Synthesis and quorum sensing inhibitory activity of key phenolic compounds of ginger and their derivatives

Food Chemistry 159 (2014) 451–457

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Synthesis and quorum sensing inhibitory activity of key phenolic compounds of ginger and their derivatives N. Vijendra Kumar, Pushpa S. Murthy, J.R. Manjunatha, B.K. Bettadaiah ⇑ Plantation Products, Spices and Flavour Technology Department, CSIR-Central Food Technological Research Institute, Mysore 570 020, India

a r t i c l e

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Article history: Received 30 October 2013 Received in revised form 6 March 2014 Accepted 8 March 2014 Available online 20 March 2014 Keywords: Quorum sensing inhibitory activity [6]-Gingerol [6]-Shogaol Zingerone [6]-Azashogaol and isoxazoline derivative

a b s t r a c t Phenolic components of ginger (Zingiber officinale Roscoe) viz. [6]-gingerol, [6]-shogaol and zingerone exhibited quorum sensing inhibitory activity (QSI) against Chromobacterium violaceum and Pseudomonas aeruginosa. The inhibitory activity of all the compounds was studied by zone inhibition, pyocyanin, and violacein assay. All the compounds displayed good inhibition at 500 ppm. [6]-Azashogaol, a new derivative of [6]-shogaol has been synthesized by Beckmann rearrangement of its oxime in the presence of ZnCl2. The structure elucidation of this new derivative was carried out by 1D (1H NMR and 13C NMR) and 2D-NMR (COSY, HSQC and NOESY) spectral studies. This compound showed good QSI activity against P. aeruginosa. An isoxazoline derivative of [6]-gingerol was prepared and it exhibited good QSI activity. Present study illustrated that, the phenolic compounds of ginger and their derivatives form a class of compounds with promising QSI activity. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The ginger rhizome (Zingiber officinale Roscoe), belonging to Zingiberaceae family, is consumed throughout the world as a spice. It is a common ingredient in foods & beverages and is valued for its pungency. It is used in food preparations both in fresh and dry forms (Zachariah, 2008, chap. 5). [6]-Gingerol is the active pungent principle of fresh ginger whereas [6]-shogaol, the dehydrated form of [6]-gingerol, is the pungent principle of dry ginger. Ginger is also valued as a medicinal herb for its bio-active attributes in the treatment of ailments like catarrh, rheumatism, nervous diseases, gingivitis, toothache, asthma, stroke, constipation, and diabetes (Rahath & Rao, 2012; Tapsell et al., 2006). The health benefits of ginger are mainly due to the presence of key phenolic compounds such as [6], [8], [10]-gingerols, [6], [8], [10]-shogaols, zingerone, paradols etc. (Ali, Bluden, Tanira, & Nemmar, 2008). [6]-Azagingerol, a derivative of [6]-gingerol, is protective against development of metabolic syndrome in mice fed a high fat diet (Okamoto et al., 2011). The phenomenon of quorum sensing or cell-to-cell communication relies on the principle that when a single bacterium releases autoinducers (AI’s) into the environment, their concentration is too low to be detected. However, when sufficient bacteria are pres⇑ Corresponding author. Address: Department of Plantation Products, Spices and Flavour Technology, Central Food Technological Research Institute, Cheluvamba Mansion, Mysore 570 020, India. Tel.: +91 821 2512352; fax: +91 821 2517233. E-mail address: [email protected] (B.K. Bettadaiah). http://dx.doi.org/10.1016/j.foodchem.2014.03.039 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

ent, AI concentrations reach a threshold level that allows the bacteria to sense a critical cell mass and to activate target genes (Vasil, 2003). In Gram negative bacteria AI’s are N-acyl homoserine lactones (AHL’s), which interact with cellular receptors and triggers the expression of genes including virulence, bio-luminescence, bio-film formation, mobility and swarming, a process called quorum sensing (Manefield et al., 2002). Quorum sensing is employed by a diverse group of bacteria including those commonly associated with food. It results in pathogenesis, cellular dissemination or dispersal, DNA transfer, metabolism and microbial biofilm development (Thiba, Wai-Fong, & Kok-Gan, 2012). Thus, food spoilage and biofilm formation by food-related bacteria is significant problem in food industry (Houdt & Michiels, 2010; Jamuna & Ravishankar, 2011). The molecules, natural and synthetic, capable of quenching QS have been an intense focus recently in combating bacterial pathogenesis (Kalia, 2013). We investigated the QSI activity of phenolic components of ginger and their derivatives as QSI is an important in the area of food processing environment. Long alkyl chain containing structural motifs resembling N-acyl-homoserine lactone are known to inhibit the LasR-dependent gene expression, which is important in quorum sensing activity, and molecules having such long chains are believed to be scaffolds for identification of new quorum sensing modulators. One such compound containing amide spacer between a phenyl ring and an alkyl chain having 12-carbons binds to LasR with IC50 at 10 lmols (Müh et al., 2006). QS inhibitory activity of ginger was identified for ginger conserve but, no active

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constituent was tested (Vattem, Mihalik, Crixell, & McLean, 2007). Since phenolic constituents of ginger posses the structural requirement of long alkyl side chain for QS inhibitory activity, we have selected [6]-gingerol and [6]-shogaol for screening studies. Zingerone, one of the most important bioactive components of ginger has also been selected for its QS inhibitory activity. Also, in this study two compounds viz., [6]-azashogaol and isoxazoline derivative of [6]-gingerol were synthesized and their QSI activity tested. The structure of compounds used in the present study is provided in Fig. 1. 2. Materials and methods

(q, 2H, J = 7.16 Hz, H-5), 2.90–2.84 (m, 4H, H-9 & H-10), 3.89 (s, 3 H, H-17), 6.10 (dt, 1 H, J = 15.81, 1.41 Hz, H-6), 6.69 (dd, 1 H , J = 8.04, 1.75 Hz, H-16), 6.72 (d, 1 H, J = 1.77 Hz, H-12), 6.86–6.80 (m, 2 H, H-15 & H-6). 13C-NMR (125 MHz, CDCl3): d = 199.54 (C8), 147.57 (C-6) , 146.13 (C-13) , 143.61 (C-14), 132.90 (C-11) , 130.00 (C-7), 120.48 (C-16), 114.04 (C-15), 110.85 (C-12), 55.55 (C-17), 41.65 (C-9), 32.12 (C-5), 31.01 (C-3), 29.57 (C-10), 27.45 (C-4), 22.09 (C-2), 13.61 (C-1). HRMS: Mass (ESI): [M++Na] for C17H25O3Na, Calculated: 299.1622; Found: 299.1682.

2.3. Synthesis of [6]-shogaol oxime (5)

2.1. Materials and equipment All the solvents and reagents used for the synthesis were of analytical grade. Zinc chloride, DIMCARB, and hydroxylamine hydrochloride were procured from Sigma Chemical Co. (St. Louis, MO, USA). NMR spectra (1H and 13C) for the compounds and intermediates were recorded on a 500 MHz NMR spectrometer (Bruker Avance, Reinstetten, Germany) using CDCl3 as solvent. The chemical shift values (ppm) and coupling constants (J) are given in d and Hz respectively. Mass spectral analyses were carried out in the ESI positive mode using HRMS mass spectrometer (Waters Q-Tof Ultima, Manchester, UK). OD of samples was measured using UV/Vis spectrophotometer, UV-1800 Shimadzu, Tokyo, Japan. Thin-layer chromatographic (TLC) analyses were performed on silica gel 60 F254 (Merck, Germany) coated on alumina sheet by eluting with 2–10% ethyl acetate in n-hexane. The crude products were purified by column chromatography on silica gel (200–400 mesh) with a mixture of ethyl acetate and petroleum ether (60–80 °C) as eluting medium. All the chemicals and petri-plates used for QS inhibitory studies were procured from Hi Media Ltd., Mumbai, India. 2.2. Synthesis of [6]-shogaol (4) To a magnetically stirred solution of zingerone (0.5 g, 2.57 mmol) and dimethylammonium dimethyl carbamate (DIMCARB, 0.52 g, 3.86 mmol), hexanal (0.51 g, 5.15 mmol) was added drop wise at 52 °C over a period of 1 h. The progress of the reaction was monitored by TLC using 20% ethyl acetate in hexane. After completion of reaction (5 h), it was acidified with 10% aqueous HCl and extracted with CH2Cl2 (10 mL  3). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The crude product thus obtained was purified by column chromatography over silica gel (200–400 mesh). Pure [6]-shogaol was obtained in 70% yield (0.46 g). 1 H-NMR (500 MHz, CDCl3); d = 0.91 (t, 3 H, J = 6.9 Hz, H-1), 1.29–1.35 (m, 4 H, H-2 & H-3), 1.46 (p, 2H, J = 7.35 Hz, H-4), 2.21

O

To a solution of [6]-shogaol (0.5 g, 1.71 mmol) in methanol (3 mL), hydroxylamine hydrochloride (0.24 g, 3.43 mmol) was added at room temperature. The mixture was magnetically stirred, and progress of the reaction was monitored by TLC using 20% ethyl acetate in hexane. After completion of reaction (12 h), the mixture was filtered and the residue was washed with methanol. The filtrate was concentrated under reduced pressure to afford a syrupy mass and it was dissolved in dichloromethane (10 mL). The organic layer was washed with water and dried over anhydrous Na2SO4. The resultant clear solution was concentrated to afford material (0.5 g, 95%). 1 H-NMR (500 MHz, CDCl3): d = 6.86 (d, 1H, J = 8.47 Hz, H-15), 6.75–6.78 (m, 2H, H-12 & 16), 6.10–5.99 (m, 2H, H-6 & H-7), 3.90 (s, 3H, H17), 2.79 (s, 4H, H-9 & H-10), 2.16 (q, 2H, J = 6.8 Hz, H5), 1.45–1.38 (m, 2H, H-4), 1.35–1.28 (m, 4H, H-2 & H-3), 0.91 (t, 3H, J = 7.05, H-1). 13C-NMR (125 MHz, CDCl3): d = 159.15 (C-8), 146.10 (C-13), 143.66 (C-14), 133.29 (C-11), 136.39 (C-6), 125.90 (C-7), 114.05 (C-15), 110.85 (C-12), 55.66(C-17), 32.56 (C-5), 31.86 (C-9), 31.04 (C-3), 28.33 (C-4), 26.36 (C-10), 22.16 (C-2), 13.68 (C-1). HRMS: Mass (ESI): [M++1] for C17H25NO3, Calculated: 292.1834; Found: 292.2115.

2.4. Synthesis of [6]-azashogaol (6) [6]-shogaol oxime (0.2 g, 0.68 mmol) was dissolved in dry acetonitrile (5 ml). To this solution was added anhydrous ZnCl2 (0.05 g, 0.34 mmol). The reaction mixture was stirred at room temperature under an inert atmosphere. The progress of the reaction was monitored by TLC using ethyl acetate in hexane (20:80). After completion of reaction (24 h), solvent was evaporated, and the residue was taken in dichloromethane (20 mL). The organic extract was washed with water (20 ml  3) followed by brine (5 mL) and then dried over anhydrous sodium sulphate. The crude product obtained was purified by column chromatography over silica gel

O

O

O

O

O

HO

HO

HO

Zingerone (3)

[6]-Gingerol

[6]-Shogaol (4) O

O

N N H

OH

O

O HO

HO [6]-Azashogaol (6)

Isoxazoline derivative of [6]-gingerol

Fig. 1. Structure of phenolic components of ginger and their derivatives.

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(200–400) by using a mixture of ethyl acetate and petroleum ether to afford [6]-azashogaol (0.16 g, 80%). 1 H-NMR (500 MHz, CDCl3): d = 6.98 (d, 1H, J = 9.85 Hz, NH), 6.84 (d, 1H, J = 8 Hz, H-15), 6.77–6.67 (m, 3H, H-7, H-12 & H-16), 5.63 (bs, 1H, –OH), 5.07 (p, 1H, J = 7.21 Hz, H-6), 3.86 (s, 3H, H-17), 2.91 (t, 2H, J = 7.53 Hz, H-10), 2.47 (t, 2H, J = 7.56 Hz, H-9), 1.99 (q, 2H, J = 6.9 Hz, H-5), 1.38–1.33 (m, 2H, H-4), 1.30–1.26 (m, 4 H, H-2 & H-3), 0.89 (t, 3H, J = 6.9 Hz, H-1). 13C-NMR (125 MHz, CDCl3): d = 168.94 (C-8), 146.21 (C-13), 143.80 (C-14), 132.30 (C11), 121.92 (C-7), 120.48 (C-16), 114.10 (C-15), 113.10 (C-6), 110.80 (C-12), 55.58 (C-17), 38.53 (C-9), 30.95 (C-10), 29.36 (C4), 29.26 (C-5), 29.18 (C-3), 22.14 (C-2), 13.68 (C-1). HRMS: Mass (ESI): [M++1] for C17H25 NO3, Calculated: 292.1834; Found: 292.2254.

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2.8. Extraction of violacein from broth cultures of C. violaceum Violacein pigment extraction was carried out according to the method of Adonizio, Downum, Bennett, and Mathee (2006). Synthesized compounds (100 lL, 500 ppm) were mixed in LB media (5 ml) along with C. violaceum culture (OD600 = 0.1) and incubated overnight at 37 °C in shaking condition (150 rpm). Tubes were then vortexed, and 200 lL of culture was taken into 1.5 ml Eppendorf tube. The cells were lysed by adding 200 lL of 10% sodium dodecyl sulfate and mixed thoroughly using a vortex mixer and incubated at room temperature for 5 min. The violacein was quantitatively extracted from the cell lysate by adding 900 lL of water saturated butanol, vortexing for 5-s and centrifuging at 13,000 rpm for 5 min. The butanol phase containing the violacein was collected, and its absorbance was measured at a wavelength of 585 nm.

2.5. Strains and culture conditions Pseudomonas aeruginosa MTCC 2297 and Chromobacterium violaceum MTCC 2656 were procured from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India. The stock cultures were maintained at 20 °C in LB broth supplemented with cryoprotectant.

2.6. Screening of QSI activity The quorum sensing inhibitory activity of the synthesized compounds was assayed by the agar-well diffusion method (Zahin et al., 2010). The bacterial cultures C. violaceum and P. aeruginosa were grown in Luria Bertani broth (Hi-media, India) and incubated for 16–18 h in an orbital incubator (Labtech, Korea) at 150 rpm, 37 °C temperature. The LB agar plates were prepared and inoculated with C. violaceum and P. aeruginosa (105 CFU/ml). The Cups/ wells (10 mm diameter) were bored using sterile borer. The compounds were dissolved in sterile DMSO and a volume of 100 ll of 500 ppm of each compound was dispensed into the wells made in triplicate against C. violaceum (105 CFU/ml) inoculated LB agar plates. The plates were incubated at 37 °C for 24–48 h, and then the results were recorded. The QS inhibition was measured for the zone of turbid halo pigment-less bacterial inhibition. Quorum sensing inhibition was calculated using the equation (r2–r1) in mm; where r2 is the total growth-inhibition zone radius and r1 is the clear zone radius (Saraf, Quereshi, Sharma, & Khan, 2011). Further, determination of the Minimum Inhibitory Concentration (MIC) of the phenolic compounds was carried out against the test bacteria by broth micro-dilution test as described by Metzler et al. (2004). The MIC value was recorded as the lowest concentration of the extract that totally inhibited the growth of the organism. Tetracycline (30 lg/ml) was used as a positive control with sterile DMSO as a negative control. All the quorum sensing assays were performed at concentrations lower than the MIC values known as sub-inhibitory concentration.

2.7. Pyocyanin assay Pyocyanin was extracted from overnight P. aeruginosa culture supernatant. The 100 lL compounds of 500 ppm concentration were dispensed in 5 ml LB broth along with fresh P. aeruginosa culture (OD600 = 0.1) and incubated overnight at 37 °C. Chloroform (3 mL) was added to the P. aeruginosa culture supernatant and mixed vigorously. The chloroform layer was mixed with 1 mL of HCl (0.2 M) and centrifuged (10 min, 28 °C, 8000 rpm). The HCl layer was separated, and the absorbance was measured at 520 nm against 0.2 M HCl using UV/Vis spectrophotometer (Antunes, Ferreira, Buckner, & Finlay, 2010).

3. Results and discussion 3.1. Synthesis of phenolic compounds of ginger and their derivatives The synthetic scheme for key phenolic compounds viz., zingerone (3), Shogaol (4), Shogaol oxime (5) and [6]-azashogaol (6) is presented in Scheme 1. The hydrogenation of dehydrozingerone (2) afforded zingerone in excellent yield. It was carried out using ethanol as a solvent and in the presence of 5% (w/w) Pd/BaSO4 catalyst (10%). Zingerone was isolated and purified by column chromatography and used in the screening studies. Dehydrozingerone was obtained in quantitative yield by condensation of vanillin with acetone in the presence of sodium hydroxide. [6]-Shogaol was prepared by condensing zingerone with hexanal using DIMCARB (Mase, Kitagawa, & Takabe, 2010). The synthetic protocol reported was simplified in respect of addition of 1-hexanal reagent as it is very long using a syringe pump (10 h at rt). We have found, alternatively, a good yield of product (70%) even with addition was carried out using pressure equalisation addition funnel (1 h at 60 °C). Also, we have observed completion of reaction was fast (5 h against 48 h). The [6]-shogaol thus obtained was confirmed by the 1H, 13C and attached proton test (SEFT) experiments. The proton and carbon correlations were confirmed by 2D HSQC. The synthesis of shogaol oxime was visualised to insert amide linker between aryl group and alkyl side chain by Beckmann rearrangement reaction (Santosh Kumar, Srinivas, Negi, & Bettadaiah, 2013). It was prepared in excellent yield (95%) using hydroxyl amine hydrochloride. The formation of oxime from [6]-shogaol is apparent from the change in the chemical shift of carbonyl carbon (199.5 ppm) to oxime carbon (159.1 ppm). Also, the oxime carbon gave HMBC correlations to four methylene protons at 2.79 ppm and two olefinic protons. The shogaol oxime was obtained as a mixture of syn- and anti-forms in 2:3 ratios by yield of isolated product. The syn-refers to the presence of hydroxyl attached to nitrogen and the double bond on the same side of carbon–nitrogen double bond whereas anti-refers to the same on the opposite side. The pure anti-oxime gave positive Beckmann rearrangement reaction in the presence of anhydrous ZnCl2 as Lewis acid catalyst and afforded a product which we named [6]-azashogaol. The formation of this product is due to the highly stereo-specific nature of Beckmann rearrangement reaction (Prakash, Mathew, & Olah, 2012). The syn-oxime did not react under similar conditions to afford the amide compound having carbonyl group in between –NH– and double bond. The [6]-azashogaol was confirmed to be having structure as showed in Fig. 1. It was confirmed from the 2D-NMR spectral studies (Fig. 2). The presence of –NH– moiety in the product was detected by the presence of doublet at 6.98 ppm in PMR spectrum and its connectivity to none of the carbons in HSQC spectrum. The NOE studies revelled

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O

O O

a

H

O

O

O

b

HO

HO

c

HO

1

3

2

N OH

O O

O

d

e HO

HO

5

4 O O

N H

HO 6 Scheme 1. Synthesis of zingerone (3) and [6]-azashogaol (6). Reagents and conditions: (a) Acetone, NaOH, rt, 12 h. (b) H2Pd-C, EtOH. (c) DIMCARB, hexanal, 50 °C, 5 h. (d) NH2OH
HCl, MeOH, rt, o/n. (e) ZnCl2, CH3CN, rt, 2 days.

ppm [6]-azashogaol- NOE

ppm

1.8

a

b

1.9

5.7 5.6

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5.4 2.2

O O

5.3 N H

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HO

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5.0 2.6

4.9

O

2.7

O

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N H

4.8 4.7

2.9

4.6 3.0

4.5 3.1

4.4 7.1

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6.9

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ppm

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7.5

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ppm 125

c

d

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167 135 140

168 145 O O

150

N H HO

169

155 160

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O 165

O

170

HO

N H

171

175 3.2

3.0

2.8

2.6

2.4

ppm

7.1

7.0

6.9

Fig. 2. NOE (a & b) and HMBC (c & d) spectra of [6]-azashogaol.

6.8

6.7

6.6

ppm

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N. Vijendra Kumar et al. / Food Chemistry 159 (2014) 451–457 Table 1 QSI activity phenolic compounds. Phenolic compounds

[6]-Gingerol [6]-Shogaol [6]-Azashogaol Isoxazoline derivative Zingerone a

QSI zone C. violaceuma (mm)

QSI zone P. aeruginosaa (mm)

100 ppm

250 ppm

500 ppm

100 ppm

250 ppm

500 ppm

13 ± 0.1 15 ± 0.1 13 ± 0.1 13 ± 0.2 14 ± 0.1

15 ± 0.1 17 ± 0.1 14 ± 0.2 16 ± 0.1 15 ± 0.1

18 ± 0.1 20 ± 0.1 17 ± 0.1 19 ± 0.2 15 ± 0.2

13 ± 0.1 14 ± 0.1 14 ± 0.1 14 ± 0.2 12 ± 0.1

15 ± 0.1 15 ± 0.2 15 ± 0.1 15 ± 0.1 13 ± 0.1

17 ± 0.2 17 ± 0.1 17 ± 0.1 16 ± 0.2 13 ± 0.1

Mean of three readings (r2–r1).

the correlation of –NH– proton with methylene proton at 2.47 ppm (Fig. 2a), this confirms the amide insertion in the alkyl side chain. The –NH– also presents a strong NOE with olefinic proton at 5.07 ppm (Fig. 2b). The HMBC correlations are seen for methylene protons at 2.91 & 2.47 ppm from carbonyl carbon (C-8) and aromatic quaternary carbon at C-11 (Fig. 2c), this confirms the presence of two methylenes between aromatic ring and carbonyl group. The HMBC correlation of carbonyl carbon was also seen for the –NH– proton at 6.98 ppm and one of the olefinic protons (Fig. 2d). The complete 2D-NMR spectra and numbering of carbons is provided as a Supplementary data. The synthesis of [6]-gingerol and its isoxazoline was executed starting from eugenol, a key constituent of clove oil by our method (Vijendra Kumar, Srinivas, & Bettadaiah, 2012). QS Inhibitory activity of pure [6]-gingerol was taken up as the studies reported are mainly for the ginger extracts (Chan et al., 2011). The isoxazolines, key structural feature of many bioactive compounds, exhibited a range of bioactive properties (Sutharchanadevi & Murugan, 1996). To our knowledge their QS inhibitory activity is not reported, hence it interested us to take this study where isoxazoline is a linker between alkyl chain and phenolic moiety.

3.2. QSI activity of phenolic compounds of ginger and their derivatives Five phenolic compounds at the concentration of 100, 250 and 500 ppm exerted inhibitory activity in the agar-well diffusion assay (Table 1). [6]-Shogaol showed highest inhibitory activity with C. violaceum with 20 mm halo inhibitory zone followed by isoxazoline derivative and [6]-gingerol. [6]-Azashogaol showed 17 mm halo inhibitory zone against both C. violaceum and P. aeruginosa while zingerone exhibited lowest halo inhibitory zones of 15 and 13 mm. The halos produced on lawns of the bio-monitor strains could be the result of either inhibition of cell growth or quenching of QS signals. After the initial screening using the qualitative agar diffusion assay, minimum inhibitory concentration (MIC) was carried out (Fig. 3). [6]-Azashogaol was the most effective and exhibited low MIC against both C. violaceum and P. aeruginosa. All the compounds exhibited same MIC at 750 ppm against P. aeruginosa. All the compounds except [6]-azashogaol showed MIC at 1000 ppm against C. violaceum. Zingerone was least effective (low molecular weight and higher values for concentrations if expressed in mmoles) against C. violaceum and P. aeruginosa and it is attributed to lack of long alkyl

Fig. 3. Minimum inhibitory concentration of phenolic compounds and derivatives against (a) C. violaceum and (b) P. aeruginosa. (Values followed by same letters for each concentration is not significantly different (p < 0.05).

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0.30

Pyocyanin Violacein

bition (35%). These observations suggest that phenolic derivatives from ginger can inhibit quorum sensing in pathogenic bacteria.

0.35

4. Conclusion

0.18 0.30 0.12 585

0.25

Violacein (OD)

Pyocyanin (OD 520)

0.24

0.40

0.06 0.20 0.00 Control

[6]-Aza shogaol

6-Isoxazoline Derivative

[6]-Shogaol [6]-Gingerol Zingerone

Phenolic compounds (500 ppm) Fig. 4. Inhibition of pyocyanin and violacein by phenolic compounds of ginger and derivatives.

side chain in zingerone compared to other phenolic compounds tested in the present study. The role of length of alkyl side chain in the phenolic compounds of ginger towards various activities is expected to be very interesting and outline the scope for detailed study. The criteria for QSI molecules with low molecular weight and high specificity are very useful. The molecules like [6]-gingerol, [6]-shogaol, zingerone, [6]-azashogaol, and isoxazoline derivative of gingerol have low molecular weight and posses a long alkyl side chain similar to N-acylhomoserine lactones. Hence, mechanistically they function as similarly to other QSI molecules (Kalia, Wood, & Kumar, 2013).

3.3. QSI activity of phenolic compounds of ginger and their derivatives by pyocyanin assay Quorum sensing is a cell density dependent expression of species in bacteria mediated by hormone like compounds called autoinducers (AI). P. aeruginosa produces a blue–green phenazine pigment called pyocyanin (Blosser & Gray, 2000) a secondary metabolite whose expression is under the control of QS. To explore anti-QS potential of the phenolic derivatives, assays were done on inhibition of pyocyanin produced by P. aeruginosa. [6]-Azashogoal showed 90% reduction in pyocyanin formation (Fig. 4). A significant decrease in pyocyanin production of P. aeruginosa to the level of 83–90% was observed after treatment with phenolic compounds and their derivatives. [6]-Gingerol showed a comparative inhibition with [6]-azashogaol and [6]-shogaol. The assay indicated that the presence of –NH– in the side chain has some positive effect on the inhibition of pyocyanin production. Thus amide linker between the long alkyl side chain and phenolic moiety plays a significant role in the activity profile of tested compounds. Hence, the structure activity relationship of these compounds for QSI activity is influenced by the presence of amide linker in the phenolic compounds.

3.4. QSI activity of phenolic compounds of ginger and their derivatives by extraction of violacein C. violaceum is a Gram-negative bacterium that produces the purple pigment violacein. The phenolic constituents of ginger were found to inhibit violacein production in C. violaceum (Fig. 4). Loss of purple pigmentation in C. violaceum in the presence of phenolic constituents of ginger is indicated as QS inhibition. Fifty percent antagonistic activity was observed with samples (500 ppm) treated with [6]-shogaol, [6]-gingerol while zingerone showed lesser inhi-

We have demonstrated the quorum sensing inhibitory activity of key phenolic constituents of ginger and some of their derivatives. Since quorum sensing plays a major role in food spoilage, biofilm formation, and food-related pathogenesis, the present investigation helps in realising the potential of phenolic compounds of ginger as possible QS inhibitors. Isoxazoline derivative of [6]-gingerol exhibited good QSI activity. The [6]-azashogaol, a new derivative of [6]-shogaol, was exhibited better QSI activity than all other compounds screened against C. violaceum. [6]-Gingerol exhibited best QSI activity against P. aeruginosa. Important structure–activity requirement for the QSI activity of phenolic compounds related to the presence of hetero linkage between long alkyl chain and phenolic moiety has been observed. The lack of long alkyl side chain in the phenolic derivatives resulted in decreased activity while its presence in the compounds influenced the activity positively. The activity of phenolic compounds of ginger is further increased with the introduction of amide linker between long alkyl side chain and phenolic moiety while heterocyclic ring linker (isoxazoline) did not influence the activity profile much. Acknowledgements Author N.V.K. is thankful to UGC, New Delhi for the award of Senior Research Fellowship. Dr. P. Srinivas, Chief Scientist, CFTRI is gratefully acknowledged for his valuable suggestions. Help from Mr. Padmere Mukund Laxman for HRMS is gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014. 03.039. References Adonizio, A. L., Downum, K., Bennett, B. C., & Mathee, K. (2006). Anti-quorum sensing activity of medicinal plants in southern Florida. Journal of Ethnopharmacology, 105, 427–435. Ali, B. H., Bluden, G., Tanira, M. O., & Nemmar, A. (2008). Some phytochemical, pharmacological and toxicological properties of ginger (zingeber officinale Roscoe): A review of recent research. Food and Chemical Toxicology, 46, 409–420. Antunes, L. C., Ferreira, R. B., Buckner, M. M., & Finlay, B. B. (2010). Quorum sensing in bacterial virulence. Microbiology, 156, 2271–2282. Blosser, S. R., & Gray, M. K. (2000). Extraction of violacein from C. violaceum provides a quantitative bio-assay for N-acyl homoserine lactone autoinducers. Journal of Microbiological Methods, 40, 47–55. Chan, K., Atkinson, S., Mathee, K., Sam, C., Chhabra, S. R., & Cámara, M. (2011). Characterization of N-acylhomoserine lactonedegrading bacteria associated with the Zingiber officinale (ginger) rhizosphere: Coexistence of quorum quenching and quorum sensing in Acinetobacter and Burkholderia. BMC Microbiology, 11(51), 1–13. Houdt, R. V., & Michiels, C. W. (2010). Biofilm formation and the food industry, a focus on the bacterial outer surface. Journal of Applied Microbiology, 109, 1117–1131. Jamuna, B. A., & Ravishankar, R. V. (2011). Bacterial quorum sensing in food industry. Comprehensive Reviews in Food Science and Safety, 10, 184–194. Kalia, V. C. (2013). Quorum sensing inhibitors; an overview. Biotechnology Advances, 31, 224–245. Kalia, V. C., Wood, T. K., & Kumar, P. (2013). Evolution of resistance to quorumsensing inhibitors. Microbial Ecology. http://dx.doi.org/10.1007/s00248-0130316-y. Manefield, M., Rasmussen, T. B., Henzter, M., Steinberg, P., Kielleberg, S., & Givsko, M. (2002). Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover. Microbiology, 148, 1119–1127.

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