Characterization of the bioactive constituents of Nymphaea alba rhizomes and evaluation of anti-biofilm as well as antioxidant and cytotoxic properties

Anti-biofilm represents an urge to face drug resistance. Nymphaea alba L. flowers and rhizomes have been traditionally used in Ayurvedic medicine for dyspepsia, enteritis, diarrhea and as an antiseptic. This study was designed to identify the main constituents of Nymphaea alba L. rhizomes and their antibiofilm activity. 70% aqueous ethanolic extract (AEE) of N. alba rhizomes was analyzed by liquid chromatography, high resolution, mass spectrometry (LC-HRMS) for its phytoconstituents in the positive and negative modes in addition to column chromatographic separation. Sixty-four phenolic compounds were identified for the first time in N. alba rhizomes. Hydrolysable tannins represent the majority with identification of galloyl hexoside derivative, hexahydroxydiphenic (HHDP) derivatives, glycosylated phenolic acids and glycosylated flavonoids. Five phenolics have been isolated and identified as gallic acid and its methyl and ethyl ester in addition to ellagic acid and pentagalloyl glucose. Minimum inhibitory concentrations (MIC) and anti-biofilm activity for the extract and the major isolated compounds were determined. Radical scavenging activity using 2.2Di (4-tert-octylphenyl)-1picryl-hydrazyl (DPPH) assay as well as cytotoxic activity using 3-(4, 5-dimethyl thiazol-2-yl)-2, 5diphenyl tetrazolium bromide (MTT) assay have also been evaluated. MIC of N. alba rhizomes against Staphylococcus aureus was 0.25 mg/mL compared with 0.1 mg/mL for methyl gallate. The best reduction in biofilm formation (84.9%) as well as the best radical scavenging (IC50 3 μg/mL) and cytotoxic (IC50 9.61 ± 0.3 μg/mL) activities were observed with methyl gallate. This is the first study for in-depth characterization of phenolic compounds in N. alba rhizomes revealing it as a valuable source of phenolic compounds and promising anti-biofilm forming agent of natural origin.


INTRODUCTION
Bacterial illnesses are caused by many virulence factors.
Author(s)agreethatthisarticleremainpermanentlyopenaccessunderthetermsoftheCreative Commons Attribution License 4.0 International License that assists the persistence of pathogens in harsh environmental conditions (Upadhyay et al., 2014).Cells in biofilms grow as communities, surrounded by a selfproduced thick layer of extracellular polymeric substances (EPS, also known as matrix or slime) (Sauer and Camper, 2001).The extracellular matrix of biofilmembedded microorganisms is capable of sequestering and concentrating environmental nutrients such as carbon, nitrogen and phosphate.In addition, they can evade multiple clearance mechanisms produced by host and synthetic sources such as antimicrobial and antifouling agents, shear stress, host phagocytic elimination and host radical and protease defenses (Archer et al., 2011).Anti-biofilms have attracted the attention of scientists for the last forty years to combat biofilms that are involved in a wide range of infections and antibioticresistant infections, in a trial to develop new and effective antimicrobial agent with high efficiency.
Gram-positive bacteria are the commonest cause of nosocomial infections with predominance of Staphylococcus aureus (Valentino et al., 2014).Staphylococcus is the most common infectious agent in skin, mucous commensal and indwelling medical devices (Otto, 2009).S. aureus biofilm-associated infections are difficult to treat with antibiotics and devices need to be replaced more frequently than those infected with Staphylococcus epidermidis (Jones et al., 2001).
Traditional medicine attracted the attention of traditional healers and scientists thousands of years ago.World Health Organization (WHO) estimated that about threequarters of the world population living in developing countries relied upon traditional remedies (mainly herbs) for the health care of its people (Gilani and Rahman, 2005).
Tannins are polyphenolic compounds with wide range of biological activities.The mode of antimicrobial action of tannins is potentially due to the inactivation of microbial adhesins and cell envelope transport proteins (Saura-Calixto and Pérez-Jiménez, 2009).
Nymphaea alba L. (Nymphaeaceae), also known as the European White Waterlily, White Lotus or Nenuphar, is an aquatic flowering plant with perennial rhizomes or rootstocks anchored with mud (Wiersema, 1987).There are approximately 50 species in this genus.The flowers are white and they have many small stamens inside.Water-lilies have extensive rhizome systems from which leaf and flower stalks emerge each year.The root of the plant was used by monks and nuns for hundreds of years as an aphrodisiac, being crushed and mixed with wine.The dried roots and rhizomes of the white water lily have been used orally to treat gastrointestinal, genital, and bronchial conditions (Khan and Sultana, 2005).Interest on rhizomes of N. alba has increased.Bose et al. (2013) proved its possible sedative as well as powerful uterotonic effects (Bose, 2014).Moderate antioxidant activity, analgesic and anti-diarrheal activities have been also proved (Bose, 2012a, b).Although of promising results, to the best of our knowledge, no scientific reports have been found concerning chemical characterization of rhizomes as well as its antimicrobial activity.Therefore, the aim of this study was to get in-depth identification of phenolic constituents of N. alba rhizome extract using LC-MS and X calibur software, in addition to the isolation of its main constituents and evaluation of their antimicrobial as well as cytotoxic activities.

Preparation of plant extract
Rhizomes of N. alba L. were collected from AL Orman garden, Giza, Egypt in November 2012 during the flowering stage.Authentication of the plant was performed by Dr. Therese Labib Youssef (consultant of plant taxonomy, Ministry of Agriculture).A voucher specimen (RS006) was deposited at the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, October University for Modern Science and Arts (MSA), Egypt.
N. alba rhizome (300 g) was dried, reduced, and sieved to obtain the powdered rhizome, extracted with 70% ethanol under reflux.The aqueous ethanolic extract (AEE) was filtered, concentrated using a rotary evaporator and dried in vacuum at 40°C, to yield 40 g (13.3% yield).

Identification of phenolic compounds of aqueous ethanol extract of N. alba rhizomes by LC-HRMS
N. alba AEE was investigated according to Hassaan et al. (2014).The mobile phase consisted of (A) 2% acetic acid (pH 2.6) and (B) 80% methanol.Gradient elution at a flow rate of 100 L/min was used from 5 to 50% B at 30°C.Pneumatically assisted electrospray ionization was used.Spectra were recorded in positive and negative ion mode between m/z 120 and 1,500 with 4000V capillary voltage.Heated dry nitrogen gas at temperature 200°C and flow rate 10 L/min was used.The gas flow to the nebulizer was set at pressure 1.6 bar.For collision-induced dissociation (CID) MS-MS measurements, the voltage over the collision cell varied from 20 to 70 eV and Argon was used as collision gas.Data analysis software was used for data interpretation.Sodium formate was used for calibration at the end of LC-MS run.Interpretation for ESI-MS was performed by Xcalibur 2.1 software from Thermo Scientific (Berlin, Germany).

Total phenolic and flavonoid content
The total phenolic content (TPC) was determined using the Folin-Ciocalteu's reagent.Concentration of phenolic content was expressed as gallic acid equivalent (GAE) (Sellappan and Akoh, 2002).Flavonoid content (FC) was estimated using aluminum chloride colorimetric assay.Concentration of flavonoid content was expressed as quercetin equivalent (QE) (Kosalec et al., 2004).

Extraction and isolation
AEE (25 g) was fractionated on a cellulose column (375 g, 110 × 7 cm) using a step-gradient from 10% MeOH in H2O to 100% MeOH, to yield 80 fractions of 100 mL each, which were further collected into 5 major collective fractions (I-IV) monitored using paper chromatography and solvents S1 and S2 visualized using UV-light.Fraction I (2.55 g) was found to be polyphenolic-free (FeCl3 spray reagent/PC).Fraction II (10% MeOH, 1.5 g) was applied on Sephadex LH-20 eluted with 50% methanol to afford 1 (15 mg each).Fraction III (70% MeOH, 400 mg) was chromatographed on PPC using S1 as solvent followed by further purification using Sephadex LH-20 affording 2 and 3. Fraction IV (100% MeOH, 2.5 g) was chromatographed on a microcrystalline cellulose column using saturated butanol as a solvent then further purified on Sephadex LH-20 affording 4 and 5.All separation processes were followed up by Comp-PC with S1 and S2 solvents.

Determination of microbial sensitivity to N. alba rhizome extract by disk diffusion method
Bacteria and Candida albicans were grown in nutrient broth and Sabarouds' dextrose broth, respectively, overnight and adjusted to a concentration of 10 8 CFU/mL by comparing it with McFarland standard 0.5.
For the disk diffusion assay, 1 mL of each bacterial suspension was uniformly spread on a solid growth medium in a Petri-dish.Four sterile paper disks (6 mm in diameter; Becton, Dickinson & Co.) were placed on the surface of each agar plate and were impregnated with 10 μL of the diluted plant extract (250 mg/mL).Plates were incubated for 24 h under appropriate cultivation conditions.Antimicrobial activity was measured as a diameter of inhibition zone around a disk following the 24 h incubation.Susceptibility was estimated by measuring the inhibition zone according to Valgas et al. (2007).Inhibition zones of 16-21 mm indicates strong activity, 12-16 mm denotes good activity, while 10-11 mm and <10 mm indicate intermediate and no activity, respectively.Disks impregnated with sterile distilled water and ethanol served as negative controls and a disk with an antibiotic (ofloxacin or Amphotericin B, Sigma-Aldrich GmbH, Steinheim, Germany) served as a positive control.Replicas at each concentration were performed.

Determination of minimum inhibitory concentrations (MIC) of N. alba rhizome extract and main constituents by microbroth dilution method
MIC was determined according to Klancnik et al. (2010), MIC of each antimicrobial was performed in flat-bottomed 96-well microplates (Greiner Bio-one, Stuttgart, Germany).This test was carried against standard strains of B. subtilis (ATCC 6633), S. aureus (ATCC 6538), E. coli (ATCC 8739), P. aeruginosa (ATCC 27853) and C. albicans (ATCC 10231).The antimicrobial activity of N. alba rhizome extract against standard Staphylococcus aureus was further confirmed by testing its activity against four clinical isolates of S. aureus (staph1-4 ).These clinical isolates were recovered from wound infection.To determine MIC of each, plant extract, and isolated compounds, dilution range was prepared one step higher than the final dilution range required from 0.15 to 2 mg/mL in Müeller Hinton broth.
The inoculum was prepared by adjusting the turbidity from an overnight microbial culture by comparing it to McFarland 0.5 and then diluting it to reach a final concentration of 10 6 CFU/mL.A volume of 75 µL of inoculum was added to equivalent volumes of the two-fold serially diluted plant extract or isolated compound in a microplate.Control wells were prepared with culture medium, bacterial suspension only, plant extracts only and ethanol in amounts corresponding to the highest quantity present.The plate was incubated for 24 h at 37°C and the MIC was recorded as the lowest concentration of antimicrobial which gave no visible growth.The average value of three replicates was taken.

Effect of N. alba rhizome extract and main constituents on bacterial biofilm
Strains, which showed sensitivity for antimicrobials of 1 mg/mL for extract were used in the next experiments as this might show promising activity (Rios and Recio, 2005).The effect of N. alba rhizome extract and its main constituents was evaluated on biofilm synthesis and the percentage reduction of biofilm was estimated.The concentration used was the MIC value for the plant extract and plant constituents against each isolate.This test was done by adding tested product, in desired concentration, after distribution of bacterial inoculum in microplate wells so that final concentration of bacteria was 5 × 10 5 cell/mL.The plates were incubated for 24 h at 37 °C.The plates were then aspirated, washed, fixed and stained with crystal violet as described by Peeters et al. (2008).Readings of optical density at 545 nm, using microplate plate reader (Stat Fax ® 2100), in the presence of different concentrations of antimicrobials was compared to the positive control wells without antimicrobials (Yassien and Khardori, 2004).
Where Ac is OD545nm for positive control wells and At is OD545nm for biofilm in the presence of antimicrobials.

Radical scavenging activity
The activity of 1, 1-diphenylpicrylhydrazil (DPPH) was estimated according to the method described by Shimada et al. (1992).Radical scavenging activity was measured at 517 nm.

Cytotoxic activity
The viability of control and treated cells were evaluated at the Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University using the MTT assay in triplicate.Liver carcinoma cell line (HepG2) was used to test the cytotoxic activity according to Fotakis and Timbrell (2006).Doxorubicin was used as the positive control drug while the untreated cells whose absorbance was considered as 100% represented the negative control.Serial twofold dilutions of the tested compound and reference compound were estimated.The results were determined by three independent experiments (Wilson, 2000).Percentage cell viability was calculated as follows: % Cell viability = (Mean Abs control -Mean Abs test metabolite) × 100/Mean Abs control Where: Abs is absorbance at 570 nm.
The graphic plots were used for estimation of the 50% inhibitory concentration (IC50).STATA statistical analysis package was used for the dose response curve drawing in order to calculate IC50.

RESULTS AND DISCUSSION
As part of ongoing effort to investigate plants from traditional medicine, N. alba represents an interesting field of study where the rhizomes have not been previously studied and preliminary testing of flavonoid and phenolic contents showed high concentration, reflecting probably promising biological activities.

LC-HRMS
HPLC-MS-MS provides a powerful tool for phytochemical analysis in crude plant extracts.It provides useful structural information and allows for tentative compound identification when standard reference compounds are unavailable (Seeram et al., 2006).HPLC-MS-MS analysis of AEE of N. alba rhizomes revealed the identification of sixty-four phenolic compounds reported for the first time (Figure 1 and Table 1).
The main fragmentation pattern from gallotannins involved the loss of one or more galloyl groups (152 amu) and/or gallic acid (170 amu) from the deprotonated molecule [M-H] -.However, the fragmentation pattern of ellagitannins was less clear than that of gallotannins as ellagitannins display enormous structural variability because of different linkages of HHDP residues with the glucose molecule and their strong tendency to form C-C and C-O-C linkages (Khanbabaee and Vanree, 2001).
Gallic Pk 1 was tentatively identified as HHDP hexoside with a precursor ion at [M-H] at m/z 481.06 and daughter ions at m/z 301.12 and 275.16.Pk 8 was tentatively identified as galloyl HHDP-hexose with [M-H] at 633.07 and daughter ion at m/z 463.19 [M-H-170] and 301.13 [M-H-170-162].Isomer with same molecular weight appeared at Pk 13 with different fragmentation pattern and daughter ion at m/z 451.12 and identified as isostrictinin (Galloyl HHDP hexose).The presence of a compound with the same molecular weight at different retention times illustrated one of its isomeric forms.Different isomeric forms of hydrolysable tannins were observed and have been reported previously in eucalyptus (Barry et al., 2001)

Isolated compounds
The 2D-PC screening of the AEE of N. alba rhizomes revealed the presence of phenolic compounds (color properties under UV-light and responses to NH 3 ).The isolated compounds were identified on the basis of their spectral data (UV, 1D NMR), co-chromatography, in addition to comparison with published references of phenolics or in family Nymphaeaceae, genus Nymphaea and N. alba flowers (Nonaka et al., 1987;Jambor and Skrzypczak, 1991;Nawwar et al., 1994;Li et al., 1999;Elegami et al., 2003).This study is the first report for isolation of phenolics from N. alba rhizomes.
Five main phenolics have been isolated and identified including, gallic acid and its methyl and ethyl ester, ellagic acid and pentagalloyl glucose.Methyl and ethyl gallate as well as pentagalloyl glucose, were tested for the differences in their response for antibiofilm, radical scavenging and cytotoxic activities.

Antimicrobial activity by disc diffusion test
The sensitivity of nine standard strains to extracts from N. alba rhizomes was tested using disc diffusion method.All standard bacterial strains used were sensitive to ofloxacin while C. albicans was sensitive to amphotericin B. Both S. aureus and Sarcina lutea showed the highest sensitivity to N. aba rhizome extract as shown in Table 2.

Minimum inhibitory concentrations of N. alba rhizome extract and isolated compounds
The MIC of rhizome extract was equivalent to 0.25 mg/mL for standard S. aureus and above 2 mg/mL with standard strains of B. subtilis, E. coli, P. aeruginosa and C. albicans (Table 3 to 5).
Standard S. aureus was chosen for next studies in addition to the four clinical strains.N. alba extract showed high activity against Standard S. aureus and clinical isolates except Staph (3) isolate.Methyl gallate showed the highest activity against all tested microorganisms compared to ethyl gallate and pentagalloyl glucose.
The effect of N. alba extract and pure compounds on biofilm formations was studied.The minimum concentration that causes inhibition of growth was used.Methyl gallate caused a significant reduction in biofilm formation (84.9%) followed by the rhizome extract (78.8%) (P<0.01).Both standard strain and the clinical isolate Staph (2) were the most affected by the extract and methyl gallate followed by isolate Staph (3).The degree of inhibition was not correlated with the MIC of extract or the pure component on different tested microorganisms.Pentagalloyl glucose showed the least effect on biofilm formation.Methyl gallate showed the best antimicrobial and anti-biofilm activity in agreement with previous reports (Kang et al., 2008).This activity was attributed to its structure; a lipophilic alkyl chain at one end is connected via an ester linkage to the galloyl group bearing the polar hydroxyl groups at the other end.This amphiphilic property makes the cell membrane of S. aureus one of the most likely target sites of the action of alkyl gallate (Shibata et al., 2005).The antibacterial effect of alkyl gallate is due to, both, membrane disruption and affecting cell division by anti-FtsZ activity (Król et al., .Their antibacterial mode of action was also suggested to be as surface-active agents affecting membrane integrity and hence affect biofilm formation (Takai et al., 2011).1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucose (β-PGG) is a prototypical gallotannin and the central compound in the biosynthetic pathway of hydrolysable tannin.β-PGG has five ester bonds formed between carboxylic groups of gallic acids and aliphatic hydroxyl groups of the glucose core.It is present in a number of medicinal herbals such as Rhus chinensis Mill and Paeonia suffruticosa and showing several biological activities (Zhang et al., 2009).In our study, PGG showed lower activity compared with methyl and ethyl gallate.
PGG cytotoxic activity against hepatocellular carcinoma was comparable to ethyl gallate with IC 50 41.2 and 41.9 µg/ml, respectively.This was in agreement with Oh et al. (2001) who isolated PGG from the root of Paeonia suffruticosa and tested its in vitro effect on human hepatocellular carcinoma SK-HEP-1 cells.Up to 50 μM PGG inhibited the growth of SK-HEP-1 cells in a dosedependent fashion and 30 μM PGG significantly induced G1 arrest.

Conclusion
This study is the first report for the identification and characterization of phenolic constituents in N. alba rhizomes and evaluation of its anti-biofilm and cytotoxic activities.N. alba rhizomes revealed a promising antibiofilm activity with suggested contribution in antibacterial therapy.

Table 1 .
Tentatively assigned structures based on HPLC/ESI-MS.

Table 2 .
N. alba rhizome AEE against different standard microorganisms.

Table 3 .
Minimum inhibitory concentrations (MIC) of N. alba rhizome AEE against standard microbial strains.

Table 4 .
Minimum Inhibitory Concentrations (MIC) of N. alba rhizome AEE and its main constituents against S. aureus standard strain and clinical isolates.

Table 5 .
Percentage reduction in biofilm formation of Staphylococcus aureus by N. alba rhizome AEE and its main constituents at MIC.