-graceum (seed) extract on β-galactosidase activity inE. coli MG4/pKDT17. All of the data are presented as mean ± SD. ∗Significance at p ≤ 0.05 and significance at p ≤ 0.005.


## 3.2. Anti-Infective Potential of Seed Extract inC. elegans Nematode Model

In the absence of plant extracts, complete mortality of the PAO1-preinfectedC. elegans was observed within 72 h, which shows the potent pathogenicity of PAO1 towards theC. elegans nematode. However,C. eleganspreinfected with PAO1 further treated withT. foenum-graceum extract (1000 μg/mL) displayed enhanced survival of 48% (Figure 4). However, the extract ofT. foenum-graceum alone demonstrated no significant effect on the mortality ofC. elegans at tested concentrations.Figure 4
Anti-infection potential of sub-MICs (1000μg/mL) ofT. foenum-graceum (seed) extract in increasing survival of preinfectedC. elegans Nematode model. Means values of triplicate independent experiments and SDs are shown.


## 3.3. GC-MS Analysis

A total of 18 chemical components were identified in methanol seed extract by GC-MS analysis. These numbers may be extended with the help of chemometric techniques. The major compounds identified were 1,3,7-trimethyl-3,7-dihydro-1h-purine-2,6-dione [caffeine] (40.82%) followed by methyl 14-methylpentadecanoate (8.22%), palmitic acid (6.41%), 1,2,3-benzenetriol (6.13%), linoleic acid, methyl ester (5.58%), and capric acid (4.2%). The remaining compounds were present in percentages from 2.01 to 0.1 as depicted in Table5.Table 5
Components ofTrigonella foenum-graceum (seed) extractas identified by GC-MS analysis.
Peak number
Components
Retention time
Area (%)
1
1,2,3-Benzenetriol
8.76
6.13
2
Capric acid
11.42
4.20
3
1,3,7-Trimethyl-3,7-dihydro-1h-purine-2, and 6-dione (Caffeine)
13.85
40.82
4
Methyl 14-methylpentadecanoate
14.29
8.22
5
Palmitic acid
14.70
6.41
6
Linoleic acid, methyl ester
15.93
5.58
7
9,12,15-Octadecatrienoic acid, methyl ester
16.00
9.17
8
Phytol
16.11
1.45
9
Methyl stearate
16.20
1.75
10
10,12-Hexadecadien-1-ol
16.34
2.56
11
cis-11-Eicosenoic acid, methyl ester
17.75
0.43
12
Palmidrol
17.89
0.10
13
Methyl 18-methylnonadecanoate
17.95
0.12
14
Hexacosane
18.54
0.11
15
1-Monopalmitin trimethylsilyl ether
19.37
0.13
16
Methyl 18-methylnonadecanoate
22.87
0.40
17
2-Hydroxy-3-(palmitoyloxy)propyl (9E)-9-octadecenoate
28.14
1.48
18
Stigmasterol
29.46
2.01
19
Stigmast-8(14)-en-3.beta.-ol
29.98
0.48


## 3.4. HPTLC Analysis

The mobile phase resolved caffeine efficiently from other components ofT. foenum-graceum(seed) extract. The Rf of caffeine was found to be 0.33 as shown in Figure 5. The findings of the HPTLC analysis clearly indicate the presence of caffeine in the extract.Figure 5
HPTLC chromatogram ofT. foenum-graceum (seed) extract: (a) methanol extract and (b) pure caffeine.


## 3.5. Evaluation of QS Inhibitory Activity of Caffeine

Caffeine (1,3,7-trimethyl-3,7-dihydro-1h-purine-2,6-dione) was tested for its possible role in contributing to the anti-QS activity of the plant extract againstChromobacterium violaceum CVO26 andP. aeruginosa PAO1. Caffeine, at a maximum concentration of 200 μg/mL, showed 87% violacein inhibition in CVO26 (Figure 6). Further, at the same concentration, the compound reduced the biofilm formation (64%), swarming motility (70%), total protease (78%), LasB elastase (68%), and pyocyanin (74%) inP. aeruginosa PAO1 (Figure 6). Moreover, caffeine showed no growth inhibitory effect on the test bacterial pathogen.Figure 6
Effect of caffeine on quorum sensing regulated functions and biofilm. All of the data are presented as mean ± SD.∗Significance at p ≤ 0.05, ∗
∗significance at p ≤ 0.005, and ∗
∗
∗significance at p ≤ 0.001.


## 4. Discussion

P. aeruginosa proteases and LasB are believed to play a major role in pathogenesis via host tissue degradation [23]. Sub-MICs ofT. foenum-graceum extract resulted in significant decrease in elastase and protease activities of PAO1 and PAF79. Similar results with other plant extracts were also reported by Adonizio et al. [24] and Packiavathy et al. [25]. It is likely that the extract downregulated LasB activity.Pyocyanin is another important virulence factor produced under QS regulation. Therefore, the effect of the extracts in reducing the pyocyanin production was assessed and a significant decrease was recorded. By comparison,Centella asiatica extract inhibited 80% pyocyanin production at 400 μg/mL [26]. Extracts of some edible plants and fruits also showed similar pyocyanin reduction in PAO1 at sub-MICs [27, 28]. Chitinase plays a major role in the pathogenesis ofP. aeruginosa in cystic fibrosis patients [29]. Chitinase production byP. aeruginosa strains (PAO1 and PAF79) was reduced considerably in a concentration dependent manner under the influence of sub-MICs ofT. foenum-graceum. The findings are in agreement with our results obtained with sub-MICs of clove oil [20].It has been well reported that AHL-dependent QS plays a major role in the formation of a biofilm with a complex wild type architecture in many bacteria. In the present study, the extract ofT. foenum-graceum inhibited the biofilm biomass considerably (p ≤ 0.005) in a dose dependent manner without affecting the bacterial growth in both strains ofP. aeruginosa. Light and scanning electron microscopic images (Figure 2) displayed disintegrated architecture and reduced the number of microcolonies during the biofilm formation of test bacterial pathogens. Therefore, it is envisaged that treatment of bacterial pathogens with the extracts resulted in the formation of weak biofilms possibly by reducing the surface adhesion and subsequent microcolony formation. The results obtained in our study are similar to those reported with the polyphenolic extract ofRosa rugosa [30] and standardized extractof Sclerocarya birrea [31]. Factors closely associated with biofilm formation ofPseudomonas aeruginosalike swarming motility and EPS production were also evaluated in this study. Since, EPS production and swarming motility are under the control of QS, interference with QS would result in the reduced production of EPS and swarming motility. In the present study, the total amount of EPS produced and motility was reduced when bacterial pathogens (PAO1 and PAF79) were treated with sub-MICs ofT. foenum-graceum extract. Our results find support from the reports onCapparis spinosa [32],Cuminum cyminum [25] that demonstrated comparable inhibition of swarming motility and EPS production inP. aeruginosa. Therefore, it is envisaged that plant extract significantly (p ≤ 0.05) reduces EPS and swarming motility which will possibly weaken biofilm formation.A number of secreted virulence factors are responsible for host tissue destruction during initiation of the infectious process byAeromonas sp. Virulence factors like production of exoproteases, EPS production, and the formation of biofilm are known to be regulated byahyRIQS system [33]. Sub-MICs of the extract demonstrated dose dependent significant reduction in the total protease (p ≤ 0.005) and EPS production (p ≤ 0.05) inAeromonas hydrophilaWAF38. Biofilm formation was also inhibited in a concentration dependent manner by sub-MICs ofT. foenum-graceum extract. Significant inhibition of biofilm was recorded at all the tested concentrations. The data obtained in our study indicates that the oil is potentially acting on the ahyRI system impairing the production of C4-HSL. Similar significant reduction of total protease, EPS, and biofilm formation ofA. hydrophila by 0.4% (v/v) of clove oil has been reported by Husain et al. [20].The addition of extract ofT. foenum-graceum decreased β-galactosidase luminescence inE. coli MG4/pKDT17 of this range 18.8–52.9% (p ≤ 0.05) at the tested sub-MICs. The results of the assay demonstrate that the reduced production of AHL under the effect of sub-MICs of plant extract inhibitslas-controlled transcription. The findings of the assay are in agreement with the above observations as reduced β-galactosidase activity is indicative of reduced AHL levels and therefore reduced expression oflasBgene. Similar observations with other plant extracts were reported by Adonizio et al. [24] in south Florida plants, Singh et al. [34] inLagerstroemia speciosafruit extract, and Zhou et al. [15] in eugenol.Further, the protective role of plant extract was assessed inC. elegans. Methanol extract ofT. foenum-graceumdisplayed an enhanced survival of 48% suggesting that the addition of the extract is affecting the production of cyanide either throughhcndirectly or indirectly via the QS genes. Aqueous extracts of three south Florida plants:Conocarpus erectus,Callistemon viminalis, andBucida buceras prevented mortality via gut infection in almost 60% of the worms and reduced death from toxins by 50–90% [35].GC-MS and HPTLC analysis of extract showed the presence of caffeine as one of the major compounds. Studies with caffeine showed significant reduction in AHL regulated production of violacein in CVO26 and QS controlled virulence factors and biofilm formation inP. aeruginosa PAO1. Our observations find support from the results demonstrated by Norizan et al. [36]. They showed that caffeine inhibited swarming and AHL production ofP.aeruginosa PA01 and confirmed that caffeine did not degrade AHLs but rather inhibited their production.Methanol fraction of theTrigonella foenum-graceum (seed) extract demonstrated broad spectrum interference of QS regulated functions and inhibited biofilm formation in the gram negative pathogens.In vitroattenuation of virulence factors correlated well with thein vivostudy. Further, caffeine and caffeine related compounds need to be investigated for their possible mechanism of QS interference.



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*Source: 879540-2015-04-27.xml*