Piper nigrum, Piper betle and Gnetum gnemon- Natural Food Sources with Anti-Quorum Sensing Properties

Various parts of Piper nigrum, Piper betle and Gnetum gnemon are used as food sources by Malaysians. The purpose of this study is to examine the anti-quorum sensing (anti-QS) properties of P. nigrum, P. betle and G. gnemon extracts. The hexane, chloroform and methanol extracts of these plants were assessed in bioassays involving Pseudomonas aeruginosa PA01, Escherichia coli [pSB401], E. coli [pSB1075] and Chromobacterium violaceum CV026. It was found that the extracts of these three plants have anti-QS ability. Interestingly, the hexane, chloroform and methanol extracts from P. betle showed the most potent anti-QS activity as judged by the bioassays. Since there is a variety of plants that serve as food sources in Malaysia that have yet to be tested for anti-QS activity, future work should focus on identification of these plants and isolation of the anti-QS compounds.


Plant Sample Identification, Deposition of Voucher Specimens and Preparation of Plant Extracts
Plant samples were purchased from a local market in Selangor, Malaysia. Voucher specimens of P. nigrum (047695), P. betle (047696) and G. gnemon (047698) have been deposited in the Herbarium of University of Malaya. Samples were washed with sterile distilled water and finally rinse with 70% (v/v) ethanol before drying in the oven at 45 °C for three days. Dried samples were grounded to fine powder and soaked sequentially in hexane, chloroform and methanol. The extracts were then filtered through Whatman No. 1 filter paper. Removal of solvents from filtrate was done using a rotary evaporator (EYELA, Tokyo, Japan). Plant extract was dissolved in 100% DMSO (v/v) and were diluted using ultrapure water prior to be used.

QS Inhibition against C. violaceum CV026
Briefly, 15 mL of overnight C. violaceum CV026 culture was added to 200 mL of molten LB agar that has been supplemented with C6-HSL(0.25 µg/mL). The C. violaceum CV026 agar suspension was poured into Petri dishes. Wells were made using sterile pipette tips once the agar solidified. Plant extract was placed in each well and DMSO (50% v/v) served as the negative control. The Petri dishes were incubated at 28 °C for 24 h. Halo formation on a purple background suggested that the plant extracts exhibited anti-QS. The violacein formed was quantified by incubating C. violaceum CV026 (supplemented with C6-HSL, 0.125 µg/mL) with plant crude extract in 96-well plate. The plate was incubated at 28 °C and after 16 h, the 96-well plate was completely dried at 60 °C. Then, DMSO (100 µL) was added onto each well and the 96-well plate was placed in the lab shaker [29]. The reading of the solubilized violacein was taken using a DYNEX MRX Elisa reader (Chantilly, VA, USA) at 590 nm.  (20 µL) were added into the well of a 96-well microtitre plate. The bioluminescence and OD 495nm were determined every 30 min for 24 h using a Tecan luminometer (Infinite M200, Mannerdorf, Switzerland). Expression of bioluminescence was given as relative light unit (RLU)/OD 495nm against time [30].

Bacterial Growth
Bacterial growth was measured using methods by Hayouni and colleagues with modifications. Briefly, overnight cultures of P. aeruginosa PA01, C. violaceum CVO26, E. coli [pSB 401] and E. coli [pSB 1075] were diluted to OD 600nm 0.001. Then, the bacteria and plant extracts were placed in a 96-well microtitre plate to make up a final volume of 250 µL in each well. The optical density OD 600nm were determined every 30 min for 24 h by a Tecan luminometer (Infinite M200). Bacteria growth was determined by plotting the OD 600nm against time [32].

Statistical Analysis
The significance differences between the mean values were tested using ANOVA test (P < 0.05) using GraphPad Prism software. All the assays were performed in triplicate.

Results and Discussion
P. nigrum, P. betle and G. gnemon used in this study had been used as food sources in the Southeast Asia region for centuries. These plants serve not only as foods, but they are also used as medicinal plants, although little is known about the anti-QS properties of these plants. This study provides a new insight on the anti-QS capabilities of these plant samples. Table 1 shows the summary of the results obtained from this study. Violacein is a purple pigment produced by C. violaceum. It is a strong antioxidant and acts by protecting the bacteria membrane against oxidative stress [33]. Three extracts of P. nigrum and two extracts of P. betle (hexane and chloroform) causes halo formation on the purple background, indicating that these extracts have anti-QS properties against C. violaceum CV026. The formation of the halo zone around the well can be observed clearly in Figure 1. Plants extracts have been shown to exhibit anti-QS activity against C. violaceum CV026. For instance, Tremella fuciformis, vanilla, peas, black olive, bottle brush and graceful sandmat have shown their ability in inhibiting violacein production in previous study [20,[34][35][36]   All three extracts of P. betle showed significant inhibition against pyocyanin formation by P. aeruginosa PA01. Quantified pyocyanin from hexane and chloroform extracts of P. betle ( Figure 2) decreases with increasing plant extract concentration. Pyocyanin is a secondary metabolite produced by P. aeruginosa PA01. Pyocyanin can be found in large quantities in the sputum of cystic fibrosis patients and it disturbs ion transports, ciliary beatings and mucus secretion in the respiratory epithelial cells [37]. Swarming usually occurs on semi-solid agar and this motion enables the cell to colonize the surrounding surfaces [38]. The swarming agar that we had optimized in this study comprised of 0.5% (w/v) agar. Swarming inhibition against P. aeruginosa PA01 can be seen in swarming agar that has been seeded with P. betle (Methanol) extracts ( Figure 3). Extend of inhibition increases as the concentration of plant extract increases. Pyocyanin formation and swarming motility are the virulent phenotypes that are regulated by QS. Compounds from P. betle were able to influence the synthesis of these virulent determinants and this makes P. betle extracts good candidate for further studies. Growth of bacteria was monitored over the course of 24 h to make sure that the inhibitions caused by the plant extracts were solely due to anti-QS and not due to inhibition of bacterial growth. All of the extracts in Figure 4 showed significant inhibition against QS and results obtained after 24 h showed that the plant extracts only have anti-QS properties and did not cause any form of inhibition in the bacterial growth. Same observation was obtained from other plant extracts when cultured together with other bacterial strains. DMSO of final concentration of 0.08%, 0.16% and 0.24% serves as the negative controls at each corresponding concentration.
Consequently, the results obtained from this study also showed that the extracts of P. betle have the most effective inhibition against QS as compared to P. nigrum and G. gnemon. We cannot be sure at which level QS has been modulated by these extracts because the extracts could be competing or disrupting the AHLs binding to the receptors by degradation of AHLs; blocking AHLs from forming AHL-receptor complex; changing the structures of the enzymes that is involved AHLs synthesis. Tertiary plants have developed some form of protective mechanism against bacterial infections through evolution. It is proven through studies which found that chestnut honey, Terminalia catappa, Chinese medicinal plants, Scorzonera sandrasica and Italian medicinal plants possess anti-QS activity [39][40][41][42][43]. Since they are food sources, the compounds derived will have lower possibility of causing any unwanted reaction in the human system. The samples used in this study can be widely found in Malaysia. Our future goal is to screen for more plants in Malaysia that possess anti-QS.

Conclusions
Usage of antibiotics has caused pathogenic bacteria to become resistant and poses a global threat to public health. QS provides an alternative solution because by targeting bacterial communication the expression of the virulence phenotype is inhibited. Our data illustrated that these plants possess compounds that can be used to quench QS-mediated virulence determinants. Currently, we are in the process of isolating these compounds using column chromatography and preparative HPLC, and molecular structures will then be determined by mass spectrometry and NMR spectroscopy.