Ruthenium Chloride—Induced Oxidative Cyclization of Trans-Resveratrol to (±)-ε-Viniferin and Antimicrobial and Antibiofilm Activity Against Streptococcus pneumoniae

Polyphenol ε-viniferin (2) is a protective phytochemical found in several plant families. Here, we report a simple and effective method for the synthesis of (±)-ε-viniferin (2) as major product and (±)-(E)-ω-viniferin (3) as a minor product. Synthesized viniferin compounds and standard viniferin were analyzed for antibacterial and antibiofilm activity against Gram-positive bacteria Streptococcus pneumoniae. The minimum inhibitory concentrations (MICs) of (±)-ε-viniferin (2) and standard viniferin were 20 µm. However, the MICs of (±)-(E)-ω-viniferin (3) and compound 8 were 40 µm. Although viniferin significantly (p < 0.05) reduced pre-established in vitro biofilms and killed bacteria within the biofilm, it was unable to prevent biofilm formation at sub-MIC concentrations. The time kill experiment revealed that viniferin killed bacteria and reduced 2.8 log10 bacteria at 2 × MIC concentration after 24 h. Scanning electron microscope (SEM) analysis and live/dead biofilm staining of pre-established biofilms revealed that viniferin treatment disrupts membrane integrity of biofilm bacteria. Crystal violet absorption, total protein, and DNA and RNA release revealed that viniferin alters bacterial cell permeability, eventually killing bacteria.


INTRODUCTION
Streptococcus pneumoniae (S. pneumoniae) asymptotically colonizes the nosocomial cavity, causing infections under immune-compressed conditions. Pneumococci initially colonize nasopharyngeal mucosa in the form of biofilms (Bogaert et al., 2004;Simell et al., 2012). Biofilms in the nasopharyngeal cavity serve as reservoirs of bacteria that transit to sterile sites, causing infections such as otitis media (OM) pneumonia, bacteremia, meningitis, and sepsis (Wardlaw et al., 2006;Bergenfelz and Hakansson, 2017). Pneumococcal biofilms have been detected in infected sites such as tympanostomy tubes, human mucosa biopsies, and resected adenoids (Hall-Stoodley et al., 2006;Hoa et al., 2009). S. pneumoniae biofilms were also detected in the middle ear and nasopharynx of experimentally infected cinchona and mice (Reid et al., 2009;Sanchez et al., 2010). Bacteria in the biofilms were found in a self-produced matrix consisting of an extracellular polymeric substance (EPS) that confers resistance against conventional antibiotics and host immune defense (Donlan and Costerton, 2002). In mature biofilms, the EPS matrix resists antibiotic penetration via: i) charged polymers that prevent drug diffusion or ii) restriction of antimicrobial agents to the surface, leaving deeper cells unaffected, causing bacteria persistence (Marsh, 2004). Also, compared to planktonic bacteria, biofilm bacteria exhibit differential gene expression by adopting metabolic pathways less sensitive to the quinolone class of antibiotics and β-lactams, which target macromolecules or metabolic pathways (Xie et al., 2005;Mascio et al., 2007;Yadav et al., 2012). Antimicrobials inhibiting normal cells are made ineffective due to bacteria in biofilms growing slowly under nutrientand aeration-depleted conditions (Davies, 2003). Resistance against commonly used antibiotics hampers treatment options (Korona-Glowniak et al., 2018). Consequently, new safe antimicrobials or antibiofilm agents that limit emergence of antibiotic-resistant bacteria are needed (Hurdle et al., 2011). Reportedly, novel phyto-compounds display potential to mitigate bacterial growth, including both pneumococcal planktonic and biofilm growth (Kwieciński et al., 2009;Ma et al., 2012;Quave et al., 2012;Burt et al., 2014;Yadav et al., 2015;Ma et al., 2018).
Few reports are available on the synthesis of ε-viniferin. However, related natural products have been synthesized. Oxidative cyclization of resveratrol using FeCl 3 ·6H 2 O and Ti (NO 3 ) 3 in methanol has been reported (Yao et al., 2004;Takaya et al., 2005). Although a method for total synthesis of ε-viniferin has been reported, it only yields low amounts of viniferin and requires more steps (Lindgen et al., 2016). To overcome these limitations, we synthesized ε-viniferin (2) and (E)-ω-viniferin (3) as racemates using ruthenium chlorideinduced oxidative cyclization of trans-resveratrol. Compounds 8 and 9 were also synthesized from ε-viniferin (2). This novel method is economical and improves yields for large-scale synthesis. Synthetic viniferins were evaluated for antimicrobial and antibiofilm activity against S. pneumoniae planktonic and biofilm states.

Synthesis of Viniferins
All chemicals were purchased commercially and used without further purification. Flash column chromatography enabled product purification, and thin layer chromatography (TLC) analysis was conducted on commercial plates coated with silica gel 60 F 254 . Visualization of spots on TLC plates was achieved via ultraviolet (UV) radiation. Mass spectra were recorded using high-resolution mass spectrometry (HRMS, electrospray ionization MS) obtained on a G2 quadrupole time-of-flight (QTOF) mass spectrometer. Proton nuclear magnetic resonance ( 1 H NMR) spectra were determined on a Varian (400 MHz) spectrometer (Varian Medical Systems, Inc., Palo Alto, CA, USA). 13 C NMR spectra were recorded on a Varian (100 MHz) spectrometer using tetramethylsilane (TMS) as an internal standard. Chemical shifts are provided in parts per million (ppm) downfield with coupling constants in hertz (Hz). Standard abbreviations s, d, t, and m refer to singlet, doublet, triplet, and multiplet, respectively. Infrared (IR) spectra were recorded on FT-IR (NICOLET-iS5). Final product purity was checked by reversed-phase high-pressure liquid chromatography (RP-HPLC) performed on the YL9100 YoungLin HPLC system equipped with an UV detector set at 280 and 254 nm. Mobile phases used were: (A) H 2 O containing 0.1% trifluoroacetic acid and (B) acetonitrile containing 0.1% trifluoroacetic acid. HPLC chromatogram for purity check was: 5-55% B for 0-12 min, 55-100% B for 12-15 min, 100% B for 15-19 min, 100-5% B for 19-22 min, and 5% B for 22-23 min. HPLC employed an Agilent Eclipse Plus C18 reversephase column (3.5 μm particle size) that was 4.6 in diameter × 100 mm in size with a flow rate of 1.0 ml/min. Starter chemicals such as resveratrol and RuCl 3 .H 2 O were purchased from Sigma Aldrich, TCI, Alfa-Aesar, and Combi Blocks. The purity of all biologically evaluated compounds was >95% (per HPLC).

Minimum Inhibitory Concentration (MIC) Detection
MICs were determined using the broth microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI, 2005). S. pneumoniae D39 colonies grown on BAP were scraped and grown in MH broth until log phase. Log-phase cells were diluted to prepare bacterial suspensions containing 1-5 × 10 5 colony-forming units (cfu)/ml. Viniferin solutions were added to Mueller Hinton Broth (MHB) cell suspension from 10 to 50 µm concentrations. Next, 200 µl of cell suspension along with viniferin solutions were inoculated in each well of a 96-well plate. Negative control samples were supplied with DMSO. Plates were incubated for 18 h at 37°C. Bacterial growth was detected by measuring optical density at 600 nm using a microplate reader. MIC was defined as the lowest concentration of viniferin solution at which no visible bacterial growth was observed. Alternately, MICs were confirmed by determining bacterial cfu counts. Cell suspensions were diluted two-fold, plated on BAP agar plates, and incubated overnight at 37°C, and cfu were counted. The experiment was repeated twice in triplicate. The inhibitory effect of 20 µm viniferin on strains resistant to erythromycin (> 512 µg/ml) and different serotypes (2, 3, 19A, 19F) of S. pneumoniae was evaluated.

Effect of Viniferin on Biofilm Inhibition
The effect of ε-viniferin (2) on S. pneumoniae biofilm growth was evaluated in vitro using static biofilm models, and biofilm biomass was quantified using crystal violet (CV) microtiter plate assays (Christensen et al., 1985;Yadav et al., 2017). Bacteria grown on blood agar plates were scraped and grown to log phase in BHI broth. Freshly prepared bacterial cell suspensions were diluted 1:200 and inoculated at 200 µl in 96-well plates or at 1 ml in 24-well plates. Viniferin solution was added to each well at concentrations from 10 to 50 µm. The control samples (vehicle) were treated with similar volumes of DMSO (0.1 to 0.5%). Plates were incubated at 37°C for 18 h. Following incubation, the medium and planktonic cells were decanted and the plate washed twice with phosphatebuffered saline (PBS). Biofilms at the bottom of the plates were stained using 50 µl of 0.1% CV for 15 min. The biofilms were washed twice with PBS and air-dried, and CV was dissolved in ethanol (200 µl ethanol for 96-well or 1 ml for 24-well plate). Absorbance of the dissolved CV was measured at 570 nm using microplate readers. To evaluate the effects of viniferin on planktonic growth, S. pneumoniae D39 were grown in different concentrations of synthetic ε-viniferin (2) (MIC, 0.5 × MIC, and 0.25 × MIC). Growth was detected by measuring absorbance at 600 nm at different time intervals.

Effect of Viniferin on Eradication of Pre-Established Biofilms
Microbial biofilms are difficult to eradicate, and several-foldhigh concentrations of antibiotics are needed to eradicate preestablished biofilms (Høiby et al., 2010;Co et al., 2018). We evaluated in vitro biofilm eradication potential of viniferin.
In vitro biofilms of S. pneumoniae were grown in 24-well plates for 18 h via procedures described above. These biofilms were treated with MIC and 2 × MIC concentrations of viniferin for 6 h. Biofilm biomasses were quantified using CV microtiter plate assays, and viable bacteria were detected by cfu counts as described above. Daptomycin, a lipopeptide antibiotic known to eradicate biofilm, was used as a positive control.
Metabolically active bacteria within pre-established biofilms were detected using resazurin staining. Resazurin is a bluecolored, non-fluorescent dye, and in the presence of the metabolically active cells, it gets reduced to a pink and highly fluorescent compound called resorufin. The fluorescent detection allows the quantitative measurement of cell viability. Resazurin staining was performed as per previously reported procedure with minor modification (Paytubi et al., 2017). 0.02% (w/v) resazurin sodium salt (Sigma, USA) solution was prepared in sterile distilled water and filter-sterilized. Pre-established biofilms grown in 24-well plates and treated with viniferin as mentioned above were dissolved in sterile water, and 100 μl was transferred to a 96-well plate. The biofilm suspensions were incubated with 25 μl (0.02%) resazurin dye and 100 μl BHI broth. The plate was incubated at 37°C for 2 h, and fluorescence was measured (excitation 530 nm, emission 590 nm) using a microplate reader (Thermo Scientific, Waltham, MA, USA).

SEM Analysis of Pre-Established Biofilms
S. pneumoniae form robust biofilms of significant depth in vitro (Moscoso et al., 2006). We evaluated changes in S. pneumoniae in vitro biofilm morphology upon viniferin treatment using SEM. Synthetic ε-viniferin (2) was the most active; therefore, experiments were performed with ε-viniferin (2). In vitro biofilms were grown in 24-well plates for 18 h via procedures described above and treated with 2 × MIC concentration of ε-viniferin (2) for 6 h. Control biofilms were treated with DMSO. Biofilms were washed with PBS pre-fixed by 2% glutaraldehyde + formaldehyde solution and post fixed with osmic acid (2%) for 2 h. The biofilms were dehydrated via increasing ethanol concentration gradients (60-95%) followed by t-butyl alcohol treatment. The samples were freeze-dried and coated with platinum. Images were captured using field emission SEM (FE-SEM; Hitachi, S-4700, Tokyo, Japan).

Confocal Microscopy Analysis of Pre-Established Biofilms
Alteration of pre-established biofilm structure and viability upon viniferin treatment was analyzed using live/dead biofilm staining and confocal microscopy. S. pneumoniae D39 biofilms were grown on micro-discs for 18 h via procedures described above, and treated with ε-viniferin (2) (2 × MIC) for 6 h. The biofilms were stained with a LIVE/DEAD biofilm viability kit (Invitrogen, California, USA) according to the manufacturer's instructions. Control biofilms were treated with similar volumes of DMSO. After washing, stained biofilms were viewed under a Nikon A1 confocal microscope (Nikon Instruments Inc., NY, USA) using fluorescein (green) and Texas red (red) band-pass filter sets.

Effect of Viniferin on Bacteria Killing
To evaluate the bactericidal or bacteriostatic effect of viniferin on S. pneumoniae, a bacteria-killing experiment was performed. An S. pneumoniae D39 cell suspension was grown in BHI broth up to early log phase, and aliquots were treated with 2 × MIC concentrations of viniferin (compounds 2, 3, and 8, and ε-viniferin standard). Cells were further grown at 37°C, and viable bacteria were detected at different time points (0, 6, 12, and 24 h) via cfu counts as described above. Control samples were treated with DMSO.

Crystal Violet Absorption Assay
Alteration of bacterial membrane permeability upon viniferin treatment was evaluated using CV absorption assays (Devi et al., 2010). S. pneumoniae D39 colonies were grown in BHI broth up to early log phase. The cells were pelleted via centrifugation at 4,500g for 5 min at 4°C. Bacterial cells were washed with PBS and treated with ε-viniferin (2) and erythromycin (MIC and 2 × MIC) for 3 h at 37°C. Erythromycin was used as the control, as it had no effect on bacterial membrane permeability (non-dividing cells). Negative control samples were treated with DMSO. Following incubation, cells were harvested via centrifugation at 9300g for 5 min and suspended in 10 µl/ml CV solution (0.01%). The cell suspension was incubated for 10 min and centrifuged at 13,400g for 15 min. The OD of the supernatant was detected at 590 nm using a microplate reader. The OD value of the original solution was considered 100%, and the percentage of CV absorption was detected as follows: OD value of the sample OD value of the crystal vi iolet solution × 100

Leakage of Total Proteins Through the Bacterial Membrane
Integrity of S. pneumoniae cell membrane upon viniferin treatment was examined by determining total protein release in supernatants. Protein concentrations in the supernatants were evaluated using the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, MA, USA). S. pneumoniae were grown to log phase, pelleted via centrifugation, and suspended in PBS. Cell suspensions (approx. 10 7 /ml) were treated with viniferin (MIC and 2 × MIC) and incubated at 37°C for 3 h. Next, cells were pelleted, and supernatant was filtered through a 0.2 µm syringe filter. The cell-free supernatants were treated with 200 µl each of solutions A and B (provided in kit) for 30 min, and absorbance at 562 nm was measured.

Leakage of DNA and RNA Through the Bacterial Membrane
The integrity of the pneumococci's cell membrane upon viniferin treatment was evaluated by monitoring release of cytoplasmic constituents such as DNA and RNA as per a previous report (Bouyahya et al., 2019). S. pneumoniae were grown up to midlog phase in BHI medium. The cell suspensions were pelleted by centrifugation and dissolved in PBS. The cells were treated with viniferin (MIC and 2 × MIC) for 3 h at 37°C. After treatment, cells were pelleted by centrifugation (10,000g for 5 min), and supernatants were filter-sterilized using a 0.2 µm syringe filter. The released DNA and RNA in supernatants were determined by measuring optical density at 260 nm.

Statistical Analysis
Experiments were performed in replicate, and means were calculated and compared with those of the control group. Statistical significance was assessed using Student's t-tests, and a P < 0.05 was considered significant.

Viniferin Is Not Effective on S. pneumoniae in Vitro Biofilms at Sub-MIC
Planktonic growth results showed no bacterial growth at MIC and slow bacterial growth at 0.25 × MIC (sub-sub-MIC) or 0.5 × MIC (sub-MIC) of ε-viniferin (2) ( Figure 3A). No significant difference was detected in the planktonic growth of bacteria between ε-viniferin (2) treated with 0.25 × MIC or 0.5 × MIC and the control following 15 h incubation ( Figure 3A). Growing S. pneumoniae in vitro biofilms in presence of 0.25 × MIC or 0.5 × MIC of viniferin did not induce significant inhibition of biofilm growth ( Figure 3B). However, at the MIC of viniferins, no biofilm growth was detected, which may have been due to complete inhibition of planktonic cell growth, which prevented the cells from reaching the number required for biofilm buildup. These results suggest that viniferin was unable to impede in vitro biofilms of S. pneumoniae at sub-MIC but hindered bacterial growth in planktonic form and biofilms at MIC.

Viniferin Treatment Distorts S. pneumoniae Within Biofilms
Changes that occur in biofilm structures following viniferin treatment were evaluated using SEM. SEM revealed that intact three-dimensional biofilms with typical EPS matrices were deposited on the surface of bacteria in control samples (Figures 5A, B). However, the analysis also revealed that the structures of biofilms treated with ε-viniferin (2) were altered (Figures 5C, D). Cells in viniferin-treated biofilms were distorted, and the cell membrane appeared shrunken. The lysed cells (Figure 5D, arrow) indicated that viniferin effectively killed bacteria within the biofilm but was unable to completely disassemble the biofilm matrix. Also, the EPS present on bacterial surfaces in biofilms of control samples (Figure 5B, arrow) was absent on viniferin-treated biofilm bacterial surfaces.

Confocal Microscopy Analysis of Pre-Established Biofilms
The LIVE/DEAD biofilm viability kit utilizes a mixture of the SYTO ® 9 green-fluorescent nucleic acid stain and the redfluorescent nucleic acid stain, propidium iodide. Bacteria with intact cell membranes (live cells) are stained green, and those with damaged membranes are stained red. Live/dead biofilm staining revealed that control (DMSO) biofilms were compact with no visible dead cells (Figures 6A, B). However, biofilms treated with ε-viniferin (2) were thin, and dead cells (red) or cells with compromised membranes (yellow) were visible (Figures 6C, D), indicating that viniferin treatment kills pneumococci in biofilm.

Viniferin Increases CV Absorption
A CV absorption assay was performed to determine alterations in bacteria cell membrane permeability due to viniferin treatment. The assay demonstrated increased CV absorption by viniferintreated bacteria, compared with untreated cells (control). CV uptake by untreated bacteria (control) was approximately 40% (Figure 8). However, the bacteria treated with ε-viniferin 2 at MIC and 2 × MIC demonstrated 61% and 74% CV uptake, respectively (Figure 8). Increased CV absorption by viniferin-treated bacteria indicates that bacterial cell membrane permeability was altered (Figure 8). Erythromycin, known for its undetectable effect on cell permeability in non-growing bacteria, was used as a negative control. The leakage of cytoplasmic proteins through the cell membrane was examined to evaluate the integrity of pneumococci membrane on viniferin treatment. Protein quantification results showed that total protein release by viniferin-treated bacteria was significantly elevated (P < 0.05) compared to that by control bacteria. Total protein released by bacteria treated with MIC and 2 × MIC viniferin was significantly increased by 43% and 59%, respectively (p < 0.05) (Figure 9A).
The leakage of genetic material (DNA and RNA) through the pneumococcal membrane on viniferin treatment was increased. Quantification of DNA and RNA results showed significantly increased (p < 0.05) DNA and RNA in viniferin-treated bacteria (Figure 9B). At MIC and 2 × MIC viniferin, DNA and DNA  release increased by 46% and 64%, respectively, in comparison to the control ( Figure 9B).

DISCUSSION
S. pneumoniae causes various infectious diseases and is a leading cause of child morbidity. Approximately 25-60% of healthy children are pneumococci carriers (Korona-Glowniak et al., 2018). Reportedly, biofilms of S. pneumoniae are established in the nasopharynx, and planktonic bacteria from biofilms may transit to sterile mucosal tissue, causing Acute Otitis Media (AOM), pneumonia, bacteremia, or local infections (Bogaert et al., 2004). The biofilm resistance paradigm obstructs treatment, resulting in recurrent and recalcitrant infections, which increase recovery time and treatment costs (Costerton et al., 1999;Thomas et al., 2006). Classical antibiotics targeting actively growing bacteria are ineffective due to the dynamic structure of biofilms. Therefore, it is necessary to find new antimicrobials that can effectively control biofilms and prevent the development of antimicrobial resistance (Chopra et al., 2002). One strategy to prevent biofilm-related infections is to inhibit or eradicate initial colonization by bacteria using natural compounds. Reportedly, various phyto-compounds such as 220D-F2 derived from Rubus ulmifolius, carvacrol (a constituent of oregano), Melaleuca alternifolia (tea tree oil), magnolol, and eugenol are effective against biofilms (Kwieciński et al., 2009;Ma et al., 2012;Quave et al., 2012;van Alphen et al., 2012;Burt et al., 2014;Yadav et al., 2015). We developed a novel method for synthesizing viniferin via oxidative coupling with ruthenium chloride and tested its antimicrobial and antibiofilm potential against S. pneumoniae.
Pneumococcal biofilms cause various infections including AOM, pneumonia, bacteremia, and meningitis (Hall-Stoodley et al., 2006;Pichichero et al., 2013;Shak et al., 2013). These biofilms act as bacterial reservoirs from where bacteria can transit in the form of planktonic bacteria to other sterile anatomical sites, causing infections (Hall-Stoodley et al., 2006;Sanchez et al., 2010;Weimer et al., 2010). Therefore, controlling pneumococcal biofilms is important for infection management. Viniferin at MIC prevented biofilm formation in all tested serotypes, including strains resistant to clindamycin >128, erythromycin >512, and tetracycline 16 µg/ml. In contrast, classical aminoglycoside antibiotics accelerate stress in bacteria, resulting in increased biofilm formation at low MICs or sub-MICs (Hoffman et al., 2005). Therefore, an ideal antimicrobial agent should not induce biofilm growth at low concentrations but be effective in preventing biofilm aggregation and eradicating pre-established biofilms. Viniferin shows potential as an ideal antimicrobial agent for pneumococcal biofilm control. Our findings demonstrate that viniferin does not induce biofilm formation at sub-MIC, although it was unable to inhibit pneumococci biofilm formation at sub-MIC. However, it completely inhibited pneumococcal growth under biofilm and planktonic states at MIC. Moreover, viniferin is toxic to bacteria, and the treatment by 2 × MIC viniferin significantly reduced bacterial load by 2.8 log 10 after 24 h. However, definitions of bactericidal and bacteriostatic indicate that viniferin was not bactericidal (a drug is bacteriostatic if it kills <3 log 10 bacteria and bactericidal if it kills >3 log 10 bacteria after 24 h of incubation) (Basri et al., 2014). Similarly, bacteriostatic effects were previously reported for methicillin-resistant Staphylococcus aureus (Basri et al., 2014). Reportedly, plant extracts containing viniferin inhibited in vitro biofilm growth of Gram-negative bacteria such as Pseudomonas aeruginosa and E. coli. However, the target of viniferin or underlying mechanisms was not reported (Cho et al., 2013;Lee et al., 2014). In this study, no effects of viniferin on in vitro biofilm growth of pneumococci were detected. This implies that viniferin activity is different for Gram-positive and Gram-negative bacteria. The difference between biofilminhibiting activities of viniferin in P. aeruginosa (Gram-negative bacteria) and S. pneumoniae (Gram-positive) may be due to a difference in mechanisms underlying biofilm regulation and biofilm matrix composition (Hall and Mah, 2017). Another reason may be cell membrane differences; the cytoplasmic membrane of Gram-positive bacteria is surrounded by a relatively simple porous cell wall, whereas the cell envelope of Gram-negative bacteria is more complex and relatively impermeable, consisting of an inner cytoplasmic membrane and an outer membrane separated by a peptidoglycan layer (Minnock et al., 2000).
The microbial biofilms are resistant to antibiotics, and the dynamic structure of mature biofilm confers resistance against antimicrobials. Typically, biofilms are surrounded by a selfgenerated extracellular polymeric matrix (Costerton et al., 1995;Donlan and Costerton, 2002). Reportedly, the charged polymer of the biofilm obstructs drug diffusion through the matrix, thereby limiting drug effects to the surface and leaving bacteria deep inside the biofilm relatively safe (Marsh, 2004). Our results showed that viniferin-treated pre-established biofilms exhibited decreased cfu counts and biofilm biomasses. These results indicated that viniferin may have killed bacteria within the biofilms. SEM analysis of pre-established biofilms treated with viniferin revealed deformed and shrunken cells in biofilms. The bacteria in viniferin-treated biofilms had lost shape, resulting in a shrunken appearance. Confocal microscopy revealed dead cells in viniferin-treated biofilms. CV absorption assay revealed that the viniferin treatment altered bacterial membrane permeability. Leakage of total protein, DNA, and RNA further confirmed that the viniferin treatment disrupted the integrity of the bacterial membrane (Bouyahya et al., 2019). Altogether, SEM analysis; live/dead biofilm staining; CV absorption; and total protein, DNA, and RNA release revealed that viniferin altered bacterial cell permeability and caused cell lysis, indicating that bacteria cell membrane was the target of viniferin. Similar activity was demonstrated by our group in the polyphonic phyto-compounds, eugenol and carvacrol (Devi et al., 2010;Yadav et al., 2013;Yadav et al., 2015;Khan et al., 2017). Classical antibiotics targeting different metabolic pathways of actively growing bacteria are often ineffective against biofilms. Bacteria in biofilm grow slowly, express different genes than planktonic bacteria, and adapt alternate metabolic pathways (Xie et al., 2005;Mascio et al., 2007;Yadav et al., 2012). Biofilms grow slowly in nutrient-depleted conditions, which makes them insensitive to antibiotics (Davies, 2003). The findings of this study and previous reports indicate that viniferin is an important, therapeutic bioactive compound that possesses antiobesity, anticancer, antimicrobial and antibiofilm properties (Piver et al., 2003;Ohara et al., 2015;Monteillier et al., 2018).

CONCLUSION
The current study proposes a simple and short procedure for the synthesis of (±)-ε-viniferin (2) and (±)-E-ω-viniferin (3) in large quantities. Furthermore, (±)-ε-viniferin (2), (±)-E-ω-viniferin (3), and compound 8 effectively inhibited S. pneumoniae growth and killed bacteria in biofilms. The killing effect of viniferin may due to alteration of cell membrane permeability. Viniferin is known for anti-obesity and anticancer activities, and our study demonstrated its antibacterial and antibiofilm activities against S. pneumoniae. Therefore, viniferin and its derivatives show potential as promising candidate molecules for the development of novel antimicrobial agents against pneumococci. August 2019 | Volume 10 | Article 890 Frontiers in Pharmacology | www.frontiersin.org

DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.