Tannin-rich fraction from Terminalia catappa inhibits quorum sensing (QS) in Chromobacterium violaceum and the QS-controlled biofilm maturation and LasA staphylolytic activity in Pseudomonas aeruginosa

Journal of Ethnopharmacology 134 (2011) 865–871

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Tannin-rich fraction from Terminalia catappa inhibits quorum sensing (QS) in Chromobacterium violaceum and the QS-controlled biofilm maturation and LasA staphylolytic activity in Pseudomonas aeruginosa Joemar C. Taganna a,∗ , Jusal P. Quanico c,1 , Rose Marie G. Perono c , Evangeline C. Amor c , Windell L. Rivera a,b a

Laboratory of Applied Microbiology, Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City, 1101, Philippines Molecular Protozoology Laboratory, Natural Sciences Research Institute, University of the Philippines, Diliman, Quezon City, 1101, Philippines c Natural Products and Bioorganic Chemistry Laboratory, Institute of Chemistry and the Natural Science Research Institute, College of Science, University of the Philippines, Diliman, Quezon City, 1101, Philippines b

a r t i c l e

i n f o

Article history: Received 6 October 2010 Received in revised form 18 January 2011 Accepted 21 January 2011 Available online 1 February 2011 Keywords: Medicinal plants Antimicrobial activity Quorum sensing Anti-quorum sensing Anti-infective potential

a b s t r a c t Aim of the study: The study aimed to test the activity of Terminalia catappa L. against bacterial quorum sensing (QS) in order to provide a potential scientific basis for the traditional use of leaf extracts of this plant as an antiseptic. Materials and methods: The anti-QS activity of the methanolic leaf extract of Terminalia catappa was detected through the inhibition of the QS-controlled violacein pigment production in Chromobacterium violaceum. Fractions resulting from size-exclusion chromatography were assayed. The most active fraction was characterized through qualitative phytochemical detection methods. The effect of this fraction on known QS-controlled phenotypes in test strains was assessed. Results: The fraction with the highest activity (labeled as TCF12) was characterized to be tannin-rich. It specifically inhibited QS-controlled violacein production in Chromobacterium violaceum with 50% reduction achieved at 62.5 ␮g mL−1 without significantly affecting growth up to about 962 ␮g mL−1 . The assessment of its effects on LasA activity of Pseudomonas aeruginosa ATCC 10145 found that the production of this virulence determinant is reduced in a concentration dependent manner with about 50% reduction at 62.5 ␮g mL−1 . Furthermore, it was found that TCF12 was able to inhibit the maturation of biofilms of Pseudomonas aeruginosa, a phenotype that has also been known to be QS-regulated. Conclusion: Therefore, tannin-rich components of Terminalia catappa leaves are able to inhibit certain phenotypic expression of QS in the test strains used. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Bacterial populations coordinate communal behavior through a process of chemical cell-to-cell signaling mediated by diffusible signal molecules (Schauder and Bassler, 2001). Through this system, bacteria are able to control gene expression in response to population density; hence, the mechanism was called quorum sensing (QS). This system has been known to control a wide array of phenotypes in bacteria ranging from simple bacterial cell motility to complex communal behaviors such as biofilm formation

∗ Corresponding author. Current address: Koningin Maria Hendrikaplein 51, Ghent, 9000, Belgium. Tel.: +32 49 381 5679. E-mail addresses: [email protected], [email protected] (J.C. Taganna). 1 Current address: University of Bergen, Faculty of Mathematics and Natural Sciences, P.O. Box 7800, N-5020 Bergen, Norway. 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.01.028

and production of virulence factors (Gambello and Iglewski, 1991; Davies et al., 1998; Labbate et al., 2004; Atkinson et al., 2006). In the past few years, inhibition of QS has become a very intensive area of research because of its applications in medicine, industry and biotechnology. By disrupting the language of bacteria, it would be possible to repress the expression of QS-regulated phenotypes, which will have great clinical significance in relation to the treatment of bacterial infections, especially those caused by antibiotic-resistant strains (Hentzer and Givskov, 2003; March and Bentley, 2004). In the quest for these inhibitors, studies have demonstrated that many eukaryotes, particularly plants, and even bacteria themselves produce anti-QS substances (Rasmussen et al., 2005; González and Keshavan, 2006). For plants, the first one to be reported was the Australian red alga (Delisea pulchra), which was found to produce halogenated furanones that inhibit the QS system of a marine bacterium, Serratia liquefaciens (Givskov et al., 1996) and other QS systems in other Gram-negative bacteria. Several species of higher

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plants, including pea seedlings, secrete a series of unidentified chemical signals that are capable of interfering with the QS of reporter strains (Teplitski et al., 2000). In another study it was found that l-canavanine, secreted by the legume alfalfa (Medicago sativa) interferes with the QS of Sinorhizobium meliloti, a nitrogen-fixing bacterium that invades its roots (Keshavan et al., 2005). The present study documents a potential scientific basis for the ethnopharmacological use of Terminalia catappa (Terminalia catappa L.). This plant has been used in many traditional preparations as an antiseptic (Burkill, 1985) and current studies showed some basis for antibiotic, antioxidant and anti-inflammatory activities (Ko et al., 2003; Babayi et al., 2004; Chitmanat et al., 2005; Chyau et al., 2006). While its antiseptic activity can be explained by direct killing of bacterial pathogens, it could also be possible that a different anti-infective mechanism is involved, which could be that of QS inhibition. 2. Materials and methods 2.1. Bacterial strains The bacterial strains used for QS inhibition assays were Chromobacterium violaceum JCM 1249 (Japan Collection of Microorganisms, Japan) and Chromobacterium violaceum CV026 (NCTC 13278) (National Collection of Type Cultures, London, U.K.). The former is the wild type strain with a functional QS system, while the latter has a mutated CviI (LuxI homologue) gene rendered incapable of producing autoinducers. Staphylococcus aureus ATCC 33591 (American Type Culture Collection, U.S.A.) was used for the LasAstaphylolytic assay and Pseudomonas aeruginosa ATCC 10145 was used in the anti-LasA and anti-biofilm assays. All of these bacterial strains were stored in 40% glycerol stocks at −20 ◦ C and were cultured in Luria–Bertani (LB) Lennox broth (Pronadisa) prior to use. 2.2. Qualitative and quantitative screening assays for QS inhibition An overnight culture of Chromobacterium violaceum JCM 1249 in LB broth (OD660 of 1.0) and appropriate dilution (2000 ␮g mL−1 for crude methanolic extracts) of the extracts were prepared. For the plate-based qualitative screening assay, LB agar plates were flooded with one milliliter of this culture to prepare a lawn. Excess inoculum was either sucked by pipette or poured to another plate. Four 10-mm diameter holes were bored in each agar plate using a flame-sterilized glass tube connected with an aspirator at the other end. Forty (40) microliters of the extracts were placed into each hole. Distilled water and 100 ␮g mL−1 chloramphenicol (Fluka) were used as negative and growth inhibition controls, respectively. These plates were then incubated at 30 ◦ C for 24 h. Inhibition of QS in Chromobacterium violaceum is manifested as the inhibition of purple pigmentation around the holes containing the extracts (McLean et al., 2004). The quantitative screening assay was done through spectrophotometric determination of bacterial cell density at 720 nm and violacein production at 577 nm using the same pool of methanolic extracts used for the qualitative plate-based assay (Pitlovanciv et al., 2006). 2.3. Bulk methanolic extraction Leaves and bark of Terminalia catappa were obtained from mature trees around the Hardin Ng Bougainvillea, University of the Philippines, Diliman, Quezon City on January 23, 2008. A voucher specimen (Bandong 10-0215) was deposited at PUH (accession number 14606). The leaves and bark were washed in running water and air-dried. Five hundred grams of pulverized leaf and bark materials were exhaustively extracted with methanol. The solvent was

drained by filtration using Whatman No. 32 filter and the filtrate was concentrated in vacuo to obtain the methanolic extract with a total yield of 37.4%. A portion of the methanolic extract was used for qualitative assay to test for its anti-QS activity. 2.4. Extraction by solvent partitioning Solvents used were single-distilled technical solvents. Ten grams of the methanol extract was exhaustively partitioned with hexane and water (1:6 water to hexane). The hexane layer was recovered and was dried under reduced pressure to obtain the hexane fraction with a yield of 1.87%. The resulting aqueous layer was then exhaustively partitioned with ethyl acetate (1:6 water to ethyl acetate). The ethyl acetate layer was recovered and dried in vacuo obtaining a yield of 6.07%. The remaining aqueous portion was lyophilized to obtain the aqueous fraction. The hexane, ethyl acetate and aqueous extracts were assayed for anti-QS activity. 2.5. Size exclusion chromatography The ethyl acetate fraction was subjected to size-exclusion chromatography (SEC) using the SephadexTM LH-20 gel in a 100 cm length and 3 cm internal diameter column. The wet-packed column was then equilibrated with methanol (the mobile phase) overnight. Two grams of the ethyl acetate fraction, dissolved in methanol, was loaded then eluted with the mobile phase at a flow rate of 2 mL per min. Twenty-milliliter fractions were collected up to complete elution of the loaded sample. The fractions collected were spotted into thin layer chromatography (TLC) plates to monitor the profiles. Fractions with similar profiles were pooled, dried through rotary evaporation and were subsequently assayed for anti-QS activity. 2.6. Quantitative QS inhibition assay The effect of the most active fraction, denoted as Terminalia catappa fraction 12 (TCF12), on the QS-controlled production of violacein was determined using the wild type pigment-producing strain of Chromobacterium violaceum JCM 1249,while the potential toxic effects was monitored using non-pigmented Chromobacterium violaceum CV026 strain to avoid inaccuracy due to light scattering of violacein at 620 nm (Demoss and Happel, 1959). This protocol is a modification of that described by Martinelli et al. (2004). A two-fold serial dilution of the sample was prepared in nine (9) eight-well lanes of a 96-well microtiter plate (1000, 500, 250, 125, 62.5, 31.25, 15.625 and 0 ␮g mL−1 ) at 150 ␮L each using LB broth as diluent. Fifty microliters per well of an overnight culture of Chromobacterium violaceum JCM 1249 in LB broth (OD660 of 1.0) were added to three lanes. The same amount of an overnight culture of CV026 in LB (OD660 of 1.0) was added to the next three lanes. The other three lanes contained serially diluted TCF12 at the indicated concentrations dissolved in LB but without any culture added to measure the absorbance of TCF12 in LB alone for normalization purposes. The plate was then incubated at 37 ◦ C for 24 h; after which, it was read at 570 nm (for violacein) and 620 nm (for cell density) using the Tecan Spectra III microplate spectrophotometer (Austria). 2.7. Anti-biofilm assays Biofilm formation in polystyrene microtiter dishes was assayed as described previously (O’Toole and Kolter, 2002), with a few modifications. An overnight culture of Pseudomonas aeruginosa ATCC 10145 was diluted 1:100 with LB broth and grown for another hour. After the addition of different concentrations of TCF12 (same dilutions as described earlier), 100 ␮l aliquots of culture were pipetted into the wells of the microtiter dishes and incubated for 48 h at 37 ◦ C. Thereafter, the medium was removed, and 100 ␮L of a 1% (w/v) aqueous solution of crystal violet (CV) were added. Following

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140 120 100 80 60 40 20 0

B Percentage (%)

Percentage (%)

A 160

250

500

1000

Extract Concentration (µg/mL)

2000

160 140 120 100 80 60 40 20 0

250

500

867

1000

Extract Concentration (µg/mL)

2000

Fig. 1. Simple spectrophotometric screening assay using the wild type Chromobacterium violaceum to determine the effect of crude methanolic extract on both bacterial cell density (at 720 nm, represented by the dark gray columns) and violacein production (at 577 nm, represented by the light gray columns): (A) Terminalia catappa leaves and (B) Terminalia catappa bark.

staining at room temperature for 20 min, the dye was removed and the wells were washed thoroughly with sterile water. For quantification of attached cells the bound CV was solubilized in dimethyl sulfoxide (DMSO) and the absorbance was determined at 570 nm. Inhibitor-mediated reduction of biofilm formation was assessed by comparing it to those without TCF12. For the assay for biofilm susceptibility to biocidal agents, a separate set-up was done. The same process was done to grow the biofilms but after discarding the medium and washing with distilled water, the wells were washed with 1% sodium dodecyl sulfate (SDS) to disrupt the biofilms that formed in the walls. The succeeding steps for staining, washing and quantification were done as described above. 2.8. Phytochemical screening The active fraction TCF12 was screened for the presence of common classes of plant secondary metabolites based on the procedures described by Harborne (1973) and Houghton and Raman (1998), unless otherwise cited: 2.8.1. Test for terpenoids Approximately 5 mg of the sample were dissolved in methanol and spotted on silica 60G TLC plates. A positive control was also dissolved in the appropriate solvent and was spotted beside the sample spot on the same plate. The chromatogram was developed in chloroform (CHCl3 ), dried, and sprayed with vanillin-sulfuric acid (H2 SO4 ). The development of red to purple spots upon heating the sprayed chromatogram indicates the presence of terpenoids. The Salkowski test was performed to confirm the result of the previous test. For this analysis, 2 mg of the sample were mixed with 2 mL of CHCl3 and concentrated H2 SO4 was carefully added to form a layer. A reddish brown coloration of the interface formed indicates the presence of terpenoids. 2.8.2. Test for saponins Five milligrams of sample were dissolved in 5 mL distilled water in a test tube. This solution was heated to boiling temperature, allowed to cool and shaken for 2 min. The existence of a persistent froth indicates the presence of saponins. 2.8.3. Tests for tannins Tannins were detected by treating 2 mg of sample with three drops of 15% ferric chloride. Formation of a blue-black precipitate indicates the presence of hydrolyzable tannins while brown precipitates show the presence of condensed tannins. The result of this test was confirmed by treating the extract with three drops of gelatin–salt solution (prepared by equal amounts of 1% gelatin and 10% sodium chloride) and any formation of gelatinous precipitates indicates the presence of tannins. 2.8.4. Tests for flavonoids About 2 mg of extract were dissolved in diluted sodium hydroxide (NaOH) and hydrochloric acid (HCl) was added. A yellow

solution with NaOH that turns colorless upon adding HCl indicates the presence of flavonoids. This test was confirmed by a twodimensional chromatographic detection test for flavonoid, which was done as follows: a spot was made 3–4 cm from the bottom left of 6 cm × 6 cm silica plate and was developed with butanol–acetic acid–water (4:1:5), upper phase, in one dimension and 5% acetic acid on the other. The same was done for a control. The developed chromatograms were fumed with ammonia after observing and noting the colors or fluorescence under light or UV. A change in color or fluorescence of the spots after fuming with ammonia indicates the presence of flavonoids. 2.8.5. Tests for alkaloids Alkaloids were detected by treating the sample with 3 drops of Wagner’s reagent (2 g of iodine and 6 g of potassium iodide in 100 mL water), observing for the formation of a blue-black precipitate (Stephenson and Parker, 1921). The result of this test was confirmed by a chromatographic detection of alkaloids by spraying the developed chromatogram of the sample and control with Marquis reagent (1 mL formaldehyde mixed with 5 mL sulfuric acid) (Makkar et al., 2007). A change or enhancement of the color of the sample spot suggests the presence of alkaloids. 2.9. Data processing and statistical analysis All the experiments were done in at least three replicates. The raw absorbance data for the spectrophotometric assays were normalized against those containing the serially diluted TCF12 alone by simply subtracting the absorbance of the dissolved TCF12 from any absorbance reading for each concentration. The normalized absorbance data were then transformed into percentages with the untreated set as 100%. Standard deviations were computed and are shown as error bars in the figures. 3. Results and discussion An initial screening from 27 species of Philippine medicinal plants (unpublished data) provided the lead to further explore the anti-QS potential of Terminalia catappa. Through inhibition of violacein pigmentation in Chromobacterium violaceum growing on agar plates, it was found that the methanolic extracts of Terminalia catappa showed the presence of QS inhibitors. The anti-QS activity of these extracts was initially tested using the tube-based qualitative and plate-based anti-QS screening assay as described in the materials and methods. The leaf extract of Terminalia catappa (Figs. 1A and 2A) had a better activity than the bark extract (Fig. 1B and 2B) but both were shown to inhibit QS in Chromobacterium violaceum JCM 1249. Both extracts of Terminalia catappa at 2000 ␮g mL−1 exhibited observable inhibition of violacein pigmentation as opposed to complete clearing of bacterial growth around the well for the antibiotic control (100 ␮g mL−1 chloramphenicol) (Fig. 2C). Though there is a growth clearing zone very close to the wells of the extracts, it can be observed that beyond this zone,

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Fig. 2. Result of anti-QS screening for Terminalia catappa and the controls. Both the leaf (A) and bark (B) extracts of Terminalia catappa at 2000 ␮g mL−1 caused observable inhibition of violacein pigmentation as opposed to complete clearing of bacterial growth around the well for the antibiotic control (C, 100 ␮g mL−1 chloramphenicol). The picture showing no clearing (D) is for the negative control using sterile distilled water.

15.625 ␮g mL−1 increased by about 15%, at which point the QS activity began to show a steep decline that eventually dropped to 50% at 62.5 ␮g mL−1 . The increase in QS activity coincides with an exponential increase in growth (Fig. 3), which could be due to the known presence of flavonoid glycosides in Terminalia catappa that can release sugars upon enzymatic action by bacteria (Lin et al., 2000; Vermerris and Nicholson, 2006). Therefore, the effective concentration of TCF12 at which 50% of the QS activity was reduced is approximately 62.5 ␮g mL−1 , which can be designated as the minimum QS inhibitory concentration (MQSIC). The assessment on the effects on growth of the test organism (Fig. 3) revealed that the growth of Chromobacterium violaceum CV026 is enhanced in the presence of 15.625 ␮g mL−1 and 450 400 Percentage (%)

where the concentration of the extract is decreasing, growth can be observed but with markedly reduced pigmentation. While this could mean that the extract causes a partial inhibition of growth, it is clear that the cells growing in this region have reduced or no pigmentation. This implies that the extract is growth inhibitory at high concentrations but QS inhibitory at lower concentrations. The leaf extracts of Terminalia catappa were subjected to a bioassay-guided fractionation scheme. The methanolic extract of Terminalia catappa leaves was first subjected to extraction by solvent partitioning to yield the hexane, ethyl acetate and aqueous extracts. Subsequent assays of these three extracts revealed a relatively more enhanced activity of the ethyl acetate extract compared to the aqueous and methanolic extract while the hexane extract did not have any activity at all (data not shown). Of the 19 fractions collected from the SEC of the ethyl acetate extract, fraction 12, labeled TCF12, had the highest activity and yield so it was chosen for characterization and quantitative assays. Based on a modification of previously described methods, a spectrophotometric assay was performed to quantify the relative amount of pigmentation of wild-type Chromobacterium violaceum treated or untreated with TCF12. Since the pigments from this wild type strain may absorb light at a wavelength used for monitoring bacterial cell density (540–700 nm), the mutant strain Chromobacterium violaceum CV026 – a CviI mutant incapable of producing the pigments – was used for the assessment of the effects of TCF12 on the growth of the test organism (Martinelli et al., 2004). Thus, in this modified assay, the effect on pigment production was assessed in the wild-type strain while the effect on growth was monitored using the mutant non-pigmented strain. In general, the concentration of TCF12 and QS activity show an inverse relationship (Fig. 3). However, the QS activity at

Bacterial Cell Density

350

Violacein Production

300 250 200 150 100 50 0 0

15.63

31.25

62.5

125

250

500

1000

Concentration (μg/mL) Fig. 3. Bacterial cell density of the non-pigmented mutant Chromobacterium violaceum CV026 and violacein production by the wild type Chromobacterium violaceum at increasing concentration of TCF12.

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Fig. 4. The effect of increasing concentration of TCF12 on the growth, biofilm formation and biofilm resilience/maturation of Pseudomonas aeruginosa ATCC 10145.

31.25 ␮g mL−1 of TCF12, corresponding to 320 and 150% growth, respectively. Although significant adverse effect on growth was observed at 125 and 250 ␮g mL−1 , these corresponded only to 18 and 21% reduction in cell density, respectively. Estimation of the minimum inhibitory concentration – 50% (MIC) by extrapolation from the slope of 500 and 1000 ␮g mL−1 gave 961.84 ␮g mL−1 . The range of concentration from MQSIC (62.5 ␮g mL−1 ) to MIC (961.84 ␮g mL−1 ) can be said to be where TCF12 has a specific and optimal QS inhibition activity with zero or minimal effect on growth in Chromobacterium violaceum. Having known that TCF12 is indeed an inhibitor of QS in Chromobacterium violaceum, the possibility that it may also be able to inhibit QS-controlled phenotypes in other Gram-negative bacteria was explored. Pseudomonas aeruginosa was chosen for this purpose because of the known QS systems that control a number of genes involved in biofilm formation and production of virulence factors (Schuster and Greenberg, 2006). While the molecular interplay between QS and biofilm development is still uncertain, it is clear now that QS is involved in the maturation and differentiation of biofilms (Favre-Bonté et al., 2003). QS mutants of Pseudomonas aeruginosa have been shown to form thick and flat biofilms while wild type strains produce biofilms with canals and mushroom structures characteristic of mature biofilms (Davies et al., 1998). The thicker biofilms in QS mutants were found to be more susceptible to treatment with biocidal agents than the biofilms formed by wild type strains. It is with this rationale that the assessment of the effects of TCF12 on the formation of biofilms in this study was a two-sided approach. One was to measure the effect of TCF12 on the biofilm growth alone while another was to quantify the biofilm growth after disruption by 1% sodium dodecyl sulfate (SDS). Both set-ups contain increasing concentration of TCF12 and both stained afterwards with CV for quantitation of adherent biofilm-associated cells. Fig. 4 shows the effect of TCF12 on biofilms of Pseudomonas aeruginosa ATCC 10145. Biofilm growth was significantly enhanced by TCF12 in a concentration-dependent manner with a peak of about 220% increase in biofilm growth at 500 and 1000 ␮g mL−1 . Upon washing with a biocidal agent (1% SDS), the biofilms formed in the presence of the extract were easily disrupted, which means that the resilience of the formed biofilms to biocidal treatment was significantly decreased, also in a concentration-dependent manner. Thus, while the treated Pseudomonas aeruginosa form thicker biofilms (higher biofilm growth) at increasing concentra-

tions of TCF12, it becomes more and more susceptible to biocidal treatment. This is an indication that maturation and differentiation of Pseudomonas aeruginosa biofilms were impaired in the presence of TCF12. Using 1% SDS as biocidal agent, a statistically significant biofilm disruption of 14% was observed in biofilms grown at 31.25 ␮g mL−1 TCF12 concentration (Fig. 4). However, reduction to almost half (approximately 46%) of the grown biofilms was only achieved at 1000 ␮g mL−1 . The results of this disruption assay can vary depending on the type and concentration of biocidal agent used. Thus, the concentration of TCF12 at which half of the biofilms can be disrupted can be a lot lower using other agents (for instance, antibiotics). In one study, tobramycin was used in addition to QSinhibitory extract from garlic to enhance the disruption of biofilms formed by Pseudomonas aeruginosa PAO1 (Rasmussen et al., 2005). The MIC-50 of TCF12 on Pseudomonas aeruginosa ATCC 10145 was determined to be 1256.51 ␮g mL−1 through linear extrapolation of the slope of 250, 500 and 1000 ␮g mL−1 . Another QS-controlled phenotype in Pseudomonas aeruginosa that was assessed was LasA activity. Pseudomonas aeruginosa is known to secrete extracellular proteases that are associated with virulence of this opportunistic pathogen. Two elastases (LasA and LasB) are among the well-characterized. Along with other extracellular enzymes (elastase, alkaline protease, lipase, phospholipase C) and toxins, LasA contribute to both acute and chronic Pseudomonas aeruginosa infections in immuno-compromised patients, including those affected by the genetic disorder cystic fibrosis (Sandoz et al., 2007). These elastases are able to degrade various substrates including elastin, collagen types III and IV, laminin, immunoglobulins A and G, and complement components (Kessler et al., 1993). Their group was able to prove that LasA can lyse Staphylococcus aureus cells and is the same protein that was reported to be staphylolytic in earlier studies. Other studies have employed this staphylolytic activity of LasA for time-lapse quantitative assays (Kong et al., 2005; Adonizio et al., 2008) to monitor and measure the expression of LasA. In the present work, this same assay is used to compare the LasA activity of Pseudomonas aeruginosa ATCC 10145 cells treated with increasing concentration of TCF12. The results show (Fig. 5) that TCF12 effectively reduced the ability of Pseudomonas aeruginosa cells to lyse Staphylococcus aureus cells. The enzyme activity (measured by Staphylococcus aureus cell density) was reduced in a concentration-dependent manner with

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Fig. 5. The effect of increasing concentration of TCF12 on LasA activity of Pseudomonas aeruginosa 10145 over time.

about 50% reduction of LasA activity at 62.5 ␮g mL−1 in 5 min. The enzyme activity was not shown to vary over with time since it was almost complete after 10 min in all treatments. The results for the phytochemical screening (Table 1) reveal that TCF12 does not contain any detectable levels of terpenoids and alkaloids. Based on the gelatin precipitation test and ferric chloride spot test, TCF12 contains an abundant level of tannins. The ferric chloride spot test yielded a blue-black precipitate indicating that hydrolyzable tannins are the predominant type of tannins present. The test for flavonoids was also positive, which can mean that flavonoids are also present in the fraction or that the tannin oligomers or polymers present in TCF12 contain flavonoids as monomeric units like the condensed tannins, or as polyol centers like many complex tannins (Vermerris and Nicholson, 2006). The finding that Terminalia catappa leaves is rich in tannins has been reported by earlier studies (Tanaka et al., 1986; Lin and Hsiu, 1999). Many of these tannins in Terminalia catappa have been isolated and their structures have been elucidated. While there are studies that showed flavonoids affect the expression of QS-controlled genes in plant-associated bacteria (Economou et al., 1989; Cubo et al., 1992), reports about more direct anti-QS activity of both flavonoids and tannins are very few. It was reported that ellagic acid and epigallocatechyl gallate (EGCG) are able to inhibit QS in Escherichia coli, Pseudomonas putida and Chromobacterium violaceum (Huber et al., 2003; Taganna and Rivera, 2008). The present work is the first report of anti-QS activity of Terminalia catappa extract. Though several tannins have been isolated from Terminalia catappa leaves including terflavin A and B, tergallagin, chebulagic acid, graniin, punicalin, punicalagin, tercatain, and others (Tanaka et al., 1986), none of these have been tested for anti-QS activity.

There are no studies yet that report any mechanism by which tannins and flavonoids can inhibit QS. Some known mechanisms of QS inhibition include competitive binding of signal-like molecules to cognate receptors, as in the case of furanones (Manefield et al., 1999) and, enzymatic degradation of QS signals, as in the case of acyl homoserine lactone (AHL) acylases (Dong et al., 2000, 2002; Zhang et al., 2002). It is unlikely for tannin/flavonoid QS inhibitors to follow the enzymatic mechanism of acylases, since tannins are not proteins and are not known to have catalytic activity. Instead, the known protein-binding ability of tannins (Vermerris and Nicholson, 2006) may be responsible for the anti-QS activity by binding to intracellular proteins including the protein receptors of QS signals or even the enzymes that catalyze the production of the signals. It has long been demonstrated that oligomeric or polymeric tannins can achieve a certain degree of binding specificity to certain peptide motifs (Hagerman and Butler, 1981), which may be naturally designed by plants for specific protein targets in invading bacterial pathogens. Terminalia catappa is a large, spreading tropical tree in the Family Combretaceae. It is now distributed throughout the tropics in coastal environments where various parts of the plant have been used in traditional medicine (Burkill, 1985). In the Philippines and South India, sap from young leaves is made into an ointment for scabies, leprosy and other cutaneous diseases. The leaves when applied externally are refreshing and sudorific, and appear to exercise an anodynal effect on pain for they are used for headache, on rheumatic joints, or in an oily ointment for breast-pain. In Nigeria the leaves macerated in palm-oil have been used as a remedy for tonsillitis. Leaf in Java has shown some antibiotic activity. Interestingly, Adonizio et al. (2006) previously identified another species of Combretaceae with anti QS activity, which could indicate, along with the result of the present study, that the production of this antiQS active metabolites is shared in at least certain members of this family.

Table 1 Results of phytochemical screening of the active TCF12 fraction.

Terpenoids

Saponins Tannins/polyphenols Flavonoids

Alkaloids

Test/spray reagent

Result

TLC with chloroform vanillin-sulfuric acid Salkowski test Froth test Gelatin test Ferric chloride test 2D-TLC with BAW and 5% acetic acid fumed with ammonia Sequential NaOH and HCl treatment Wagner’s reagent Marquis reagent

Negative Negative Negative Positive Positive Positive Positive Negative Negative

4. Conclusions In this study, the anti-quorum sensing (QS) activity of an active fraction obtained from the methanol leaf extract of Terminalia catappa was detected through the inhibition of the QS-controlled violacein pigment production in Chromobacterium violaceum. Various chemical characterization methods employed demonstrated that this fraction is rich in tannins and probably mixed with other polyphenolic compounds. The effect of this fraction on known QS-controlled phenotypes was assessed. On the LasA activity of Pseudomonas aeruginosa ATCC 10145, it was found that

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