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Rhodomyrtone: A new candidate as natural antibacterial drug from Rhodomyrtus tomentosa
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Phytomedicine 16 (2009) 645–651 www.elsevier.de/phymed
Rhodomyrtone: A new candidate as natural antibacterial drug from Rhodomyrtus tomentosa Surasak Limsuwana,b, Erik N. Tripc, Thijs R.H.M. Kouwenc, Sjouke Piersmac, Asadhawut Hiranratd, Wilawan Mahabusarakamd,e, Supayang P. Voravuthikunchaib,e, Jan Maarten van Dijlc, Oliver Kaysera, a
Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, NL-9713 AV Groningen, the Netherlands b Department of Microbiology and Natural Products Research Center, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c Department of Medical Microbiology, University Medical Center Groningen (UMCG) and University of Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands d Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand e Natural Products Research Center, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand
Abstract Rhodomyrtone [6,8-dihydroxy-2,2,4,4-tetramethyl-7-(3-methyl-1-oxobutyl)-9-(2-methylpropyl)-4,9-dihydro-1Hxanthene-1,3(2H)-di-one] from Rhodomyrtus tomentosa (Aiton) Hassk. displayed significant antibacterial activities against Gram-positive bacteria including Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Streptococcus gordonii, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus salivarius. Especially noteworthy was the activity against MRSA with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) ranging from 0.39 to 0.78 mg/ml. As shown for S. pyogenes, no surviving cells were detected within 5 and 6 h after treatment with the compound at 8MBC and 4MBC concentrations, respectively. Rhodomyrtone displays no bacteriolytic activity, as determined by measurement of the optical density at 620 nm. A rhodomyrtone killing test with S. mutans using phase contrast microscopy showed that this compound caused a few morphological changes as the treated cells were slightly changed in color and bigger than the control when they were killed. Taken together, the results support the view that rhodomyrtone has a strong bactericidal activity on Gram-positive bacteria, including major pathogens. r 2009 Elsevier GmbH. All rights reserved. Keywords: Rhodomyrtone; Rhodomyrtus tomentosa; Medicinal plant; Antibacterial activity; Methicillin-resistant Staphylococcus aureus; Antibiotic-resistant bacteria
Introduction Corresponding author. Tel.: +31 50 363 3299;
fax: +31 50 363 3000. E-mail address: [email protected] (O. Kayser). 0944-7113/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.01.010
Medicinal plants are interesting as a source of pharmacologically active compounds. Many plants have been used by the world population for their basic health
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care needs including the treatment of infections. It has been recognized by WHO that they are the main medicinal source used to treat infectious diseases in most developing countries and also become more important in a number of developed countries (WHO 2002a). It is of interest that scientific and clinical studies for alternative treatment of infections with medicinal plants as a source of antibacterial compounds has increased worldwide (Chusri and Voravuthikunchai 2008; Mativandlela et al. 2008; Nazemiyeh et al. 2008; Pesewu et al. 2008). There is an increasing concern that bacteria are becoming resistant to clinically used drugs and there is a high demand to discover new antibiotics to fight against resistant bacterial species (WHO 2002b). Many studies try to find alternative ways to reduce and prevent the problem of antibiotic resistance in bacteria. A number of plants may have a strong activity and valuable medical potential to be developed into an effective drug. Such plants may substitute antibiotic consumption or decrease antibiotic resistant bacteria. Downy rose myrtle, Rhodomyrtus tomentosa (Aiton) Hassk., is an evergreen shrub native to Southeast Asia (Latiff 1992), and has been reported as serious invader of native plant communities in Florida (Winotai et al. 2005). This plant is one of the components in traditional medicines used to treat urinary tract infections (Wei 2006b) for example rhodomyrtone with an amount of 0.3% (dry weight). Our preliminary screening test demonstrated that the leaf extract from R. tomentosa possessed significant antibacterial activity against Gram-positive bacteria (Voravuthikunchai et al. 2007). Therefore, the aim of this study was to further investigate the antibacterial activities of rhodomyrtone, a potent bioactive compound from this plant species.
Materials and methods
Department of Medical Microbiology, University Medical Center Groningen (UMCG), The Netherlands. Staphylococcus epidermidis ATCC 12228, S. epidermidis ATCC 35984, and Staphylococcus aureus ATCC 25923 were used as a reference strains. All strains were cultured on blood agar (BA, Difco, France) plates incubated at 37 1C for 24 h.
Antibacterial activities A broth microdilution method was carried out according to Clinical and Laboratory Standards Institute Guidelines (CLSI 2006). The minimum inhibitory concentration (MIC) was recorded as the lowest concentration that produced a complete suppression of visible growth. Aliquots from the broth with no growth were plated onto BA plates by using a sterile loop and incubated at 37 1C for 24 h. The minimum bactericidal concentration (MBC) was defined as the lowest concentration of the extracts completely preventing bacterial growth.
Time-kill assay The bactericidal activity of rhodomyrtone on Streptococcus pyogenes was studied using a time-kill assay. The bacterial culture (5 105 cfu/ml) was added to brain heart infusion broth (BHI, Difco, France) containing rhodomyrtone at 2MBC, 4MBC and 8MBC and cultures were incubated with 5% CO2 at 37 1C. The samples were collected at hourly intervals for 7 h and at 24 h. A control incubation was performed with 1% DMSO. Surviving bacteria were cultured on BA.
Bacteriolysis
A voucher specimen of R. tomentosa was deposited at the Herbarium of Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand. Dried leaves were extracted with 95% ethanol. The extract was completely dried and dissolved in dimethylsulfoxide (DMSO, Merck, Germany) before use. The fractions were made and further purified by MPLC; bioactive compounds were isolated using antibacterial bioguided fractionation. Penicillin G and vancomycin (Sigma, France) were used as reference antibiotics. HPLC-grade solvents were purchased from Merck (Germany).
A modified method from Carson et al. (2002) was used in this experiment. Suspensions of S. pyogenes (5 105 cfu/ml) in 0.85% normal saline solution were prepared from the culture on BHI agar. The suspensions were supplemented with rhodomyrtone at concentrations equivalent to 2MBC, 4MBC, and 8MBC. The suspensions were mixed with a vortex mixer, and the Optical Density at 620 nm (OD620) was measured for 0, 2, 4, 6, 8, 10, 12, and 24 h to detect cell lysis as indicated by a decrease in OD. Corresponding dilutions of test agents were used as blanks. 1% DMSO was used as a control. The results were expressed as a ratio of the OD620 at each time interval versus the OD620 at 0 min (in percent).
Bacterial strains and culture conditions
Time-lapse microscopy
Clinical bacterial isolates were taken from the collection of the Laboratory of Molecular Bacteriology,
Time-lapse phase contrast microscopy to monitor bacterial killing by rhodomyrtone was performed as
Plant extraction
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follows. Melted BHI agar supplemented with rhodomyrtone at a concentration of 8MBC was applied to the surface of a sterile glass slide and covered with a cover slip. After 10 min, the cover slip was removed, leaving a very thin film of solidified agar coated on the slide. A small drop of an overnight culture of Streptococcus mutans was placed onto the agar film and covered with a new cover slip. Observations were made under phase contrast microscopy at 37 1C using a Leica DM5500 B bright field microscope. Images at a magnification of 630-fold were taken every 5 min for 20 h.
Results and discussion The crude ethanolic extract from R. tomentosa showed good antibacterial activities against all tested Gram-positive bacteria (Table 1). Interestingly, the tests on MRSA strains yielded MIC values equal to the MIC value measured for the laboratory strain S. aureus SH1000. Different fractions were prepared and tested
Table 1. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of ethanolic extract from Rhodomyrtus tomentosa on some pathogenic bacteria. Bacterial strains
MIC MBC (mg/ml) (mg/ml)
Bacillus subtilis (non pathogenic) Enterococcus faecalis Staphylococcus aureus MRSA1 Methicillin-resistant clinical isolate Staphylococcus aureus MRSA2 Methicillin-resistant clinical isolate Staphylococcus aureus MRSA3 Methicillin-resistant clinical isolate Staphylococcus aureus MRSA4 Methicillin-resistant clinical isolate Staphylococcus aureus MRSA27 Methicillin-resistant clinical isolate Staphylococcus aureus MRSA28 Methicillin-resistant clinical isolate Staphylococcus aureus SH1000 Staphylococcus epidermidis biofilm negative (ATCC 12228) Staphylococcus epidermidis biofilm positive (ATCC 35984) Streptococcus gordonii Streptococcus mutans Streptococcus pneumoniae capsule negative (D39) Streptococcus pneumoniae capsule positive (D39Dcps) Streptococcus pyogenes Streptococcus salivarius
0.06 0.25 0.03
0.06 1.00 0.03
0.25
1.00
0.06
0.50
0.06
0.25
0.06
0.25
0.06
0.12
0.06 0.25
0.12 1.00
0.12
0.50
0.06 0.06 0.03
0.12 0.06 0.03
0.03
0.03
0.06 0.25
0.12 0.50
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for antibacterial activity. We found that only hexane and ethyl acetate fractions had significant antibacterial activity (Table 2). Both active fractions were further fractionated by MPLC to identify an active compound at the retention time of 40.26 min (data not shown). The isolated peak was structure elucidated by H NMR and the peak in both fractions was confirmed with reference material as rhodomyrtone (data not shown). The antibacterial activities of the MPLC fractions and rhodomyrtone itself are shown in Table 3. Seven fractions from the hexane phase (A–G), especially fractions D, E, F, and G, displayed good antibacterial activities (MICo12.5 mg/ml) and eight fractions from the ethyl acetate phase (H–O), especially fractions M and N, also revealed good activity. All active fractions from MPLC displayed lower activities than rhodomyrtone, because these fractions still contained some impurities. The fractions were made and further purified by MPLC; bioactive compounds were isolated using antibacterial bioguided fractionation. Rhodomyrtone (Fig. 1) [6,8-dihydroxy-2,2,4,4-tetramethyl-7-(3-methyl1-oxobutyl)-9-(2-methylpropyl)-4,9-dihydro-1Hxanthene-1,3(2H)-di-one] was isolated and the elucidated structure was in accordance with the previously published data (Salni et al. 2002). It exhibited significant antibacterial effects, expressed as very low MIC (MIC ¼ 0.19–1.56 mg/ml) and MBC (MBC ¼ 0.39–25 mg/ml) values against all Gram-positive bacteria (Table 4). This compound possessed remarkable activity against MRSA with MIC and MBC values ranging from 0.39 to 0.78 mg/ml. Notably, rhodomyrtone provided stronger antibacterial activity than the reference antibiotic vancomycin, which showed 2–3 times higher MIC values and 160–320 times higher MBC values than rhodomyrtone. In addition, rhodomyrtone also displayed good activity against biofilm-forming and capsulated bacteria, including S. epidermidis ATCC 35984 (biofilm positive) and S. pneumoniae (capsule positive). The killing activity of rhodomyrtone was determined against S. pyogenes (Fig. 2). Time-kill assays were performed by determining the number of colony forming units (cfu) which yielded a Dlog10 cfu/ml of 1 (90% killing), – 2 (99% killing), and – 3 (99.9% killing) at each time point tested as compared to cfu counts at time zero when rhodomyrtone was added (Pankuch et al. 1996). Bactericidal activity was defined as a decrease of X3 log units in cfu/ml. From the results, 90% killing was observed within 4, 4, and 3 h at 2MBC, 4MBC, and 8MBC, respectively. Meanwhile, 99% killing was noticed within 7, 5, and 4 h when treated with 2MBC, 4MBC, and 8MBC, respectively. Rhodomyrtone exerted the most significant bactericidal activity (X99.9% killing) within 24, 6, and 5 h at 2MBC, 4MBC, and 8MBC, respectively.
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Table 2. Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the fractions from Rhodomyrtus tomentosa extract on Streptoccocus spp. Bacterial strains
MIC/MBC (mg/ml)
Streptococcus mutans Streptococcus salivarius Streptococcus gordonii
Crude extract
Hexane fraction
Chloroform fraction
Ethanol fraction
Ethyl acetate fraction
Aqueous fraction
Penicillin G
62.5/62.5
7.8/62.5
41000/41000
3.9/62.5
250/500
15.6/31.2
41000/41000
62.5/125
62.5/62.5
41000/41000
41000/ 41000 41000/ 41000 41000/ 41000
41000/ 41000 41000/ 41000 41000/ 41000
0.031/ 0.031 0.062/ 0.062 0.031/ 0.031
Table 3. Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the fractions from MPLC and Rhodomyrtone on Streptococcus mutans. MPLC fractions
MIC/MBC (mg/ml)
n-hexane phase Fraction A Fraction B Fraction C Fraction D Fraction E Fraction F Fraction G
50/4100 12.5/4100 12.5/4100 1.56/12.5 1.56/25 6.25/25 6.25/6.25
Ethyl acetate phase Fraction H Fraction I Fraction J Fraction K Fraction L Fraction M Fraction N Fraction O Rhodomyrtone Penicillin G
25/4100 25/4100 100/4100 25/4100 50/4100 3.12/25 1.56/25 3.12/100 0.19/1.56 0.031/0.031
O
HO
O
OH
O
O
Fig. 1. Structure of rhodomyrtone.
The lack of bacteriolytic activity of rhodomyrtone is demonstrated in Fig. 3. Treatment with rhodomyrtone at the 2MBC, 4MBC, and 8MBC had no effect (OD620 range, 82–99% of the original) on the cell lysis within 24 h. Some natural compounds that induce cell lysis
62.5/62.5 15.6/31.2
have been reported previously, such as essential oils from oregano, rosewood, and thyme (Horne et al. 2001). Carson et al. (2002) reported the delayed lysis effect of tea tree oil on S. aureus, and this may be associated with the treatment-induced release of membrane-bound cell wall autolytic enzymes when the cell suspensions were reexamined after several hours. Our results did not reveal delayed lysis, and thus the antibacterial activity of rhodomyrtone may not relate to the activation of cell wall autolytic enzymes. The killing test against S. mutans under phase contrast microscopy is shown in Fig. 4. In this experiment, we would expect to see swelling or exploding cells if killing activity would involve cell wall autolytic enzymes. However, after treatment with rhodomyrtone at 8MBC for 5 h the morphology of cells was only slightly changed. Killed cells were grey and slightly bigger than the control cells at 20 h (Figs. 3E, F). Meanwhile, the cells in the control were growing (Figs. 3B, C). The effects of tea tree oil and its components on the cell wall and cell membrane of S. aureus have been studied previously (Carson et al. 2002). The predisposition to lysis, the loss of 260-nmabsorbing material, the loss of tolerance to NaCl, and the altered morphology seen by electron microscopy all suggest that tea tree oil and its components compromise the cytoplasmic membrane of S. aureus. In addition, Oonmetta-aree et al. (2006) reported that galangal extract caused membrane damage, and cytoplasm coagulation in S. aureus. The failure of rhodomyrtone to lyse S. pyogenes cells suggests that the primary mechanism of action is not gross cell envelope damage. Rhodomyrtus tomentosa has been used in the traditional medicine for a long time. The ripe fruits are eaten raw to treat diarrhea (Ong and Nordiana 1999). The liquid that is present in this plant can be used to treat gynaecopathy including morbid leucorrhoea, menoxenia, dysmenorrhoea, endometritis, appendagitis, and pelvic inflammation (Wei 2006a). Recent studies demonstrated that the ethanolic extract showed good activity against some Gram-positive bacteria, but not Gramnegative bacteria (Voravuthikunchai et al. 2007). The present studies show that the extract has strong activity
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Table 4. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of rhodomyrtone from Rhodomyrtus tomentosa against some pathogenic bacteria. Bacterial strains
MIC/MBC (mg/ml)
Bacillus cereus Bacillus subtilis Enterococcus faecalis Methicillin-resistant Staphylococcus aureus (n ¼ 4) Staphylococcus aureus ATCC 25923 Staphylococcus epidermidis ATCC 35984 (biofilm positive) Streptococcus gordonii Streptococcus mutans Streptococcus pneumoniae capsule positive Streptococcus pyogenes (n ¼ 2) Streptococcus salivarius
Rhodomyrtone
Antibiotics
0.39/0.39 0.39/0.78 1.56/12.50 0.39–0.78/0.39–0.78 0.39/0.39 0.39/25 0.19/1.56 0.19/1.56 0.39/1.56 0.39–0.78/1.56 0.39/1.56
0.062/0.062 (penicillin G) 0.062/0.062 (penicillin G) ND 1.25/125 (vancomycin) 0.62/1.25 (vancomycin) 0.78/1.56 (vancomycin) 0.031/0.031 (penicillin G) 0.031/0.031 (penicillin G) 0.015/0.062 (penicillin G) 0.015/0.015 (penicillin G) 0.062/0.062 (penicillin G)
ND ¼ not done.
12 1%DMSO
11
2MBC
10
4MBC 8MBC
Log (cfu/ml)
9 8 7 6 5 4 3 2 1 0
1
0
2
3
4 Times (h)
5
6
7
24
Fig. 2. Time-kill determination of Streptococcus pyogenes after treatment with rhodomyrtone at Minimal Bactericidal Concentration (MBC). 2MBC (’), 4MBC (’), 8MBC (’), and 1% DMSO (&). Each bar indicates the mean7SD for at least one duplicates. The lowest detection threshold was 102 cfu/mL. 100
Relative absorbance %
80 1%DMSO
60
8MBC 4MBC
40
2MBC
20
0 0
2
4
6
8
10
12 14 Times (h)
16
18
20
22
24
Fig. 3. Lack of bacteriolytic activity of rhodomyrtone against S. pyogenes at 2MBC (K), 4MBC (m), 8MBC (’), and 1% DMSO (&).
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Fig. 4. The killing test of Streptococcus mutans with rhodomyrtone at Minimal Bactericidal Concentration (MBC) as monitored by time-lapse phase contrast microscopy at 37 1C on BHI agar. (A) 8MBC 10 min. (B) 8MBC 5 h. (C) 8MBC 20 h. (D) 1% DMSO 10 min. (E) 1% DMSO 5 h. (F) 1% DMSO 20 h. Magnification 630 .
against all Gram-positive bacteria tested (Table 1). Salni et al. (2002) isolated this compound from the same plant and suggested the trivial name rhodomyrtone which had significant antibacterial activity against Escherichia coli and S. aureus. Salni et al. (2002) did not mention the concentrations of this compound in the bioassay and the method used for antibacterial assay. Therefore, we could not compare the published data with our results. Moreover, in this communication we do not only report antibacterial activities of rhodomyrtone, but also expanded the studies to investigate whether this compound would cause cell wall damage. In conclusion, the present studies show that rhodomyrtone has powerful in vitro activity against a broad range of Gram-positive bacteria, including antibiotic resistant strains. This compound therefore has potential to be used as therapeutic control agent against bacterial infections. Unanswered questions concern the mechan-
ism of action, the in vivo efficacy, and toxicity of rhodomyrtone. Future research towards these objectives should resolve these issues.
Acknowledgements We thank Jan Arends, Monika Chlebowicz, Jetta Bijlsma and other members of the Department of Medical Microbiology for strains and technical support. 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 Basic Research Grant (DBG5180021; Thailand), the Van Leersumfonds, The Netherlands (VLF/DA/3689), and the CEU projects LSHM-CT2006-019064 and LSHG-CT-2006-037469.
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References Carson, C.F., Mee, B.J., Riley, T.V., 2002. Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy. Antimicrob. Agents Chemother. 46, 1914–1920. Chusri, S., Voravuthikunchai, S.P., 2008. Quercus infectoria: a candidate for the control of methicillin-resistant Staphylococcus aureus infections. Phytother. Res. 22, 560–562. Clinical and Laboratory Standards Institute (CLSI), 2006. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard, seventh ed. Clinical and Laboratory Standards Institute Document M7-A7. Clinical and Laboratory Standards Institute, 900 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087–1898 USA. Horne, D.S., Holm, M., Oberg, C., Chao, S., Young, D.G., 2001. Antimicrobial effects of essential oils on Streptococcus pneumoniae. J. Essent. Oil Res. 13, 387–392. Latiff, A.M., 1992. Rhodomyrtus tomentosa (Aiton) Hassk. In: Coronel, R.E., Verheij, E.W.M. (Eds.), Plant Resources of South-East Asia: Edible Fruits and Nuts. Prosea Foundation, Indonesia, pp. 276–277. Mativandlela, S.P., Meyer, J.J., Hussein, A.A., Houghton, P.J., Hamilton, C.J., Lall, N., 2008. Activity against Mycobacterium smegmatis and M. tuberculosis by extract of South African medicinal plants. Phytother. Res. 22, 841–845. Nazemiyeh, H., Rahman, M.M., Gibbons, S., Nahar, L., Delazar, A., Ghahramani, M.A., Talebpour, A.H., Sarker, S.D., 2008. Assessment of the antibacterial activity of phenylethanoid glycosides from Phlomis lanceolata against multiple-drug-resistant strains of Staphylococcus aureus. Nat. Med. 62, 91–95. Ong, H.C., Nordiana, M., 1999. Malay ethno-medico botany in Machang, Kelantan, Malaysia. Fitoterapia 70, 502–513. Oonmetta-aree, J., Suzuki, T., Gasaluck, P., Eumkeb, G., 2006. Antimicrobial properties and action of galangal (Alpinia galanga Linn.) on Staphylococcus aureus. LWT 39, 1214–1220. Pankuch, G.A., Jacobs, M.R., Appelbaum, P.C., 1996. MIC and time-kill study of antipneumococcal activities of RPR 106972 (a new oral streptogramin), RP 59500 (quinupristindalfopristin), pyostacine (RP 7293), penicillin G, cefotaxime, erythromycin, and clarithromycin against 10 penicillin-
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susceptible and -resistant pneumococci. Antimicrob. Agents Chemother. 40, 2071–2074. Pesewu, G.A., Cutler, R.R., Humber, D.P., 2008. Antibacterial activity of plants used in traditional medicines of Ghana with particular reference to MRSA. J. Ethnopharmacol. 116, 102–111. Salni, D., Sargent, M.V., Skelton, B.W., Soediro, I., Sutisna, M., White, A.H., Yulinah, E., 2002. Rhodomyrtone, an antibiotic from Rhodomyrtus tomentosa. Aus. J. Chem. 55, 229–232. Voravuthikunchai, S.P., Limsuwan, S., Chusri, S., 2007. New perspectives on herbal medicines for bacterial Infection: natural products II. In: Govil, J.N., Singh, V.K., Siddqui, N.T. (Eds.), Recent Progress in Medicinal Plants. Studium Press, USA, pp. 41–101. Wei, F., 2006a. Manufacture of oral liquid containing traditional Chinese medicine extract for treating gynecopathy. (Guangxi Huahong Pharmaceutical Co., Ltd., Peop. Rep. China; Shanghai Fosun Pharmaceutical (Group) Co., Ltd.), Faming Zhuanli Shenqing Gongkai Shuomingshu. China Patent No. CN1846715. Wei, F., 2006b. Manufacture of traditional Chinese medicine composition for treating urinary tract infection. (Gungxi Huahong Pharmaceutical Co., Ltd., Peop. Rep. China; Shanghai Fosun Pharmaceutical (Group) Co., Ltd.), Faming Zhuanli Shenqing Gongkai Shuomingshu. China Patent No. CN1853687. Winotai, A., Wright, T., Goolsby, J.A., 2005. Herbivores in Thailand on Rhodomyrtus tomentosa (Myrtaceae), an invasive weed in Florida. Fl. Entomol. 88, 104–105. World Health Organization (WHO), 2002a. WHO Traditional Medicine Strategy 2002–2005. /http://www.who.int/medicines/publications/traditionalpolicy/en/index.htmlS. World Health Organization (WHO). 2002b. Antimicrobial resistance, Fact Sheet No. 194, /http://www.who.int/ mediacentre/factsheets/fs194/en/S. Reference for S. aureus SH1000: Horsburgh MJ, Aish JL, White IJ, Shaw L, Lithgow JK, et al., 2002. sigmaB modulates virulence determinant expression and stress resistance: characterization of a functional rsbU strain derived from Staphylococcus aureus 8325-4. J Bacteriol 184, 5457–5467. Reference for S. pneumonia D39: Lanie JA, et al., 2007. Genome sequence of Avery’s virulent serotype 2 strain D39 of Streptococcus pneumoniae and comparison with that of unencapsulated laboratory strain R6. J Bacteriol 189, 38–51.
Phytomedicine 16 (2009) 645–651 www.elsevier.de/phymed
Rhodomyrtone: A new candidate as natural antibacterial drug from Rhodomyrtus tomentosa Surasak Limsuwana,b, Erik N. Tripc, Thijs R.H.M. Kouwenc, Sjouke Piersmac, Asadhawut Hiranratd, Wilawan Mahabusarakamd,e, Supayang P. Voravuthikunchaib,e, Jan Maarten van Dijlc, Oliver Kaysera, a
Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, NL-9713 AV Groningen, the Netherlands b Department of Microbiology and Natural Products Research Center, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c Department of Medical Microbiology, University Medical Center Groningen (UMCG) and University of Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands d Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand e Natural Products Research Center, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand
Abstract Rhodomyrtone [6,8-dihydroxy-2,2,4,4-tetramethyl-7-(3-methyl-1-oxobutyl)-9-(2-methylpropyl)-4,9-dihydro-1Hxanthene-1,3(2H)-di-one] from Rhodomyrtus tomentosa (Aiton) Hassk. displayed significant antibacterial activities against Gram-positive bacteria including Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Streptococcus gordonii, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus salivarius. Especially noteworthy was the activity against MRSA with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) ranging from 0.39 to 0.78 mg/ml. As shown for S. pyogenes, no surviving cells were detected within 5 and 6 h after treatment with the compound at 8MBC and 4MBC concentrations, respectively. Rhodomyrtone displays no bacteriolytic activity, as determined by measurement of the optical density at 620 nm. A rhodomyrtone killing test with S. mutans using phase contrast microscopy showed that this compound caused a few morphological changes as the treated cells were slightly changed in color and bigger than the control when they were killed. Taken together, the results support the view that rhodomyrtone has a strong bactericidal activity on Gram-positive bacteria, including major pathogens. r 2009 Elsevier GmbH. All rights reserved. Keywords: Rhodomyrtone; Rhodomyrtus tomentosa; Medicinal plant; Antibacterial activity; Methicillin-resistant Staphylococcus aureus; Antibiotic-resistant bacteria
Introduction Corresponding author. Tel.: +31 50 363 3299;
fax: +31 50 363 3000. E-mail address: [email protected] (O. Kayser). 0944-7113/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.01.010
Medicinal plants are interesting as a source of pharmacologically active compounds. Many plants have been used by the world population for their basic health
ARTICLE IN PRESS 646
S. Limsuwan et al. / Phytomedicine 16 (2009) 645–651
care needs including the treatment of infections. It has been recognized by WHO that they are the main medicinal source used to treat infectious diseases in most developing countries and also become more important in a number of developed countries (WHO 2002a). It is of interest that scientific and clinical studies for alternative treatment of infections with medicinal plants as a source of antibacterial compounds has increased worldwide (Chusri and Voravuthikunchai 2008; Mativandlela et al. 2008; Nazemiyeh et al. 2008; Pesewu et al. 2008). There is an increasing concern that bacteria are becoming resistant to clinically used drugs and there is a high demand to discover new antibiotics to fight against resistant bacterial species (WHO 2002b). Many studies try to find alternative ways to reduce and prevent the problem of antibiotic resistance in bacteria. A number of plants may have a strong activity and valuable medical potential to be developed into an effective drug. Such plants may substitute antibiotic consumption or decrease antibiotic resistant bacteria. Downy rose myrtle, Rhodomyrtus tomentosa (Aiton) Hassk., is an evergreen shrub native to Southeast Asia (Latiff 1992), and has been reported as serious invader of native plant communities in Florida (Winotai et al. 2005). This plant is one of the components in traditional medicines used to treat urinary tract infections (Wei 2006b) for example rhodomyrtone with an amount of 0.3% (dry weight). Our preliminary screening test demonstrated that the leaf extract from R. tomentosa possessed significant antibacterial activity against Gram-positive bacteria (Voravuthikunchai et al. 2007). Therefore, the aim of this study was to further investigate the antibacterial activities of rhodomyrtone, a potent bioactive compound from this plant species.
Materials and methods
Department of Medical Microbiology, University Medical Center Groningen (UMCG), The Netherlands. Staphylococcus epidermidis ATCC 12228, S. epidermidis ATCC 35984, and Staphylococcus aureus ATCC 25923 were used as a reference strains. All strains were cultured on blood agar (BA, Difco, France) plates incubated at 37 1C for 24 h.
Antibacterial activities A broth microdilution method was carried out according to Clinical and Laboratory Standards Institute Guidelines (CLSI 2006). The minimum inhibitory concentration (MIC) was recorded as the lowest concentration that produced a complete suppression of visible growth. Aliquots from the broth with no growth were plated onto BA plates by using a sterile loop and incubated at 37 1C for 24 h. The minimum bactericidal concentration (MBC) was defined as the lowest concentration of the extracts completely preventing bacterial growth.
Time-kill assay The bactericidal activity of rhodomyrtone on Streptococcus pyogenes was studied using a time-kill assay. The bacterial culture (5 105 cfu/ml) was added to brain heart infusion broth (BHI, Difco, France) containing rhodomyrtone at 2MBC, 4MBC and 8MBC and cultures were incubated with 5% CO2 at 37 1C. The samples were collected at hourly intervals for 7 h and at 24 h. A control incubation was performed with 1% DMSO. Surviving bacteria were cultured on BA.
Bacteriolysis
A voucher specimen of R. tomentosa was deposited at the Herbarium of Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand. Dried leaves were extracted with 95% ethanol. The extract was completely dried and dissolved in dimethylsulfoxide (DMSO, Merck, Germany) before use. The fractions were made and further purified by MPLC; bioactive compounds were isolated using antibacterial bioguided fractionation. Penicillin G and vancomycin (Sigma, France) were used as reference antibiotics. HPLC-grade solvents were purchased from Merck (Germany).
A modified method from Carson et al. (2002) was used in this experiment. Suspensions of S. pyogenes (5 105 cfu/ml) in 0.85% normal saline solution were prepared from the culture on BHI agar. The suspensions were supplemented with rhodomyrtone at concentrations equivalent to 2MBC, 4MBC, and 8MBC. The suspensions were mixed with a vortex mixer, and the Optical Density at 620 nm (OD620) was measured for 0, 2, 4, 6, 8, 10, 12, and 24 h to detect cell lysis as indicated by a decrease in OD. Corresponding dilutions of test agents were used as blanks. 1% DMSO was used as a control. The results were expressed as a ratio of the OD620 at each time interval versus the OD620 at 0 min (in percent).
Bacterial strains and culture conditions
Time-lapse microscopy
Clinical bacterial isolates were taken from the collection of the Laboratory of Molecular Bacteriology,
Time-lapse phase contrast microscopy to monitor bacterial killing by rhodomyrtone was performed as
Plant extraction
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follows. Melted BHI agar supplemented with rhodomyrtone at a concentration of 8MBC was applied to the surface of a sterile glass slide and covered with a cover slip. After 10 min, the cover slip was removed, leaving a very thin film of solidified agar coated on the slide. A small drop of an overnight culture of Streptococcus mutans was placed onto the agar film and covered with a new cover slip. Observations were made under phase contrast microscopy at 37 1C using a Leica DM5500 B bright field microscope. Images at a magnification of 630-fold were taken every 5 min for 20 h.
Results and discussion The crude ethanolic extract from R. tomentosa showed good antibacterial activities against all tested Gram-positive bacteria (Table 1). Interestingly, the tests on MRSA strains yielded MIC values equal to the MIC value measured for the laboratory strain S. aureus SH1000. Different fractions were prepared and tested
Table 1. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of ethanolic extract from Rhodomyrtus tomentosa on some pathogenic bacteria. Bacterial strains
MIC MBC (mg/ml) (mg/ml)
Bacillus subtilis (non pathogenic) Enterococcus faecalis Staphylococcus aureus MRSA1 Methicillin-resistant clinical isolate Staphylococcus aureus MRSA2 Methicillin-resistant clinical isolate Staphylococcus aureus MRSA3 Methicillin-resistant clinical isolate Staphylococcus aureus MRSA4 Methicillin-resistant clinical isolate Staphylococcus aureus MRSA27 Methicillin-resistant clinical isolate Staphylococcus aureus MRSA28 Methicillin-resistant clinical isolate Staphylococcus aureus SH1000 Staphylococcus epidermidis biofilm negative (ATCC 12228) Staphylococcus epidermidis biofilm positive (ATCC 35984) Streptococcus gordonii Streptococcus mutans Streptococcus pneumoniae capsule negative (D39) Streptococcus pneumoniae capsule positive (D39Dcps) Streptococcus pyogenes Streptococcus salivarius
0.06 0.25 0.03
0.06 1.00 0.03
0.25
1.00
0.06
0.50
0.06
0.25
0.06
0.25
0.06
0.12
0.06 0.25
0.12 1.00
0.12
0.50
0.06 0.06 0.03
0.12 0.06 0.03
0.03
0.03
0.06 0.25
0.12 0.50
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for antibacterial activity. We found that only hexane and ethyl acetate fractions had significant antibacterial activity (Table 2). Both active fractions were further fractionated by MPLC to identify an active compound at the retention time of 40.26 min (data not shown). The isolated peak was structure elucidated by H NMR and the peak in both fractions was confirmed with reference material as rhodomyrtone (data not shown). The antibacterial activities of the MPLC fractions and rhodomyrtone itself are shown in Table 3. Seven fractions from the hexane phase (A–G), especially fractions D, E, F, and G, displayed good antibacterial activities (MICo12.5 mg/ml) and eight fractions from the ethyl acetate phase (H–O), especially fractions M and N, also revealed good activity. All active fractions from MPLC displayed lower activities than rhodomyrtone, because these fractions still contained some impurities. The fractions were made and further purified by MPLC; bioactive compounds were isolated using antibacterial bioguided fractionation. Rhodomyrtone (Fig. 1) [6,8-dihydroxy-2,2,4,4-tetramethyl-7-(3-methyl1-oxobutyl)-9-(2-methylpropyl)-4,9-dihydro-1Hxanthene-1,3(2H)-di-one] was isolated and the elucidated structure was in accordance with the previously published data (Salni et al. 2002). It exhibited significant antibacterial effects, expressed as very low MIC (MIC ¼ 0.19–1.56 mg/ml) and MBC (MBC ¼ 0.39–25 mg/ml) values against all Gram-positive bacteria (Table 4). This compound possessed remarkable activity against MRSA with MIC and MBC values ranging from 0.39 to 0.78 mg/ml. Notably, rhodomyrtone provided stronger antibacterial activity than the reference antibiotic vancomycin, which showed 2–3 times higher MIC values and 160–320 times higher MBC values than rhodomyrtone. In addition, rhodomyrtone also displayed good activity against biofilm-forming and capsulated bacteria, including S. epidermidis ATCC 35984 (biofilm positive) and S. pneumoniae (capsule positive). The killing activity of rhodomyrtone was determined against S. pyogenes (Fig. 2). Time-kill assays were performed by determining the number of colony forming units (cfu) which yielded a Dlog10 cfu/ml of 1 (90% killing), – 2 (99% killing), and – 3 (99.9% killing) at each time point tested as compared to cfu counts at time zero when rhodomyrtone was added (Pankuch et al. 1996). Bactericidal activity was defined as a decrease of X3 log units in cfu/ml. From the results, 90% killing was observed within 4, 4, and 3 h at 2MBC, 4MBC, and 8MBC, respectively. Meanwhile, 99% killing was noticed within 7, 5, and 4 h when treated with 2MBC, 4MBC, and 8MBC, respectively. Rhodomyrtone exerted the most significant bactericidal activity (X99.9% killing) within 24, 6, and 5 h at 2MBC, 4MBC, and 8MBC, respectively.
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Table 2. Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the fractions from Rhodomyrtus tomentosa extract on Streptoccocus spp. Bacterial strains
MIC/MBC (mg/ml)
Streptococcus mutans Streptococcus salivarius Streptococcus gordonii
Crude extract
Hexane fraction
Chloroform fraction
Ethanol fraction
Ethyl acetate fraction
Aqueous fraction
Penicillin G
62.5/62.5
7.8/62.5
41000/41000
3.9/62.5
250/500
15.6/31.2
41000/41000
62.5/125
62.5/62.5
41000/41000
41000/ 41000 41000/ 41000 41000/ 41000
41000/ 41000 41000/ 41000 41000/ 41000
0.031/ 0.031 0.062/ 0.062 0.031/ 0.031
Table 3. Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the fractions from MPLC and Rhodomyrtone on Streptococcus mutans. MPLC fractions
MIC/MBC (mg/ml)
n-hexane phase Fraction A Fraction B Fraction C Fraction D Fraction E Fraction F Fraction G
50/4100 12.5/4100 12.5/4100 1.56/12.5 1.56/25 6.25/25 6.25/6.25
Ethyl acetate phase Fraction H Fraction I Fraction J Fraction K Fraction L Fraction M Fraction N Fraction O Rhodomyrtone Penicillin G
25/4100 25/4100 100/4100 25/4100 50/4100 3.12/25 1.56/25 3.12/100 0.19/1.56 0.031/0.031
O
HO
O
OH
O
O
Fig. 1. Structure of rhodomyrtone.
The lack of bacteriolytic activity of rhodomyrtone is demonstrated in Fig. 3. Treatment with rhodomyrtone at the 2MBC, 4MBC, and 8MBC had no effect (OD620 range, 82–99% of the original) on the cell lysis within 24 h. Some natural compounds that induce cell lysis
62.5/62.5 15.6/31.2
have been reported previously, such as essential oils from oregano, rosewood, and thyme (Horne et al. 2001). Carson et al. (2002) reported the delayed lysis effect of tea tree oil on S. aureus, and this may be associated with the treatment-induced release of membrane-bound cell wall autolytic enzymes when the cell suspensions were reexamined after several hours. Our results did not reveal delayed lysis, and thus the antibacterial activity of rhodomyrtone may not relate to the activation of cell wall autolytic enzymes. The killing test against S. mutans under phase contrast microscopy is shown in Fig. 4. In this experiment, we would expect to see swelling or exploding cells if killing activity would involve cell wall autolytic enzymes. However, after treatment with rhodomyrtone at 8MBC for 5 h the morphology of cells was only slightly changed. Killed cells were grey and slightly bigger than the control cells at 20 h (Figs. 3E, F). Meanwhile, the cells in the control were growing (Figs. 3B, C). The effects of tea tree oil and its components on the cell wall and cell membrane of S. aureus have been studied previously (Carson et al. 2002). The predisposition to lysis, the loss of 260-nmabsorbing material, the loss of tolerance to NaCl, and the altered morphology seen by electron microscopy all suggest that tea tree oil and its components compromise the cytoplasmic membrane of S. aureus. In addition, Oonmetta-aree et al. (2006) reported that galangal extract caused membrane damage, and cytoplasm coagulation in S. aureus. The failure of rhodomyrtone to lyse S. pyogenes cells suggests that the primary mechanism of action is not gross cell envelope damage. Rhodomyrtus tomentosa has been used in the traditional medicine for a long time. The ripe fruits are eaten raw to treat diarrhea (Ong and Nordiana 1999). The liquid that is present in this plant can be used to treat gynaecopathy including morbid leucorrhoea, menoxenia, dysmenorrhoea, endometritis, appendagitis, and pelvic inflammation (Wei 2006a). Recent studies demonstrated that the ethanolic extract showed good activity against some Gram-positive bacteria, but not Gramnegative bacteria (Voravuthikunchai et al. 2007). The present studies show that the extract has strong activity
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Table 4. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of rhodomyrtone from Rhodomyrtus tomentosa against some pathogenic bacteria. Bacterial strains
MIC/MBC (mg/ml)
Bacillus cereus Bacillus subtilis Enterococcus faecalis Methicillin-resistant Staphylococcus aureus (n ¼ 4) Staphylococcus aureus ATCC 25923 Staphylococcus epidermidis ATCC 35984 (biofilm positive) Streptococcus gordonii Streptococcus mutans Streptococcus pneumoniae capsule positive Streptococcus pyogenes (n ¼ 2) Streptococcus salivarius
Rhodomyrtone
Antibiotics
0.39/0.39 0.39/0.78 1.56/12.50 0.39–0.78/0.39–0.78 0.39/0.39 0.39/25 0.19/1.56 0.19/1.56 0.39/1.56 0.39–0.78/1.56 0.39/1.56
0.062/0.062 (penicillin G) 0.062/0.062 (penicillin G) ND 1.25/125 (vancomycin) 0.62/1.25 (vancomycin) 0.78/1.56 (vancomycin) 0.031/0.031 (penicillin G) 0.031/0.031 (penicillin G) 0.015/0.062 (penicillin G) 0.015/0.015 (penicillin G) 0.062/0.062 (penicillin G)
ND ¼ not done.
12 1%DMSO
11
2MBC
10
4MBC 8MBC
Log (cfu/ml)
9 8 7 6 5 4 3 2 1 0
1
0
2
3
4 Times (h)
5
6
7
24
Fig. 2. Time-kill determination of Streptococcus pyogenes after treatment with rhodomyrtone at Minimal Bactericidal Concentration (MBC). 2MBC (’), 4MBC (’), 8MBC (’), and 1% DMSO (&). Each bar indicates the mean7SD for at least one duplicates. The lowest detection threshold was 102 cfu/mL. 100
Relative absorbance %
80 1%DMSO
60
8MBC 4MBC
40
2MBC
20
0 0
2
4
6
8
10
12 14 Times (h)
16
18
20
22
24
Fig. 3. Lack of bacteriolytic activity of rhodomyrtone against S. pyogenes at 2MBC (K), 4MBC (m), 8MBC (’), and 1% DMSO (&).
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Fig. 4. The killing test of Streptococcus mutans with rhodomyrtone at Minimal Bactericidal Concentration (MBC) as monitored by time-lapse phase contrast microscopy at 37 1C on BHI agar. (A) 8MBC 10 min. (B) 8MBC 5 h. (C) 8MBC 20 h. (D) 1% DMSO 10 min. (E) 1% DMSO 5 h. (F) 1% DMSO 20 h. Magnification 630 .
against all Gram-positive bacteria tested (Table 1). Salni et al. (2002) isolated this compound from the same plant and suggested the trivial name rhodomyrtone which had significant antibacterial activity against Escherichia coli and S. aureus. Salni et al. (2002) did not mention the concentrations of this compound in the bioassay and the method used for antibacterial assay. Therefore, we could not compare the published data with our results. Moreover, in this communication we do not only report antibacterial activities of rhodomyrtone, but also expanded the studies to investigate whether this compound would cause cell wall damage. In conclusion, the present studies show that rhodomyrtone has powerful in vitro activity against a broad range of Gram-positive bacteria, including antibiotic resistant strains. This compound therefore has potential to be used as therapeutic control agent against bacterial infections. Unanswered questions concern the mechan-
ism of action, the in vivo efficacy, and toxicity of rhodomyrtone. Future research towards these objectives should resolve these issues.
Acknowledgements We thank Jan Arends, Monika Chlebowicz, Jetta Bijlsma and other members of the Department of Medical Microbiology for strains and technical support. 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 Basic Research Grant (DBG5180021; Thailand), the Van Leersumfonds, The Netherlands (VLF/DA/3689), and the CEU projects LSHM-CT2006-019064 and LSHG-CT-2006-037469.
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