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Potential antibiotic and anti-infective effects of rhodomyrtone from Rhodomyrtus tomentosa (Aiton) Hassk. On Streptococcus pyogenes as revealed by proteomics.

doi: 10.1016/j.phy med.2011.02.007


Rhoclomyrtone from Rhodomyrtus tornentosa (Aiton) Hassk. leaf extract has a strong antibacterial activity against the bacterial pathogen Streptococcus pyogenes. Our previous studies indicated that the bactericidal activity of rhodomyrtone might involve intracellular targets. In the present studies we followed a proteomics approach to investigate the mode of action of rhodomyrtone on S. pyogenes. For this purpose, S. pyogenes was cultivated in the presence of 0.39 [micro]g/ml rhodomyrtone, which corresponds to 50% of the minimal inhibitory concentration. The results show that the amounts of various enzymes associated with important metabolic pathways were strongly affected, which is consistent with the growth-inhibiting effect of rhodomyrtone. Additionally, cells of S. pyogenes grown in the presence of rhodomyrtone produced reduced amounts of known virulence factors, such as the glyceraldehyde-3-phosphate dehydrogenase, the CAMP factor, and the streptococcal pyrogenic exotoxin C. Taken together, these findings indicate that rhodomyrtone has both antimicrobial and anti-infective activities, which make it an interesting candidate drug.

[c] 2011 Elsevier GmbH. All rights reserved.


Keywords: Rhoclomyrtone Rhodomyrtus tornentosa Glycolysis Proteomics Streptococcus pyogenes Two-dimensional gel electrophoresis


Streptococcus pyogenes is the causative agent of a wide range of invasive and non-invasive infections in humans. These include life-threatening infections, such as streptococcal toxic shock syndrome (STSS). The global emergence of antibiotic resistance in S. pyogenes (Gracia et al. 2009; Michos et al. 2009; Malli et al. 2010), and the increasing failure rates of antibiotic therapy against S. pyogenes infections (Pichichero et al. 2000; Kaplan and Johnson 2001; Pichichero and Casey 2007) are therefore causes of serious concern. Natural products, mainly medicinal plants, have been extensively studied for alternative treatment of S. pyogenes infections. These include plants from Thailand with strong antibacterial activities that can potentially be developed into effective drugs (Limsuwan and Voravuthikunchai 2008; Limsuwan et al. 2009a). Moreover, such plants may play important roles in the development of new lead drugs with thus far unknown modes of action that can replace or substitute currently used antibiotics to combat resistant bacterial pathogens like S. pyogenes in the future.

Intensive studies on Rhodomyrtus tomentosa (Aiton) Hassk. as an anti-infective agent have been reported by our research groups. We showed that the leaf extract of Rhodomyrtus tomentosa has a very strong antibacterial activity (Voravuthikunchai et al. 2007; Limsuwan and Voravuthikunchai 2008; Saising et al. 2008; Limsuwan et al. 2009a,b). Rhodomyrtone, a pure compound isolated from this plant was claimed to possess significant antibacterial activity against Escherichia coli and Staphylococcus aureus (Dachriyanus et al. 2002). Notably, our recent studies revealed that this compound displays significant bactericidal activities against many Gram-positive bacteria, but not Gram-negative bacteria. Especially noteworthy was the activity against S. pyogenes with a very low minimal inhibitory concentration (MIC = 0.39-0.78 [micro]g/ml) (Limsuwan et al. 2009b). Furthermore, our studies showed that rhodomyrtone did not cause lysis of S. pyogenes, suggesting that the primary mechanism of bactericidal action is not related to gross cell envelope damage (Limsuwan et al. 2009b). Therefore, the present studies were aimed at pinpointing the antibacterial and anti-infective mechanism(s) of rhodomyrtone on S. pyogenes. A proteomics approach was employed to define alterations in the production of cellular and secreted S. pyogenes proteins after treatment with a sub-minimal inhibitory concentration (50% of MIC) of rhodomyrtone.

Materials and methods

Plant material and compound

Plant extract preparation and rhodomyrtone isolation were performed according to our previous published data (Hiranrat and Mahabusarakam 2008; Saising et al. 2008; Limsuwan et al. 2009b). The purified rhodomyrtone was dissolved in dimethyl sulfoxide (DMSO, Merck, Germany) before use.

Bacterial strain and culture condition

A clinical isolate of S. pyogenes was taken from the collection of the Laboratory of Molecular Bacteriology, Department of Medical Microbiology, University Medical Center Groningen (UMCG), the Netherlands. This strain was cultured on blood agar plates (BA, Difco, France), or in brain heart infusion (BHI) broth (Difco, France) at37[degrees]C

Determination of bacterial growth

For growth experiments, S. pyogenes was cultured in BHI broth with rhodomyrtone at 50% MIC (0.39 [micro]g/ml). The bacterial growth was recorded by optical density readings at 600 nm ([OD.sub.600]). The period of growth that produced the same numbers of the bacterial cells in the presence of rhodomyrtone or the control culture with 1% DMSO was determined. This period of growth was used to study protein production by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional (2D) PAGE.

One-dimensional SDS-PAGE analysis of secreted proteins and cellular proteins

S, pyogenes was cultured in 2 ml of BHI broth with rhodomyrtone or 1 % DMSO. The cultures at late stationary phase were adjusted to [OD.sub.600] 1.0. The culture was centrifuged at 20,800 x g for 10min. Next, the cell supernatant was collected and proteins were precipitated with trichloroacetic acid (final concentration 10%). The precipitated proteins were further centrifuged at 20,800 xg for 20min and the supernatant was removed. The precipitate was washed with cold acetone and centrifuged at 20,800 x g for again lOmin. After acetone washing, the precipitate was dried and dissolved in SDS-PAGE loading buffer (Invitrogen, USA).To analyze the bacterial cellular proteins, the cell pellets were disrupted by violent agitation for 2 min with a Mini-BeadBeater-16 (Biospec Products, UK) in screw-cap microvials containing glass beads and SDS-PAGE buffer. The samples were heated to 95 [degrees] C for 10 min prior to gel loading. 20 [micro]l of sample and 8 [micro]l of SDS-PAGE marker (Fermentas, Germany) were separated by electrophoresis using a NuPage system with a 4-12% Bis-Tris gel (Invitrogen, USA) in combination with MESSDS Running Buffer (Invitrogen, USA). After electrophoresis, the gels were stained with Coomassie brilliant blue to visualize protein bands (Nakamura et al. 2004; Tanaka et al. 2005).

Two-dimensional PACE analysis of secreted proteins and cellular proteins

S. pyogenes was cultured in 1000 ml of BHI broth with rhodomyrtone at 50% MIC or 1% DMSO. After the incubation, the cultures were adjusted to [OD.sub.600] 0.5. The culture was centrifuged 11,180 x g at 15 C for 20 min, the supernatant was collected and precipitated with 10% trichloroacetic acid and placed overnight on ice. The precipitated proteins were centrifuged 22,600 x g at 4[degrees]C for 60min and the supernatant was removed. The protein pellets were washed 3 times with 70% ethanol. After washing, the precipitate was dried and dissolved in 8 M urea/2 M thiourea. The dissolved proteins were precipitated again with 80% acetone and centrifuged 11,180 x gat room temperature for 10 min. After centrifugation, the precipitated proteins were air-dried and kept at -80 C until use. For bacterial cellular proteins, the cell pellets were washed twice with 20 mM Tris (10,600 x g 10min). After removal of the washing liquid, the bacterial cells were resuspended in lysis buffer (50 mM Tris, 0.3% SDS, 200 mM DTT, 50 mM magnesium chloride, 1 mg/ml DNAse, 150 units/ml mutanolysin). Glass beads were added and a FastPrep (Qbiogene, UK) was used to disrupt the cells (30 s for 2 times). The samples were centrifuged at 10,600 and 20,800 x g at 4 [degrees] C for 5 and 8 min, respectively. Protein-containing supernatant was stored at -80 [degrees] C Aliquots (70-140 [micro]l) of the samples were prepared for 20-PAGE and differentially expressed protein spots were excised as described by Mateo Leach et al. (2009). The molecular weight was estimated according to the internal running calibration marker. The experiments were repeated at least three times to confirm their reproducibility.

In-gel trypsin digestion and MALDI-TOF peptide mapping

The Coomassie brilliant blue G-250 stained gels were scanned and a comparison between gel images of protein profiles obtained fromS. pyogenes cells treated with rhodomyrtone and the untreated control cells was performed. Spots of up-regulated and down-regulated proteins were excised from the gels, carefully cut to small pieces, transferred to Eppendorf tubes, and washed with 70% ammonium bicarbonate (25 mM) and 30% acetonitrile. Tubes were shaken for 30min and the washing solution was removed. Samples were washed again with an equal volume of 25 mM ammonium bicarbonate and acetonitrile. Tubes were shaken again for 30min, the supernatants were removed, acetonitrile (100%) was added, and samples were again shaken for 5min. After washing, the gel pieces were dried, digested with 10ng/|xl trypsin, and incubated overnight at 37 C. The trypsin-digested proteins were spotted with a-cyano-4-hydroxy-cinnamic acid on a target for matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS). Mass spectra were recorded with a Proteomics analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, USA). All of the tandem mass spectra were analyzed against a public database (National Centre for Biotechnology Information) with MALDI fingerprint data by MASCOT for web search (


Growth ofS. pyogenes in the presence of rhodomyrtone

The growth of S. pyogenes in the presence of rhodomyrtone at 50% MIC was determined by optical density readings at 600 nm (Fig. 1). Under these conditions, rhodomyrtone caused an extended lag phase of 10 h in the bacterial growth after which the cells started to grow, reaching stationary phase after 14 h. The optical density of cells in the stationary phase was about twofold lower than that of control cells which were cultured without rhodomyrtone up until 24 h of cultivation. Although growth inhibition activity of rhodomyrtone was observed at 50% MIC, we decided to select this concentration for our further studies, because the bacteria were still able to grow and achieve a substantial cell density in stationary phase.

Proteomics analysis of the effects of rhodomyrtone on S. pyogenes

Alterations in the secreted and bacterial cell protein patterns of S. pyogenes following treatment with 50% MIC of rhodomyrtone were first analyzed by one-dimensional SDS-PAGE. However, to the limited resolution of the proteins, it was not possible to detect clear differences in the amounts of cellular or secreted proteins of cells grown in the presence of rhodomyrtone (data not shown). Therefore, a 2D PAGE analysis was performed. Clear alterations in the cellular and secreted protein patterns of S. pyogenes cells after treatment with 50% MIC of rhodomyrtone are shown in Figs. 2 and 3, respectively. Interestingly, more spots of cellular and secreted S. pyogenes proteins were detectable in the samples of cells treated with rhodomyrtone (50% MIC). Unique protein spots (54 spots) of cellular proteins from the untreated (10 spots) and the rhodomyrtone-treated cells (33 spots), as well as secreted proteins from the growth media of untreated cells (6 spots) and the rhodomyrtone-treated cells (5), were isolated and identified by MALDI-TOF MS. Twenty-three spots of proteins from untreated (7 spots) and treated (16 spots) S. pyogenes cells, together with 6 spots of secreted proteins from the untreated (3 spots) and treated cells (3), were identified as known proteins from S. pyogenes. The Identified proteins with significant Mascot scores (P>0.05) and their functions are listed in Table 1. Nearly all of the proteins altered in significant amounts were identified as enzymes associated with important metabolic pathways, such as alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Xaa-His dipeptidase, ornithine carbamoyltransferase, putative O-acetylserine lyase, enolase (2-phosphoglycerate dehydratase), fructose-bisphosphate aldolase, and cysteine synthase. Four of these enzymes including alcohol dehydrogenase, GAPDH, enolase, and fructose-bisphosphate aldolase, are involved in the glycolysis and gluconeogenesis pathways (Fig. 4), while other enzymes such as the Xaa-His dipeptidase, ornithine carbamoyl-transferase, a putative O-acetylserine lyase and cysteine synthase are involved in pathways of amino acid metabolism. It should be noted that GAPDH was identified as one spot in samples of untreated cells, whereas in samples of rhodomyrtone-treated cells one extracellular and three cellular and spots were detected for this protein. Judged by their electrophoretic mobility, two of the GAPDH spots from the cytoplasmic fraction of rhodomyrtone-treated cells are probably degradation products of this protein. Likewise, two spots for the Xaa-His dipeptidase were detected in rhodomyrtone-treated cells, one of which is most likely a degradation product.



Table 1
Functions of S. pyogenes proteins that were detectable in different
amounts when cells were grown in the presence or absence of
rhodomyrtone at 50% MIC

Spot No.     Protein name/Mascot score    Organism


4            Alcohol dehydrogenase/89     S.
             (78)                         pyogenes

5            Glyceraldehyde-3-phosphate   S.
             dehydrogenase/71 (67)        pyogenes
1            Xaa-His dipeptidase/78       S.
             (78)                         pyogenes

4            60 kDa chaperonin (protein   S.
             Cpn60) (groEL protein )/85   pyogenes
             (67)                         M3

8            Ornithine                    S.
             carbamoyltransferase/75      pyogenes
             (67)                         M28

10           Putative O-acetylserine      S.
             lyase/171 (67)               pyogenes
                                          Ml 8

12           Enolase (2-phosphoglycerate  S.
             dehydratase)/75(67)          pyogenes

13           Peptide deformylase/106      S.
             (67)                         pyogenes

16           Fructose-bisphosphate
             aldolase/78 (67)             pyogenes

4            CAMP factor/89 (78)          S.

6            Streptococca1 pyrogenic      S.
             exotoxin C/l 14 (78)         pyogenes

1            Cysteine synthase/68 (67)    S.

2            Putative O-acetylserine      S.
             lyase/146 (78)
Spot No.     Protein name/Mascot score    Related pathway or function
             (b)                          (a)

4            Alcohol dehydrogenase/89     Glycolysis/gluconeogenesis
                                          Fatty acid metabolism
                                          Tyrosine metabolism
                                          1-and 2-Methylnaphthalene
                                          3-Chloroacrylic acid

5            Glyceraldehyde-3-phosphate   Glycolysis/gluconeogenesis
             dehydrogenase/71 (67)

1            Xaa-His dipeptidase/78       Arginine and proline
             (78)                         metabolism
                                          Histidine metabolism
                                          [beta]-Alanine metabolism
                                          Glutathione metabolism

4            60 kDa chaperonin (protein   Prevents misfolding and
             Cpn60) (groEL protein )/85   promotes the refolding and
             (67)                         proper assembly of unfolded
                                          polypeptides generated
                                          under stress conditions

8            Ornithine                    Arginine and proline
             carbamoyltransferase/75      metabolism
                                          Urea cycle and metabolism
                                          of amino groups

10           Putative O-acetylserine      Cysteine and methionine
             lyase/171 (67)               metabolism
                                          Selenoamino acid
                                          Sulfur metabolism

12           Enolase (2-phosphoglycerate  Glycolysis/gluconeogenesis

13           Peptide deformylase/106      Removes the formyl group
             (67)                         from the N-terminal Met of
                                          newly synthesized proteins.
                                          Requires at least a
                                          dipeptide for an efficient
                                          rate of reaction.
                                          L-methionine is a
                                          prerequisite for activity
                                          but the enzyme has broad
                                          specificity at other

16           Fructose-bisphosphate
             aldolase/78 (67)
                                          Pentose phosphate pathway

4            CAMP factor/89 (78)          Bacterial toxins, Type II
                                          toxins: membrane damaging
                                          toxins, pore-forming

6            Streptococca1 pyrogenic      Causative agent of the
             exotoxin C/l 14 (78)         symptoms associated with
                                          scarlet fever, has been
                                          associated with
                                          streptococcal toxic
                                          shock-like disease and may
                                          play a role in the early
                                          events of rheumatic fever

1            Cysteine synthase/68 (67)    Cysteine and methionine
                                          Selenoamino acid
                                          Sulfur metabolism

2            Putative O-acetylserine      Cysteine and methionine
             lyase/146 (78)               Selenoamino acid
                                          Sulfur metabolism

(a.) GenomeNet: a network of database and computational services for
genome research and related research areas in biomedical sciences,
operated by the Kyoto University Bioinformatics Centre
( and the Universal Protein Resource
(UniProt): a comprehensive resource for protein
sequence and annotation data (
(b.) Mascot significant matching scores (P > 0.05) are
indicated below the respective protein names.


In the present studies, we have attempted to explain the actions ofRhodomyrtus tomentosa extract and purified rhodomyrtone on S. pyogenes. Our previous results showed that Rhodomyrtus tomentosa extract and pure rhodomyrtone have no effect on the cell wall, cell membrane or activation of autolytic enzymes (Limsuwan et al. 2009b). Other effects such as interference with the cell wall, protein, DNA or RNA synthesis might thus be responsible for the respective antibacterial activities of this extract and compound. The addition of rhodomyrtone at 50% MIC decreased the growth rate of S. pyogenes and the maximal cell density reached was lower than under the control condition with no added rhodomyrtone. Judged by the growth-suppressing effect of rhodomyrtone, it can be concluded that this compound acts as a stress factor on S. pyogenes. Proteomic studies have the potential to provide new knowledge concerning the working mechanism of antimicrobial drugs, bacterial stress or starvation responses, evolution of antibiotic resistance mechanisms, and the discovery of new drug targets in pathogenic bacteria (Hecker et al. 2003; Sibbald et al 2006). Our present proteomics studies revealed that significant alterations of the cellular and secreted protein patterns of S. pyogenes occurred after treatment with 50% MIC of rhodomyrtone. In response to the presence of rhodomyrtone, S. pyogenes produced several additional cellular and secreted proteins. Nearly all of the proteins of which the amounts were significantly altered are enzymes associated with important metabolic pathways. Some of these enzymes are from the glycolytic/gluconeogenesis pathways and others are involved in amino acid metabolic pathways. These findings suggest that rhodomyrtone interferes with the functionality of glycolysis, gluconeogenesis and amino acid metabolism in S. pyogenes, though certain indirect effects on the respective enzymes due to growth inhibition cannot be entirely excluded. For example, the reduced growth may indirectly give rise to increased turnover of GAPDH and the Xaa-His dipeptidase. Alterations of bacterial glycolysis by parabens and grape polyphenols have been previously reported. Parabens were shown to inhibit glycolysis in the cariogenic dental plaque bacterium S. mutans GS-5. The bactericidal actions of parabens can thus be interpreted as being due to irreversible damage to key enzymes, such as those of the phosphotransferase system (Ma and Marquis 1996). Grape polyphenols were found to cause S. mutans to produce significantly less acid. This effect can be attributed to the inhibition of glycolytic pathway, the process by which the bacteria turns sugar into energy also produces acid (Thimotheetal. 2007).

In many pathogenic bacteria, the expression of virulence factors is regulated in response to diverse environmental changes (Read et al. 1989). Using a 2D PAGE analysis, it was found that the expression of S. pyogenes virulence factors was regulated in response to environmental signals, such as streptococcal pyrogenic exotoxin B (SPE B), streptococcal pyrogenic exotoxin F (SPE F), streptococcal inhibitor of complement (Sic), endo-[beta]-N-acetylglucosaminidase (EndoS), [alpha]-amylase, and mitogenic factor 3 (Mf3) (Nakamura et al. 2004). Here we show that some of the virulence factors of S. pyogenes, including enzymes and toxins such as the streptococcal pyrogenic exotoxin C (SPE C) and the CAMP factor, were present in severely decreased amounts after incubation with rhodomyrtone. SPE C is a superantigen produced by many strains of S. pyogenes that is highly associated with streptococcal toxic shock syndrome (STSS) and other invasive streptococcal diseases. Moreover, SPE C causes the symptoms associated with scarlet fever and may play a role in the early events of rheumatic fever. Effects of some plant extracts on bacterial toxins have been report (Braga et al. 2005; Ifesan and Voravuthikunchai 2009). Interestingly, Brijesh et al. (2006) reported that a decoction from Pongamia pinnata leaves prevented the production of cholera toxin from Vibrio cholerae due to its effects on bacterial metabolism rather than to a reduction in bacterial growth. Thus, it is conceivable that the inhibition of S. pyogenes toxin production by rhodomyrtone could be associated with the observed alterations in the metabolic pathways of this bacterium. In addition, we found that rhodomyrtone caused important changes in the expression of GAPDH, one of the key enzymes in glycolysis but also a major surface protein of S. pyogenes with multiple binding activities. The surface-located form of this enzyme is tightly attached to the streptococcal cell and, in addition, it binds to fibronectin, lysozyme, as well as the cytoskeletal proteins myosin and actin (Pancholi and Fischetti 1992). GAPDH is thus involved in the initial steps of mucosal colonization. Accordingly, the prevention of GAPDH localization to the S. pyogenes cell surface affects important virulence mechanisms, such as host cell adherence and antiphagocytic properties, in a pharmacologically desirable way (Boel et al. 2005). Notably, the cell surfaces of many Gram-negative and Gram-positive bacteria have been demonstrated to contain molecules that bind to fibronectin and lysozyme (Abraham et al. 1983; Courtney et al. 1986). In S. aureus, a fibronectin-binding protein has been implicated in the adherence of this organism to fibronectin on the surface of mammalian cells (Caparon et al. 1991). The observed inhibition of the expression of the S. pyogenes fibronectin-binding protein GAPDH by rhodomyrtone may thus affect the ability of S, pyogenes to adhere to mammalian cells and mucosal surfaces. This could be an important anti-infective activity that can at least partially explain the previously observed antibiofilm or anti-adherence activities of rhodomyrtone (Limsuwan and Voravuthikunchai 2008) and the pharmacological properties of leaf extract of Rhodomyrtus tomentosa (Voravuthikunchai et al. 2007; Limsuwan and Voravuthikunchai 2008; Saising et al. 2008; Limsuwan et al. 2009a,b).


Taken together, our proteomics analyses revealed significant alterations in the secreted and cellular protein patterns of S. pyogenes when this bacterium was cultured in the presence of rhodomyrtone. The results indicate that rhodomyrtone interferes with metabolic pathways such as glycolysis, gluconeogenesis and amino acid metabolism. In addition, the expression of streptococcal toxins such as the CAMP factor and streptococcal pyrogenic exotoxin C of S. pyogenes was inhibited when cells were exposed to rhodomyrtone. The combined effects of rhodomyrtone on metabolic pathways and the expression of toxins have a high potential to reduce the virulence of S. pyogenes significantly. We therefore conclude that our proteomic analyses have provided important leads towards the definition of the bactericidal and potential anti-infective effects of rhodomyrtone on S. pyogenes.


We thank Jan Arends, and members of the Department of Medical Microbiology and Department of Molecular Genetics for strains and technical support. We thank Assoc. Prof. Dr. Wilawan Mahabusarakam and Mr. Asadhawut Hiranrat for rhodomyrtone isolation. The work was funded by the Thailand Research Fund through the Royal Golden Jubilee, Ph.D. Program (PHD/0029/2548). Funding was furthermore provided by the National Research University Project of Thailand's Office of the Higher Education Commission, the Van Leersumfonds, The Netherlands (VLF/DA/3689), and the CEU projects LSHM-CT-2006-019064 and LSHC-CT-2006-037469.


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Surasak Limsuwana (a), Anne Hesseling-Meinders (b), Supayang Piyawan Voravuthikunchai (c), Jan Maarten van Dijl (d), Oliver Kayser (e), *

(a.) Faculty of Traditional Thai Medicine and Natural Pivducts Research Center, Faculty of Science, Prince ofSongkla University, Hat Yai, Songkhla 90112, Thailand

(b.) Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University ofGroningen, Nijenborg 7,9747 AG Groningen, The Netherlands

(c.) Department of Microbiology and Natural Products Research Center, Faculty of Science, Prince of Songlda University, Hat Yai, Songkhla 90112, Thailand

(d.) Department of Medical Microbiology, University Medical Center Groningen (UMCG) and University ofGroningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands

(e.) Department of Bio-and Chemical Engineering, Technical University of Dortmund, Technical Biochemistry, Emil-Figge-Strasse 66-68, D-44227 Dortmund, Germany

* Corresponding author. Tel.: +49 2317557487; fax: +49 2317557489. E-mail address: (O. Kayser). doi: 10.1016/j.phy med.2011.02.007

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Title Annotation:ELSEVIER
Author:Limsuwana, Surasak; Hesseling-Meinders, Anne; Voravuthikunchai, Supayang Piyawan; Dijl, Jan Maarten
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUGE
Date:Aug 15, 2011
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