Reversing [beta]-lactam antibiotic resistance of Staphylococcus aureus with galangin from Alpinia officinarum Hance and synergism with ceftazidime.
The purpose of this investigation was to extract and identify the bioactive phytochemicals from smaller galanga (Alpinia officinarum Hance). The antibacterial, synergy effects and primary mechanism of action of galangin and ceftazidime against S. aureus DMST 20651 are also investigated by minimum inhibitory concentration (MIC), checkerboard, killing curve determinations, enzyme assay and electronmicroscopy method. The rhizomes chloroform extract of this plant showed that these compounds were galangin, kaempferide and kaempferide-3-O-[beta]-D-glucoside, which had not been previously reported in this species. Synergistic FIC indices were observed in the combination of test flavonoids (galangin, quercetin and baicalein) and all selected [beta]-lactams (methicillin, ampicillin, amoxicillin, cloxacillin, penicillin G and ceftazidime) (FIC index, <0.02-0.11). The combination of ceftazidime at 5 [mu]g/ml and 5 [mu]g/ml of test flavonoids (galangin, quercetin and baicalein) exhibited synergistic effect by reduced the cfu/ml of this strain to 1 x [10.sup.3] over 6 and throughout 24 h. Galangin showed marked inhibitory activity against penicillinase and [beta]-lactamase. Eiectronmicroscopy clearly showed that the combination of galangin and ceftazidime caused damage to the ultrastructures of the cells of this strain. It was concluded that galangin, quercetin and baicalein exhibited the potential to reverse bacterial resistance to [beta]-lactam antibiotics against penicillin-resistant S. aureus (PRSA). This may involve three mechanisms of action that galangin inhibit protein synthesis and effect on PBP 2a, interact with penicillinase and cause cytoplasmic membrane damage. These findings lead us to develop a new generation of phytopharmaceuticals that may use galangin, quercetin and baicalein in combination with ceftazidime to treat PRSA that currently almost unbeatable microorganism. The anti-PRSA activity and mode of action of galangin is reported for the first time. These in vitro results have to be still confirmed in an animal test or in humans.
Keywords: Alpinia officinarum Hance Galangin Quercetin Baicalein The synergism with ceftazidime Penicillin-resistant Staphylococcus aureus
[C] 2010 Elsevier GmbH. All rights reserved.
Bacterial resistance to [beta]-lactam antibiotics is a global problem. Today around 90-95% and 70-80% of Staphylococcus aureus (S. aureus) strains are resistant to penicillin, methicillin around the word and in most of the Asian countries (Casal et al. 2005; Chambers 2001) Strains of [beta]-lactam-resistant S. aureus including methicillin-resistant S. aureus (MRSA) now pose serious problem to hospitalized patients, and their care providers (Mulligan et al. 1993). Antibiotics available for the treatment of MRSA infection are fairly toxic and their use is frequently associated with unwanted side-effects(Brumfitt and Hamilton-Miller 1989]. Novel antibiotics and/or new generation of phytopharmaceuticals approaches that can reverse the resistance to well tried agents which have lost their original effectiveness or enable their use to treat diseases instead of synthetic drugs alone are research objectives of far reaching importance (Reading and Cole 1977; Wagner and Ulrich-Merzenich 2009).
Smaller galanga (Alpinia officinarum Hance) is a pungent and aromatic rhizome, which is a member of the ginger family (Zin-giberaceae). The rhizome is cultivated in India, Vietnam, Southern China and Thailand because of its use as a spice and as a traditional medicine for several purposes such as treatment for pyogenic diseases (infectious acne, carbuncles, sty, pyoderma, pustular impetigo in Thailand), ring worm, venereal diseases, carminative, abdominal discomfort (Athamaprasangsa et al. 1994). The chemical and pharmacological studies of the rhizomes of small galanga have three groups of important chemical constituents, flavonoids, glycosides and diarylheptanoids. It has been reported that smaller galanga has biological activities, including antitumor, antiulcer, antibacterial, and antifungal properties (Itokawa et al. 1985; Newman et at. 2003; Ly et al. 2003; Matsuda et al. 2006). The purpose of this investigation was to separate and identify the bioactive compounds from the rhizome of smaller galanga. We have also investigated the in-vitro activity of galangin, a major bioactive constituent isolated from smaller galanga, and other test flavonoids (quercetin and baicalein) against [beta]-lactam-resistant S. aureus when used alone and in combination with [beta]-lactam antibiotics.
Materials and methods
General experimental procedures
The UV spectra were obtained with a Hewlett Packard 8452A diode array UV-vis spectrophotometer, whereas the IR spectra were measured with a Perkin-Elmer FT-IR 2000 spectrophotometer (by a KBr disk method). The [sup.1.H] and[sup.13.C] NMR spectra were recorded with a Bruker DRX 400 spectrometer in CD30D solution and chemical shifts are expressed in [delta] (ppm) with reference to the solvent signals. Silica gel 60 (70-230 mesh) and silica gel 60 PF 254 were used for column chromatography and preparative thin-layer chromatography, respectively. Solvents of technical grade were used for chromatographic purposes.
Plant material, [beta]-lactam antibiotics and bacterial strains sources
The fresh rhizomes of smaller galanga were digged from Saengduan Konekratoke's paddy field located in Chokchai District, Nakhonratchasima Province in July and December 2007, June and November 2008. The plant specimen has been deposited at the National Herbarium after it was identified by Dr. Paul J. Grote, School of Biology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima Province. The rhizomes of smaller galanga was separated from the stems, washed thoroughly, and dried in an oven at 50[degrees]C for three days. The dried samples were then ground to powder. Quercetin and baicalein were obtained from Indofine Chemical Company (USA). Ceftazidime, methicillin, ampicillin, amoxicillin, cloxacillin, penicillin G (benzylpenicillin), penicillinase ([beta]-lactamase) and clavulanic acid were obtained from Sigma (Sigma-Aldrich, UK). Mueller-Hinton broth was obtained from Oxoid (Basingstoke, UK). Seven clinical isolates of penicillins-resistant S. aureus DMST 20651-655, 20661-2 (PRSA), were obtained from Department of Medical Sciences, Ministry of Public Health, Thailand. S. aureus ATCC 29213, positive control, was purchased from American Type Culture Collection (ATCC).
Extraction and isolation
The 2 kg of dried powder of rhizomes of smaller galangal were extracted consecutively with hexane, chloroform and methanol by soxhlet extraction apparatus. The extracted solutions were then filtered. The filtrates were concentrated by evaporation under reduced pressure to afford 13.79g of hexane crude extract as dark yellow oil, 22.38 g of chloroform crude extract as dark yellow slush, and 30.12 g of methanol crude extract as dark brown gum.
The chloroform crude extract was separated using a column packed with hexane slurry of silica gel. Then, was dissolved in chloroform and loaded onto the column. Four major fractions (13.24 g, II 5.76g, III 9.72 g and IV 13.93 g) were separated by TLC.
A portion of fraction III (1.0085g) was further separated by preparative TLC to give crude compound 1 (0.0105g), which was recrystallized from chloroform-methanol mixed solvents to obtain pure compound 1 (0.0007 g).
Fraction IV (13.93g) was further separated using a column with hexane, then by preparative TLC to afford three fractions (C 0.0109 g, D 0.0127 g and E 0.0155 g). Fraction C and D were further purified by preparative TLC to give crude compounds 2 and 3 which were recrystallized from methanol to obtain pure compounds 2 (0.0027g) and 3 (0.0031 g).
Structures of compounds 1-3
Structural elucidation of the isolated compounds was carried out on the basis of spectral analyses, including UV, IR, MS, [.sup.1.H] NMR and [.sup.13.C] NMR, as well as comparison with reported values in the literature.
Bacterial suspension standard curve
To select bacterial suspensions with a known viable count, the method of Liu et al. (2000) was followed.
Minimum inhibitory concentration (MIC) and checkerboard determinations
MIC and checkerboard determinations of selected [beta]-lactam drugs against eight S. aureus strains were performed by following Liu et al. (2000) and Matthew et al. (2006).
Killing curve determinations
Viable counts for the determination of killing-curves were performed as previously described by Richards and Xing (1993).
The penicillinase of Bacillus cereus (B. cereus) and [beta]-lactamase of Enterobacter cloacae (E. cloacae) were obtained from Sigma (Poole, England). Enzymes activities were followed Richards et al. (1995).
Ceftazidime and galangin that dramatically decreased the MICs against S. aureus DMST 20651 (PRSA) were chosen for electronmicroscopy study when used singly and in combination. Subculture of this strain was prepared to examine by TEM following Richards et al.(1995).
Results and discussion
Compounds 1, 2 and 3 were obtained and the UV, IR, [.sup.1.H] NMR and [.sup.13.C] NMR spectrum showed that compounds 1, 2 and 3 are 3,5,7-trihydroxyflavone (galangin), 3,5,7-trihydroxy-4'-methoxyflavone (kaempferide) and kaempferide-3-O-[beta]-D-glucoside, respectively (Fig. 1). All spectronic data agree with those reported in the literature (Rubens and Wangner 2005; Eunjung et al. 2008; Agrawal 1992; Juha-Pekka et al. 2004). Keampferide and kaempferide-3-O-[beta]-D-glucoside had not been previously reported in this species.
[FIGURE 1 OMITTED]
MIC and checkerboard determinations
The MICs for test flavonoids (galangin, quercetin and baicalein) and ceftazidime against six clinical isolates strains of PRSA are shown in Table 1. The MICs of ceftazidime, test flavonoids (galangin, quercetin and baicalein) and clavulanic acid alone against six strains of S. aureus DMST were 50, 200 to >400 and >128 [mu]g/ml, respectively. In the ceftazidime plus test flavonoids or clavulanic acid combination, the FICs of ceftazidime and test flavonoids or clavulanic acid were 5-50 [mu]g/ml and 5 or >128 [mu]g/ml, respectively. The FIC indices of ceftazidime plus test flavonoids or clavulanic acid were calculated as being between <0.11 and 0.22 or 2.0, respectively, in these combination against all isolates strains. It has been proposed that synergy be declared when the FIC index [less than or equal to] 0.5 (Johnson et al. 2004). Thus, the ceftazidime plus test flavonoids combination was synergistic against all isolates strains. S. aureus ATCC 29213 and clavulanic acid were used as positive control. Table 2 shows MICs and FIC index from checkerboard assay of [beta]-lactam use alone and in combination with test flavonoids (galangin, quercetin and baicalein) or clavulanic acid against clinical isolates of S. aureus DMST 20651. Synergistic FIC indices were observed in the combination of test flavonoids and all selected [beta]-lactams (methicillin, ampicillin, amoxicillin, cloxacillin, penicillin G and ceftazidime) (FIC index, <0.02-0.11). However, no interaction FIC indices of combination between amoxicillin or ceftazidime and clavulanic acid against this strain were observed (FIC index, 2.0).
Table 1 Minimum inhibitory concentration (MIC) of ceftazidime, clavulanic acid and test flavonoids alone and fractional inhibitory concentration (FIC) from checkerboard assay of ceftazidime plus clavulanic acid or test flavonoids against clinical isolates of Staphylococci aureus (PRSA). Strain MIC ([mu]g/ml) FIC ([mu]g/ml) cef gal que bai cla cef + gal cef + que cef + bai DMST 50 300 >400 >400 >128 10 + 5 10 + 5 10 + 5 20652 DMST 50 300 >400 >400 >128 10 + 5 10 + 5 10 + 5 20653 DMST 50 300 >400 >400 >128 10 + 5 10 + 5 10 + 5 20654 DMST 50 300 >400 >400 >128 10 + 5 10 + 5 10 + 5 20655 DMST 50 200 >300 >400 >128 5 + 5 5 + 5 5 + 5 20661 DMST 50 200 >300 >400 >128 5 + 5 5 + 5 5 + 5 20662 ATCC 0.12 100 >200 >300 4 N/D N/D N/D 29213 (a) Strain FIC ([mu]g/ml) FIC index cef + cla cef + gal cef + que cef + bai cef + cla DMST 50 + >128 0.22 <0.21 <0.21 2.0 20652 DMST 50 + >128 0.22 <0.21 <0.21 2.0 20653 DMST 50 + >128 0.22 <0.21 <0.21 2.0 20654 DMST 50 + >128 0.22 <0.21 <0.21 2.0 20655 DMST 50 + >128 0.13 <0.12 <0.11 2.0 20661 DMST 50 + >128 0.13 <0.12 <0.11 2.0 20662 ATCC N/D N/D N/D N/D N/D 29213 (a) cef = ceftazidime, gal = galangin, que = quercetin, bai = baicalein, cla = clavulanic acid. N/D, no data. Each compound was measured three times. (a). S. aureus ATCC 29213 and clavulanic acid were used as positive control. Table 2 Minimum inhibitory concentration (MIC) of [beta]-lactams, clavulanic acid and test flavonoids alone and fractional inhibitory concentration (FIC) from checkerboard assay of [beta]-lactams plus test flavonoids or clavulanic acid against clinical isolates of S. aureus DMST 20651. Compound MIC FIC ([mu]g/ml) FIC index ([mu]g/ml) ([beta]-lactam + alone flavonoid) Methicillin >1000 - - Galangin 400 20 + 5 <0.03 Quercetin >400 20 + 5 <0.03 Baicalein >400 20 + 15 <0.06 Ampicillin >1000 - - Galangin 400 15 + 5 <0.03 Quercetin >400 15 + 5 <0.03 Baicalein >400 15 + 10 <0.05 Amoxycillin 250 - - Galangin 400 10 + 5 0.05 Quercetin >400 10 + 5 <0.05 Baicalein >400 10 + 10 <0.07 Clavulanic acid (a) >128 250 + >128 2.0 Cloxacillin >1000 - - Galangin 400 10 + 5 <0.02 Quercetin >400 10 + 5 <0.02 Baicalein >400 10 + 5 <0.02 Penicillin G 250 - - Galangin 400 10 + 5 0.05 Quercetin >400 10 + 5 <0.05 Baicalein >400 10 + 15 <0.08 Ceftazidime 50 - - Galangin 400 5 + 5 0.11 Quercetin >400 5 + 5 <0.11 Baicalein >400 5 + 5 <0.11 Clavulanic acid (a) >128 50 + >128 2.0 Each compound was measured three times. (a) Clavulanic acid was used as positive control.
Killing curve determinations (viable counts)
Fig. 2 shows that viable counts for S. aureus DMST 20651 were slight reduced by ceftazidime at 20 [mu]g/ml, 50 [mu]g/ml of test flavonoids and 128 [mu]g/ml clavulanic acid alone when compared with the level of the untreated control culture between 6 and 24 h period. Ceftazidime at 5 [mu]g/ml in combination with 5 [mu]g/ml of test flavonoids reduced the cfu/ml by 5 x [10.sup.3] over 6 h. The reduced counts did not recover in 24 h. Whereas, the combination of 20 [mu]g/ml ceftazidime plus 128 [mu]g/ml clavulanic acid was slightly lower than control cells.
[FIGURE 2 OMITTED]
The ability of flavonoids to inhibit the in vitro activity of penicillinase and [beta]-lactamases varied considerably. Fig. 3a indicates that galangin has an inhibitory activity against penicillinase I from B. cereus. Galangin had some activity and tectochrysin and 6-chloro-7-methylflavone showed greater activity. Fig. 3b shows the effects of galangin against penicillinase ([beta]-lactamase) type IV from E. cloacae. Galangin showed marked inhibitory activity. These results indicated that in addition to the direct effect on cell structure and cell division, the resistance reversing activity of galangin against PRSA might also include inhibition of penicillinase activity.
[FIGURE 3 OMITTED]
Electronmicroscope investigations clearly showed that the combination of ceftazidime antibiotic with galangin caused damage to the ultrastructures of S. aureus DMST 20651 cells. Fig. 4 indicates that galangin 50 [mu]g/ml reduced the thickness of the cell walls compared with the cell walls of the control cells and also apparently delayed cell division. The galangin treated cells were considerably bigger than the normal cells. Ceftazidime 25 [mu]g/ml alone apparently had no effect on the cell wall structure but the combination of ceftazidime 5 [mu]g/ml plus galangin 5 [mu]g/ml was observed to have affected the integrity of the cell walls and led to an increase in cell size. This latter effect could due to inhibition of cell division.
[FIGURE 4 OMITTED]
The present study showed that the chloroform extract of the rhizomes of smaller galanga (Alpinia officinarum Hance) were separated to afford three pure compounds. They were characterized as 3,5,7-trihydroxy flavone (galangin) 1,3,5,7-trihydroxy-4'-methoxy flavone (kaempferide) 2 and 5,7-dihydroxy-4'-methoxy-3-O-[beta]-D-glucopyranoside flavone (kaempferide-3-O-[beta]-D-glucoside) 3, which had not been previously reported in this species.
The results of MICs of test flavonoids (galangin, quercetin and baicalein) against all PRSA strains (200 to >400 [mu]g/ml) are in substantial correspondence with Pepeljnjak and Kosalec (2004) that galangin isolated from propolis showed MIC of 160 [+ or -] 30 [mu]g/ml against ten clinical isolates of MRSA strains. The results of checkerboard and viable counts of S. aureus DMST 20651 indicated that synergistic effects between test flavonoids (galangin, quercetin and baicalein) and ceftazidime or selected [beta]-lactam against this strain were occurred. These findings are in substantial agreement with those of Hemaiswarya et al. (2008) reported that flavonoids and synthetic drugs exhibited synergistic activity against bacteria. The results from enzyme assay can be explained by assuming that galangin interact with penicillinase. Consequently, free benzylpenicillin remainder can overcome bacteria. These results are similar to those of Denny et al. (2002) that galangin inhibited metallo-[beta]-lactamase by orientation at the active site of the enzyme. Moreover, epigallocatechin gallate, a flavan-3-ole flavonoid, showed penicillinase (from S. aureus) inhibition (Zhao et al. 2002). Furthermore, Cushnie and Lamb (2005) found that galangin caused potassium loss from S. aureus cells due to cytoplasmic membrane damage.
These results indicated that galangin not only have weak activity of their own against [beta]-lactam-resistant staphylococci but also have the ability to reverse the resistance of such bacterial strains to the activity of the primary antibiotics. This may involve three mechanisms of action by galangin. The first is on the integrity of the cell wall and on septum formation prior to cell division. This implies an effect on protein synthesis including an effect on penicillin-binding protein 2a (PBP 2a). The second mechanism of galangin activity is via inhibition of the activity of certain penicillinase enzyme by interaction with this enzyme. The third is galangin causes cytoplasmic membrane damage results in potassium loss. Galangin was found that there was no cross-resistance between it and the 4-quinolone drugs (Cushnie and Lamb 2006).
In the last two decades, [beta]-lactamase inhibitors like clavulanic acid have played an important role in fighting [beta]-lactam-resistant bacteria. These inhibitors work as suicide compounds to react with the enzymes since they share the same key structure with [beta]-lactam antibiotics (Coulton et al., 1994). Recent studies demonstrated that clavulanate caused a considerable induction of [beta]-lactamase expression and an increase of clavulanate concentration was followed by an elevation in [beta]-lactamase production (Tzouvelekis et al. 1997; Stapleton et al. 1995). This indicates that the presently available [beta]-lactamase inhibitors can also lose their activity by the same mechanism as the [beta]-lactam antibiotics. Our research provides a unique example that galangin, quercetin and baicalein without a [beta]-lactam structure can reverse bacterial resistance to [beta]-lactams via multiple mechanisms. Because of this structural dissimilarity, these compounds are unlikely to induce [beta]-lactamase production. It should also be remembered that conventional [beta]-lactamase inhibitors, unlike flavonoids, cannot reverse the resistance of penicillin resistant S. aureus, which is one of the most dangerous bacterial pathogens. Galangin, quercetin and baicalein as a new generation of phytopharmaceuticals, may be used with ceftazidime or [beta]-lactam drugs for treating PRSA infection that cannot treat with this drugs alone. The anti-PRSA activity and mode of action of galangin is reported for the first time.
From the study, it was concluded that galangin, quercetin and baicalein have the potential to reverse bacterial resistance to [beta]-lactam antibiotics against PRSA. In view of their limited toxicity, These test flavonoids offer for the development of a valuable adjunct to [beta]-lactam treatments against otherwise resistant strains of currently almost untreatable microorganisms. These in vitro results have to be still confirmed in an animal test or in humans.
The authors are indebted grateful to the following persons and institutions for their invaluable assistance in carrying out this study: The Thailand Research Fund for grant support, Suranaree University of Technology Research and National Research Council of Thailand for research fund, Prof. R.M.E. Richards for encouragement and suggestion, Department of Medical Sciences, Ministry of Public Health, Thailand, for bacterial strains support, Department of Chemistry, Mahidol University for the NMR data acquisition.
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Griangsak Eumkeb (a), * Santi Sakdarat (b), Supatcharee Siriwong (a)
(a) School of Biology, Institute of Science, Suranaree University of Technology. Nakhonrachasima 30000, Thailand
(b) School of Chemistry, Institute of Science, Suranaree University of Technology. Nakhonratchasima 30000. Thailand
* Corresponding author at; School of Pharmacology/Biology, Institute of Science, Suranaree University of Technology, 111 University Avenue, Suranaree Subdistrict, Muang District, Nakhonratchasima 30000, Thailand. Tel.: +66 44 224260; fax: +66 44 224633.
E-mail address: email@example.com (G. Eumkeb).
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|Author:||Eumkeb, Griangsak; Sakdarat, Santi; Siriwong, Supatcharee|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Dec 15, 2010|
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