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In this study, an attempt has been made to isolate and identify the bioactive compounds from hydroalcoholic extract of Terminalia chebula fruits effective against multidrug-resistant uropathogens and also to elucidate the influence of metal ions on the growth inhibitory activity of isolated compounds against the studied bacteria, if any.
Methods and Results
Bioassay-guided fractionation and extensive spectrometric analyses (FT-IR, 1H NMR, 13C NMR and ESI-MS) were used to isolate and characterize the bioactive compound. Growth inhibitory activities of isolated compound were studied by agar well diffusion and microbroth dilution assay methods. Checkerboard titration method was used for combination study between antibiotics and isolated compound. Influence of metal ions on growth inhibitory activity of this bioactive compound against the test isolates were also studied by INT [P-iodonitrotetrazolium violet; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride] colorimetric assay. The isolated bioactive compound 1, 2, 6-tri-O-galloyl-β-d-glucopyranose was found to be responsible for antibacterial activity against multidrug-resistant uropathogens and showed synergy with trimethoprim and gentamicin. This antibacterial activity of bioactive compound was counteracted by the supplementation of iron in the medium.
Terminalia chebula fruit extract contains bioactive compound effective against multidrug-resistant uropathogens, and this antibacterial activity may be due to its iron-complexing property.
Significance and Impact of the Study
To the best of our knowledge, the antibacterial activity exhibited by isolated gallotannin against multidrug-resistant uropathogens is first time reported by us. Besides, these promising findings may lead to the development of antimicrobial agents from T. chebula fruits for the treatment of urinary tract infections caused by these pathogens.
Infectious diseases are the leading cause of premature death (Morens et al. 2004). One of the more alarming recent trends in infectious diseases has been the increasing frequency of antimicrobial resistance among microbial pathogens. Numerous classes of antimicrobial agents have become less effective as a result of the emergence of antimicrobial resistance due to overuse or misuse of existing antimicrobial drugs and have become a global public health problem (Neu 1992).
Among the infectious diseases, urinary tract infections (UTIs) pose serious health problems affecting millions of people each year. Infections in the urinary tract are the second most common type of infections in the body. Escherichia coli, the main aetiological agent of UTIs, causes about 80% of UTIs in adults (Foxman 2002). Despite the recent introduction into clinical practice of highly potent newer antibiotics such as the aminoglycosides, fluoroquinolones and the third generation of cephalosporins, multidrug-resistant uropathogens pose a major therapeutic problem for clinicians worldwide (Sahm et al. 2001; Karlowsky et al. 2002). This resistance problem demands that a renewed effort should be made to seek antimicrobial agents from other sources effective against bacterial pathogens that are resistant to current antibiotics (Cowan 1999; Soulsby 2005). Medicinal plants stand out as veritable sources of potential resistance-modifying agents, and the Indian biosphere promises to be a potential source of such compounds owing to its rich plant species diversity.
Terminalia chebula Retz. (Fam. Combretaceae) called the ‘King of Medicine’ in Tibet and is always listed at the top of the list of ‘Ayurvedic Materia Medica’ because of its extraordinary power of healing (Sato et al. 1997; Malckzadeh et al. 2001; Saleem et al. 2002; Kim et al. 2006). During the course of our screening on antimicrobial potential of different solvent extracts of T. chebula fruits against multidrug-resistant uropathogens, hydroethanol extract of T. chebula fruits was found to be most effective against these pathogens. These important findings motivated us to isolate and identify the bioactive compounds responsible for this antibacterial activity and also to elucidate the role of metal ions on growth inhibitory activity of isolated compounds against the studied bacteria.
Materials and methods
Collection, processing and extraction of plant material
The dried fruits of T. chebula were purchased from local herbalist (Bowbazar, Kolkata, India). The plant material was identified and authenticated by a botanist Prof. Sunanda Chanda, Agricultural and Ecological Research Unit, Indian Statistical Institute, Kolkata. A voucher specimen (No. AERU/TC01/10) was deposited in the Department of Agricultural and Ecological Research Unit, Indian Statistical Institute, Kolkata. The fruits were washed with tap water, dried at 40°C and seeds were separated from the pericarp. The pericarps of the fruits were then finely powdered and used for extract preparation.
The hydroalcoholic (70% aqueous ethanol) extract of T. chebula fruits, which was reported to be most effective against multidrug-resistant uropathogens (Bag et al. 2012), was prepared by immersing T. chebula fruit powder (200 g) in 1200 ml 70% aqueous ethanol at room temperature for 24 h with occasional shaking and filtered through Whatman No. 1 filter paper. The process was repeated twice with the remaining residue, and the pooled filtrate was centrifuged at 5000 g for 15 min and concentrated under reduced pressure in a rotary evaporator (yield = 60·5 g).
For bioassay-guided fraction, 30 g of hydroalcoholic extract of T. chebula fruits was suspended in distilled water (750 ml) and partitioned (3×) successively with equal volumes of n-hexane, chloroform, ethyl acetate and 1-butanol. All the solvent fractions were concentrated under reduced pressure in a rotary evaporator, and the aqueous fraction was lyophilized. The yield of n-hexane, chloroform, ethyl acetate, 1-butanol and aqueous fractions was 1·24, 1·84, 8·18, 2·74 and 11·4 g, respectively. Antibacterial potency of each fraction was studied against E. coli the main aetiological agent of UTIs to get the most effective fraction. Ethyl acetate fraction was found to be most effective and was then subjected to column chromatography as shown in Scheme 1 for the isolation of bioactive compounds.
TLC of the fractions from column chromatography was performed on precoated Kieselgel 60 F254 (0·25 mm, thick; Merck, Darmstadt, Germany) with toluene/ethylacetate/formic acid (6 : 4 : 1), and spots were detected by spraying vanillin/sulfuric acid/ethanol reagents. The isolated compound (Ia) was subjected to HPLC analysis on Shimadzu D-7000 HPLC system (Shimadzu Co., Columbia, SC, USA) with water/acetonitrile gradient at the rate 0·6 ml min−1, monitoring at 254 nm.
The melting point of the isolated compound (Ia) was determined with Yamato melting point apparatus and was uncorrected. The isolated compound was used for recording UV spectrum at 200–500 nm in Shimadzu UV–Vis spectrometer (Shimadzu Co.,). IR(KBr) spectra were recorded on a Perkin-Elmer FT-IR spectrometer (PerkinElmer, Waltham, MA, USA) at ν 500–4000 cm−1. ESI-MS was recorded with JEOL D-300 mass spectrometer (JEOL, Peabody, IL, USA). NMR spectra were recorded with a BRUKER AVANCE II 400 (International Equipment Trading Ltd., Vernon Hills, IL, USA) spectrometer using tetramethylsilane as an internal standard. Chemical shifts were given in δ (ppm).
Antimicrobial susceptibility testing
Micro-organisms used and sensitivity towards antibiotics
Fifty-two multidrug-resistant clinical bacteria – 21-E. coli (amoxicillin, gentamicin, trimethoprim, ceftazidime resistant; ciprofloxacin, aztreonam sensitive), 16-Klebsiella pneumoniae (ampicillin, trimethoprim, gentamicin resistant; aztreonam, imipenem, ciprofloxacin sensitive), 9-Pseudomonas aeruginosa (sulfamethoxazole, gentamicin, novobiocin resistant; ciprofloxacin, imipenem sensitive) and 6-Staphylococcus aureus (oxacillin, gentamicin, cefoxitin resistant; erythromycin, ciprofloxacin, vancomycin sensitive) – collected from samples of urinary tract–infected outdoor patient were kindly provided by the Department of Microbiology, Institute of Post Graduate Medical Education and Research, Kolkata, India. Internal quality assurance was ensured using reference E. coli (ATCC 8739) and S. aureus (ATCC 6538P) strains, procured from National Chemical Laboratory, Pune, India. The isolates were maintained on nutrient agar slants at 4°C.
The inoculum size of the test isolates was standardized according to the National Committee for Clinical Laboratory Standards guidelines (NCCLS 1993). The bacterial isolates were inoculated in Mueller Hinton Broth (MHB; HiMedia, Mumbai, India) and incubated at 37°C in a shaker water bath for 3–6 h until the culture attained a turbidity of 0·5 McFarland Unit. For experimental purposes, inoculum size of test isolates was adjusted to 5 × 105 CFU ml−1.
Determination of inhibition zone diameter
Susceptibility tests were performed by a modified agar well diffusion method (Okeke et al. 2001). One millilitre of standard suspension of each bacterial isolate was spread evenly on Mueller Hinton Agar (Himedia) plates using a sterile glass rod spreader, and the plates were allowed to dry at room temperature. Subsequently 6-mm-diameter wells were bored on the surface of different plates into which 100 μl of pure isolated compound from 1 mg ml−1 stock solution reconstituted in 0·5% dimethyl sulfoxide (DMSO) was added. After holding the plates at room temperature for 2 h to allow diffusion of the test materials into the agar, they were incubated at 37°C for 18 h. Inhibition zone diameter (IZD) was measured to the nearest millimetre (mm). Ciprofloxacin (1 μg ml−1) (Himedia) was used as experimental positive control and 0·5% DMSO as negative control. The tests were performed in triplicate for each micro-organism used.
Determination of minimum inhibitory concentration
Microbroth dilution susceptibility test was performed for minimum inhibitory concentration (MIC) determination in flat-bottom 96-well microtitre plates containing MHB medium (90 μl) in each well. The pure isolated compound reconstituted in 0·5% DMSO was diluted twofold serially with MHB ranging from 24·3 to 390 μg ml−1. One hundred microlitres of diluted solution was given in each well containing broth. Ten microlitres of working inoculum suspension (5 × 105 CFU ml−1) was added to the wells. A number of wells were reserved in each plate for control of sterility (no inoculum added), inoculum viability (no sample solution added) and DMSO inhibitory effect. The plates were then incubated for 18 h at 37°C. After incubation, 10 μl of Alamar Blue was added in each well and further incubated for 4 h for a colour change from blue to pink. A blue colour in the well was interpreted as no growth, and a pink indicated growth. MICs were determined as the lowest concentration of the drug that prevented the colour change from blue to pink (CLSI 2005).
Determination of MIC50
For MIC50 determination, the following formula of geometric means was used:
where M < 50 is the MIC of highest cumulative percentage below 50%, M > 50 is the MIC of lowest cumulative percentage above 50%, n is 50% of the number of organisms tested, x is the number of organisms in the group at M < 50 and y is the number of organisms in the group at M > 50 (Smith et al. 1986).
Combination effect with antibiotics
Combination effects of isolated pure compound with antibiotics were studied using checkerboard titration method (Leclercq et al. 1991). This method utilized an inoculum of 5 × 105 CFU ml−1 on MHB with the pure isolated compound and the antibiotics in combination ranging from 1/32 × MIC to 4 × MIC. The fractional inhibitory concentration (FIC) was derived from the lowest concentration of antibiotic and pure isolated compound combination putting no visible growth of the test organisms after incubation for 24 h at 37°C. FIC indices were calculated using the formula: FICindex = (MIC of antibiotic in combination/MIC of antibiotic alone) + (MIC of pure isolated compound in combination/MIC of pure isolated compound alone). The results were interpreted according to FICindices as follows: ‘synergy’ (FICI ≤ 0·5), ‘additive’ (FICI > 0·5–4) and ‘antagonism’ (FICI > 4). All the experiments were independently repeated thrice, and the data were expressed as arithmetic average.
Influence of metal ions on inhibitory activity of isolated gallotannin
Influence of metal ions on the inhibitory activity of isolated gallotannin was studied against drug-resistant E. coli isolates as indicator strain by a rapid INT (P-iodonitrotetrazolium violet) colorimetric assay (Eloff 1998). Briefly, MHB media (90 μl) were supplemented with 30 μl of ferric chloride, manganese sulfate, magnesium sulfate and calcium chloride to the concentrations ranging from 0·15 to 1·25 mmol l−1. Ten microlitres of working inoculum suspension (5 × 105 CFU ml−1) was added to each well. Serial twofold dilutions (24·3–390 μg ml−1) of isolated gallotannin 1,2,6-tri-O-galloyl-β-d-glucopyranose in MHB were prepared. One hundred microlitres of gallotannin solution was then given in each well. The plates were then incubated for at 37°C for 18 h. After incubation, 40 μl of INT was added in each well and further incubated for 30 min. Viable bacteria reduced the yellow dye to purple. The MIC was defined as the lowest sample concentration that prevented this change and that resulted in the complete inhibition of microbial growth (Eloff 1998).
Statistical analysis was performed using SPSS software version 18.0. Tukey's test was applied for statistical analysis with the level of significance set at P < 0·05.
The identified compound obtained as a yellowish brown amorphous powder, m.p. 229–232°C possessed chromatographic properties and colour reactions similar to those of galloyl glucose derivatives (intense blue colour with FeCl3 and reddish colour with KIO3) (Haddock et al. 1982), which was supported by UV spectral analysis, λmax at 220 and 280 nm. IR(KBr) spectrum indicated hydroxyl absorption at ν 3335 cm−1 and carbonyl absorption at ν 1712 cm−1. ESI-MS spectra gave peaks at m/z 659 and 675 attributed to [M + Na]+ and [M + K]+ corresponding to the molecular formula C27H24O18. The structure of this gallotannin was finally achieved by 1H- and 13C-NMR analysis as well as comparing its 1H- and 13C-NMR data with those reported in the literature (Nonaka et al. 1981; Haddock et al. 1982). 1H-NMR (CD3OD) spectrum revealed three aromatic singlets, each integrating for two protons at δ 7·10, 7·15 and 7·18, assignable to the protons of the three galloyl moieties in the molecule. In the sugar region, the spectrum showed three clearly downfield-shifted proton resonances. One 1H signal as doublet at δ 5·85 with large coupling constant could be attributed to a β-configurated glucose anomeric proton. A 1H triplet at δ 5·31 and two 1H signals at δ 4·61 (d, J = 12 Hz) and 4·47 (dd, J = 12, 4·4 Hz) were assigned to H-2 and H-6 glucose protons (Nawwar et al. 1994). The signals of these protons are significantly downfield compared with those in β-d-glucopyranose, suggesting the location of galloyl units at these centres (De Bruyn et al. 1977). 13C-NMR (CD3OD) analysis showed signals at δ 95·9 (anomeric C); 110·3, 110·5, 110·7 (galloyl C-2, 6); 120·1, 121·3, 121·6 (galloyl C-1); 139·8 and 140·5 (galloyl C-4); 146·4–146·6 (galloyl C-3, C-5); 166·9, 168·2 and 168·3 (galloyl C=O); 62·5 (glu C-6); 69·7, 72·6 (glu C-2, C-4); 76·4 (glu C-3);and 78·9 (glu C-5), thus indicating that the hydroxyl groups at C-1, C-2 and C-6 are galloylated (De Bruyn et al. 1977) and conforming the structure to be 1, 2, 6-tri-O-galloyl-β-d-glucopyranose, which represents, to the best of our knowledge, the first report of this gallotannin from T. chebula fruit (Table 1; Fig. 1).
Table 1. Spectral data of 1, 2, 6-tri-O-galloyl-β-d-glucopyranose
220, 280 nm (MeOH) (Ar-OH)
ν 3335 cm−1 (OH stretch), ν 1712 cm−1 (Carbonyl C)
In antimicrobial activity testing, the isolated compound showed varying degrees of strain-specific antibacterial activity against all 52 test strains. The mean IZDs of isolated compound against the studied clinical bacteria were ranging from 15·77 ± 0·97 to 28·33 ± 1·03 mm, and ciprofloxacin showed inhibition ranging from 18·00 ± 0·70 to 25·00 ± 1·09 mm (Table 2). MIC50 values of isolated compound against E. coli, K. pneumoniae, P. aeruginosa and S. aureus were found to be 15·15, 67·00, 27·78 and 12·10 μg ml−1, respectively (Table 3).
Table 2. Inhibition zone diameter of isolated compound from Terminalia chebula fruit against multidrug-resistant uropathogens
Table 3. MIC range and MIC50 of isolated compound from Terminalia chebula fruit against multidrug-resistant uropathogens
MIC range (μg ml−1)
MIC50 (μg ml−1)
MIC, minimum inhibitory concentration.
Escherichia coli (MDR) (n = 21)
Pseudomonas aeruginosa (MDR) (n = 9)
Klebsiella pneumoniae (MDR) (n = 16)
Staphylococcus aureus (MDR) (n = 6)
E. coli (ATCC 8739) (n = 1)
S. aureus (ATCC 6538P) (n = 1)
In combination study, trimethoprim and gentamicin exerted ‘synergy’ with the isolated compound against most of the test isolates, whereas amoxicillin, ceftazidime and ciprofloxacin showed an additive effect. No antagonistic effect was observed with the test antibiotics (Table 4).
Table 4. Combination effect of isolated pure compound from Terminalia chebula fruit with selected antibiotics against multidrug-resistant uropathogenic Escherichia coli
Presence of iron in the medium counteracted the growth inhibitory activity of isolated gallotannin but other metal (magnesium, calcium and manganese) ions had no effect (Fig. 2).
Tannins are water-soluble polyphenols that are commonly found in higher herbaceous and woody plants (Scalbert 1991). They can be classified into two categories: hydrolysable and nonhydrolysable (condensed). A gallotannin is a class of molecules belonging to the hydrolysable tannins. Gallotannins are polymers formed when gallic acid esterifies and binds with glucose (Miranda et al. 1996). Tannins and gallotannins have been reported to have antimicrobial property against a number of microbes (Scalbert 1991; Chung et al. 1993). The antimicrobial mechanisms of these compounds may be related to their action on the membranes of the micro-organisms or complexation of metal ions (Chung et al. 1998a,b).
Iron is an essential element for the growth and development of all the scale of living organisms, and acquiring iron is crucial for the development of any pathogens. Iron participates in a large number of processes, the most important of which are oxygen transport, ATP generation, cell growth and proliferation, and detoxification. It is a coenzyme or enzyme activator of ribonucleotide reductase, a key enzyme for DNA synthesis, which catalyses the conversion of ribonucleotides to deoxyribonucleotides (Thelander et al. 1983).
Chung et al. (1998a,b) reported that inhibitory effect of tannic acid (a gallotannin) on the growth of intestinal bacteria may be caused by its strong iron-binding capacity. They also reported that tannic acid inhibited the growth of all 15 of the bacteria tested, but gallic acid or ellagic acid did not inhibit any of them. They concluded that the ester linkage (to form gallotannin) was important to the antimicrobial potential of these compounds (Chung et al. 1993). Other workers also have reported that the complexation of iron contributes to the antibacterial mode of action of gallotannins (Cho et al. 2010; Engels et al. 2010).
From our foregoing findings, it was observed that the isolated gallotannin (Fig. 1) exhibited promising antibacterial activity against the studied bacteria (Table 2). MIC50 values showed the strong susceptibility of test clinical isolates towards the isolated gallotannin ranged from 12·10 to 67·00 μg ml−1 (Table 3). Besides antibacterial activity of isolated compound against the test isolates, the results obtained in the combination study that showed either synergy or additivity, but no evidence of antagonistic effects, are very encouraging. The isolated compound has the importance to control the test isolates under very low concentration of test antibiotics (trimethoprim and gentamicin) and thus minimizing the possible toxic effect. Furthermore, synergistic activity of the isolated compound enables the use of respective antibiotics when it is no longer effective by itself during therapeutic treatment. Results further revealed that the supplementation of iron in the MHB medium decreased the antimicrobial activity of isolated gallotannin against E. coli, but magnesium, manganese and calcium had no effects (Fig. 2). These findings indicated that mineral complexation by isolated gallotannin is selective and strongly supports the view that the antibacterial activity of isolated gallotannin against the studied bacteria may be due to its strong iron-complexing property, which may be one of the possible mechanisms of antibacterial activity of this gallotannin.
The overall results of this study can be considered as very promising in the perspective of new drug discovery from plant sources and suggest the feasibility of producing powerful antimicrobial drug from T. chebula fruits against the multidrug-resistant uropathogens.
Acknowledgements and funding
The authors wish to acknowledge the Heads, Sophisticated Analytical Instruments Facility (SAIF) Units, Central Drug Research Institute, Lucknow, North-Eastern Hill University, Shillong and Bose Institute, Kolkata, for their kind help in performing the spectroscopic analysis of samples in their laboratories and also to Dr. Sukdeb Bandopadhyay, Chemistry Division, Indian Institute of Chemical Biology, Kolkata, for interpreting the spectral data. This work was supported by Indian Statistical Institute, Kolkata, India [Internally funded Project: A/C No. 5613 (2008-2011)].