Full Length Research Paper
ABSTRACT
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 anti-biofilm 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)-1-picryl-hydrazyl (DPPH) assay as well as cytotoxic activity using 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl 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.
Key words: Hydrolysable tannins, Nymphaeaceae, LC-HRMS.
INTRODUCTION
Bacterial illnesses are caused by many virulence factors. Biofilm forming capacity is an additional virulence factor that assists the persistence of pathogens in harsh environmental conditions (Upadhyay et al., 2014). Cells in biofilms grow as communities, surrounded by a self-produced thick layer of extracellular polymeric substances (EPS, also known as matrix or slime) (Sauer and Camper, 2001). The extracellular matrix of biofilm-embedded 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 anti-fouling 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 antibiotic-resistant 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 three-quarters 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.
MATERIALS AND METHODS
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.
Phytochemical investigation
The TPC of N. alba AEE was estimated as 32.96 ± 0.86 mg/g GAE (standard curve equation: y = 0.0011x+ 0.0009, r2 = 0.9867) while the FC was evaluated as 0.43 ± 0.59 mg/g QE (standard curve equation: y = 0.005x- 0.0198, r2 = 0.9774). The considerable high phenolic and moderate flavonoid contents have a great impact on 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).
Identified compounds include, caffeic acid hexoside, syringic acid hexoside, p-coumaroyl quinic acid and protocatechuic acid. Hydrolysable tannins including, gallotannins and ellagitannins, in addition to epicatechin, flavone and flavonol aglycone and glycosides have also been identified (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 acid (Pk 33) was tentatively identified with [M-H] at m/z 169.01 and a characteristic daughter ion at m/z 125.06. While ellagic acid was identified with a precursor ion peak at [M-H] at m/z 301 and characteristic fragments at m/z 257, 229 and 185.
Digalloyl hexoside (pk 4, [M-H] at m/z 483.08), trigalloyl (pk 21, [M-H] at m/z 635.09), tetragalloyl (pk 42, [M-H] at m/z 787.1), pentagalloyl (pk 17, [M-H] at m/z at 939.02), hexagalloyl (Pk 59, M-H at m/z 1091.12), as well as heptagalloyl (pk 43, [M-H] at 1243.1) hexose were tentatively identified by sequential losses of galloyl moieties and appearance of daughter ion peaks at m/z 169 and 125 in addition to comparison with literature.
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).
Pk 7 was tentatively identified as digalloyl HHDP-hexose (pedunculagin II) with [M-H] at m/z 785.11 and daughter ion at m/z 633.29 [M-H-152], 481.19 [M-H-152-152], 301.13 [HHDP]. Isomer appeared at Pk 38 with [M-H] at m/z 785.08 and daughter ion at m/z 483.28 [M-H-HHDP], 633.28 [M-H-152], 615.05 [M-H-152-H2O] and base peak at m/z 301.11 was tentatively identified as tellimagrandin I isomer. Pk 51 was tentatively identified as trigalloyl HHDP hexose (tellimagrandin II) with a precursor ion at m/z 937.09 and daughter ion at 633.22 [M-H-digalloyl], 785.18 [M-H-152] and 301.15. Pk 46 with a precursor ion peak at 935.08 [M-H] was tentatively identified as casuarinin or galloyl–bis-HHDP-hexose, with daughter ions at m/z 633.23 [M-H-HHDP] and 301.14.
Flavonoids have been also tentatively identified as myricetin hexoside (Pk 6) with [M-H] at m/z 479.05 and characteristic peak at 317.17; its pentoside (Pk 10) with a precursor ion at m/z 449.04, while the aglycone was observed at Pk 52 with typical fragmentation pattern (Table 1). Quercetin pentoside (Pk 47) was tentatively identified with [M-H] at m/z 433.04 while Pk 50 with [M-H] at m/z 463.05 and daughter ions at 343.16 [M-H-120], 373.17 [M-H-90] and characteristic base peak 301.15 was tentatively identified as quercetin hexoside. Kaempferol (Pk 53) was identified with its hexoside derivative (Pk 58). Pk 52 was tentatively identified as kaempferol glucuronide hexoside with m/z 623.13 [M-H], base peak 285.17 and daughter ion at 447.29 [M-H-176]. Apigenin (Pk 56) and its glycosylated derivative have been also identified (Pk 57, Pk 64).
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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 NH3). 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.
1, 2, 3, 4, 6 Penta-O- galloyl β-D-glucose (1), failed to crystallize and was obtained as off-white amorphous powder. It gave violet blue color using short UV light and intense blue color with FeCl3. UV λmax (nm): 272 nm.1H-NMR (300 MHz, DMSO-d6): δ ppm 6.89, 6.84, 6.83, 6.82, 6.78 (each 2H, s, galloyl H-2 & H-6), 6. 32 (1H, d, J=8 Hz, glc, anomeric H-1), 13C (75MHz, DMSO-d6): δ-ppm 165.29, 165.2, 164 (-COO-), 146.18, 145.73 (galloyl C-3, C-5), 140.1, 140.15, 140.2,(galloyl C4), 120.5, 120, 119.2, 119.1 (galloyl, C-1), 109.19 (galloyl C-2 & C-6), 92.35 (anomeric glc. C-1), 63.24 (glc. C-6), 76.66 (glc. C-5), 76.65 (glc. C-3), 72.1 (glc. C-2), 67 (glc. C-4), 63.24 (glc.C-6). Methyl gallate (2), cream colored needles, Rf values 0.72 (S1), 0.58(S2) on PC. It gave violet blue color using short UV light and intense blue color with FeCl3. UV λmax (nm): (MeOH) 218, 272.1H-NMR (300 MHz, DMSO-d6): δ 6.96 (2H, s, H-2 & H-6), δ 3.39 (3H, s, OOCH3).
Ethyl gallate (3), white amorphous powder, Rf values 0.9 (S1), 0.72 (S2) on PC. It gave violet blue color using short UV light and intense blue color with FeCl3. UV λmax (nm): (MeOH) 225, 274. 1H-NMR (300 MHz, DMSO-d6): δ ppm 6.93 (2H, s, H-2 & H-6), 4.20 (2H, q, J=7 Hz, CH2), 1.26 (3H, t, J=7 Hz, CH3). Gallic and ellagic acid have been tentatively identified by Co-chromatography.
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., 2015). 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.
Radical scavenging activity
Methyl gallate showed the best radical scavenging activity with IC50 3±0.36 µg/mL followed by ethyl gallate 4.7±0.23 µg/mL while IC50 of pentagalloyl glucose was 12±0.54 µg/mL compared with vitamin C 12±3.5 µg/mL. Antioxidant activity of methyl and ethyl gallate was proved by Wang et al. (2014) and Kalaivani et al. (2011), respectively. While moderate activity of ethyl gallate was previously reported by Kalaivani et al. (2011)
Other report showed that PGG showed an EC50 of scavenging 1, 1-diphenyl-2-picrylhydrazil (DPPH) free radical at about 1 μg/mL (1.1 μM) in test tubes, which was more potent than vitamin E (Abdelwahed et al., 2007).
Cytotoxic activity
In term of antiproliferative/cytotoxic IC50 values, Methyl gallate showed the highest activity against HepG2- cell line with IC50= 9.61±0.3 µg/ml, while ethyl gallate and pentagalloyl glucose values were 41.9±0.23 and 41.2±0.41 µg/ml, respectively compared with Doxorubicin 0.56 µg/ml, standard cytotoxic.
PGG cytotoxic activity against hepatocellular carcinoma was comparable to ethyl gallate with IC50 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 dose-dependent 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 anti-biofilm activity with suggested contribution in antibacterial therapy.
CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest.
ACKNOWLEDGEMENT
The authors are thankful to L.A. Mahmoud Omar for providing the plant material.
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