- Email: [email protected]
Screening of Chinese medicinal plants for inhibition against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA)
Journal of Ethnopharmacology 120 (2008) 287–290
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communications
Screening of Chinese medicinal plants for inhibition against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) G.Y. Zuo a,∗ , G.C. Wang a , Y.B. Zhao a , G.L. Xu a , X.Y. Hao b , J. Han c , Q. Zhao c a
Research Center for Natural Medicines, Kunming General Hospital, PLA, Kunming, China Guiyang Medical College, Guiyang, China c Yunnan Traditional Chinese Medical College, Kunming, China b
a r t i c l e
i n f o
Article history: Received 7 April 2008 Received in revised form 7 August 2008 Accepted 20 August 2008 Available online 28 August 2008 Keywords: Anti-MRSA activity Crude extract Herbal medicine Betulinic acid
a b s t r a c t Ethnopharmacological relevance: Traditional herbs are a valuable source of novel antibacterials in combating pathogenic isolates of methicillin-resistant Staphylococcus aureus (MRSA), a global nosocomial problem. Aim of the study: To assess in vitro anti-MRSA activity of extracts from Chinese herbs. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were determined in the setting of clinical MRSA isolates. Materials and methods: A collection of 19 plant extracts were obtained and bioassay-guided phytochemical analysis performed. Antibacterial susceptibilities were screened for inhibitory zone and MICs/MBCs determined by serial dilution with a standardized microdilution broth methodology. 9 MRSA isolates and a standard control strain (ATCC 25923) were cultured and exposed to the plant extract and isolated compound. Vancomycin was used as a positive control agent. Results: All the presented 19 plants showed anti-MRSA activity with MIC of 1.25–3.07 mg/ml. The most active antimicrobial plants were Dendrobenthamia capitata, Elsholtzia rugulosa, Elsholtzia blanda, Geranium strictipes and Polygonum multiflorum (MIC ≤ 1.43 mg/ml), and betulinic acid isolated from the active ethyl acetate fraction of Dendrobenthamia capitata extract was determined with MIC/MBC values as 62.5/125.0 mg/ml. Conclusions: Dendrobenthamia capitata, Elsholtzia rugulosa, Elsholtzia blanda, Geranium strictipes Polygonum multiflorum and betulinic acid demonstrate promising anti-MRSA potential. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Methicillin-resistant Staphylococcus aureus (MRSA) which is resulted from the selective pressure of antibiotics currently used has increased relentlessly and well recognized as a global
Abbreviations: Ap, aerial parts; ATCC, American type culture collection; CFU, colony forming unit; CLSI, Clinical and Laboratory Standards Institute; DMSO, dimethyl sulfoxide; EtOH, ethanol; F, fruits; HIV, human immunodeficiency virus; ICU, intensive care unit; KIB, Kunming Institut of Botany; KGH, Kunming General Hospital; MBC, minimal bactericidal concentration; MHA, Mueller–Hinton agar; MHB, Mueller–Hinton broth; MIC, minimal inhibitory concentration; MRSa, methicillin resistant Staphylococci aureus; MSSa, methicillin resistant Staphylococcus aureus; NA, not active; NCCLS, National Committee for Clinical Laboratory Standards; ND, not determined; NMR, nuclear magnetic resonance; NSFC, National Natural Science Foundation of China; Rh, rhizomes; R, roots; SEM, standard error of mean; TCM, traditional Chinese medicine; TLC, thin layer chromatography; VLC, vacuum liquid chromatography; Wp, whole plants; ZD, zone diameter. ∗ Corresponding author. Tel.: +86 871 5359434; fax: +86 871 5414186. E-mail address: [email protected] (G.Y. Zuo). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.08.021
nosocomial problem in recent years. It has now emerged as the predominant and serious pathogenic bacterium, leading to high morbidity and mortality. MRSA normally possesses a multidrug-resistant genotype which causes it resistant to lactams, aminoglycosides, fluoroquinolones and macrolides. It is only sensitive to the glycopeptides, i.e. vancomycin and teicoplanin that are considered as the last few effective agents. But resistant isolate has also been reported (Barrett, 2005; Berger and Rohrer, 2002; Deurenberg et al., 2007; Goldstein, 2007). Clinical isolates of MRSA are often encountered, especially in patients of intensive care units (ICU) admission and of those who are elderly and repeatedly hospitalized. There is an urgent need to develop anti-MRSA agents with novel mechanisms of action (Enright, 2003). The importance of Chinese herbal medicines has now been increasingly recognized. These herbal medicines are integrated into the traditional Chinese medicine (TCM), together with multinationalities’ medical treatment. In this study, the 80% ethanol extracts of different parts of 19 Chinese herbal plants which have
288
G.Y. Zuo et al. / Journal of Ethnopharmacology 120 (2008) 287–290
Table 1 Species of 19 Chinese medicinal plants and their folk usage No.
Species
Family
Speciesmen number
Folk usage
1
Anemone rivalry Buch.-Ham.
Ranunculaceae
KUN 0259076
2 3
Biota orientalis (L.) Endl. Conyza Blinii Levl.
Cupressaceae Compositae
KGH 0509006 KUN 0865179
4
Dendrobenthamia capitata (Wall.) Hutch.
Cornaceae
KUN 789239
5 6 7 8 9
Dichrocephala chrysanthemifolia (Bl.) DC. Duchesnea indica (Arulr.) Forke Elsholtzia blanda Benth. Elsholtzia rugulosa Hemsl. Gaultheria yurmaneiisis (Fr.)Rehd.
Compositae Rosaceae Labiatae Labiatae Ericaceae
KUN 0488566 KUN 652421 KUN 0215408 KUN 0090675 KGH 0509001
10 11
Geranium strictipes K.Kunth. Keineckea carnea (Andr.) Kunth.
Geraniaceae Liliaceae
KGH 0509002 KGH 0509003
12
Physalis alkekengi L.
Solanaceae
KUN 0789117
13 14
Polygonum multiflorum Thunb. Potentilla fulgens Wall.
Polygonaceae Rosaceae
KUN 814035 KGH 0509005
15 16
Rosa laevigata Michx. Rubia cordifolia L.
Rosaceae Rubiaceae
KUN 684647 KUN 0328292
17 18 19
Schizandra spaeraridra Stapf. Senecio scandens Buch.-Ham. Tetrastigma hypoglaucum PI.
Shisandraceae Compositae Vitaceae
KGH 0509004 KUN 0724534 KUN 800216
Cholagogue, chronic hepatitis, hepatocirrhosis, laryngitis Haematemesis cough and hemoptysis Tympanitis, stomatitis, diminish inflammation, clearing away heat, faucitis, and poison Ascarid, leprosy, hepatitis, cholagogue, insecticidal Acesodyne, diarrhea, hepatitis, piles, verruca Pharyngitis, acute quinsy enteritis Pyelitis Cold and rheum, fever, detoxification Vas inflammation, invigorate the circulation of blood Enteritis, diarrhea, chronic gastritis Asthma and cough, cholecystitis, cystitis, gastralgia Clearing away heat and poison, rhinitis, tracheitis, rheum, pharyngitis Chronic hepatitis Constringency, diarrhea, enteritis, tuberculosis and emptysis Diarrhea Gastroenteritis, gastric ulcer, hemostasia, acesodyne Rheumatism and lumbago, injuries from falls Sore and furuncle, eczema, acute conjunctivitis Puff, scald, malignant pustule
been described in herbal books and folklore medicine of China and possess the properties of clearing away heat and poison (Here “poison” is a term of TCM generally refer to infectious factor of the body) and anti-inflammation (Anonymous, 1970; Zeng, 1999) were screened for their anti-MRSA activity. The isolation of an active compound betulinic acid from Dendrobenthamia capitata extract was also included.
2.3. Extract preparation The air-dried and ground plant material (30 g) was macerated with 80% ethanol (500 ml) for 5 days, filtered and the mare was further macerated twice with the same solvent overnight and filtered after sonicated for 30 min. The filtrate was combined and the solvent was evaporated at 40 ◦ C in vacuum. The extracts were prepared for each plant part.
2. Materials and methods 2.4. Antibacterial screening 2.1. Plant materials The selected plants were collected in the mountainous area of Lijiang, Yunnan province, the southwest of China, at the altitudes of 2100–3000 m in September 2005. They were identified at the Botany Department, Kunming Institute of Botany (KIB), the Chinese Academy of Sciences. Voucher specimens are preserved at the herbarium of KIB. 2.2. Microorganisms tested MRSA strains were clinical isolates from infectious samples of critically ill patients in Kunming General Hospital (KGH). Pathogen purification and identification (including colonial morphology, Gram staining and coagulase testing) were conducted in our clinical microbiology laboratory and further confirmed by standard cefoxitin disk diffusion test following CLSI (Clinical and Laboratory Standards Institute) standard procedures (CLSI, 2006b; CLSI, 2007). Standard MSSA (Methicillin-resistant Staphylococcus aureus) strain ATCC25923 was used as a control strain. Vancomycin (Eli Lilly Japan K.K., Seishin Laboratories) was used as a control anti-MRSA agent. Standard Mueller–Hinton agar and Mueller–Hinton broth (MHA and MHB, Tianhe Microbial Agents Co., Hangzhou, China) were used as culture media in testing the inhibition zones (inoculums 0.5 McFarland suspension, 1.5 × 108 CFU/ml) and MIC/MBC (inoculums 5 × 105 CFU/ml) for strains, measured as described in CLSI/NCCLS standards (CLSI, 2006a; CLSI, 2007; NCCLS, 1999), respectively. Nine of MRSA strains were tested.
Antimicrobial activities of the crude extracts were firstly screened for their inhibitory zone by the agar diffusion method as described previously (Dickson et al., 2006). Briefly, Crude extracts were prepared at concentrations of 30 mg/ml using 50% aqueous DMSO, sonicated and filter sterilized using a 0.25 m Millipore filter. Wells of 6 mm diameter were made in 20 ml MHA seeded with 20 l of a suspension of test organisms under aseptic conditions. 70 l (2.1 mg) of extracts were fed into wells, allowed to diffuse for 1 h and then incubated at 37 ◦ C for 24 h, after which time they were examined for zones of inhibition. The solvent value was deducted accordingly to get the final results of activity. All experiments were carried out in triplicate. The extracts with zone diameter (ZD) ≥ 10 mm were further subjected to determination of their MICs (minimal inhibitory concentrations) by serial dilution method according to the procedures reported previously (Dickson et al., 2006; CLSI, 2006a). Briefly, stock solutions (30 mg/ml in 50% DMSO) of all extracts were passed through a pyrogenic filter to sterilize the solution and serially diluted to arrive at concentrations between 30 mg/ml and 0.1 mg/ml (100 g/ml and 0.2 g/ml for vancomycin). The 96-well plates were prepared by dispensing into each well 100 l each of an appropriate medium, test extracts and 20 l of the inoculum. A standard nutrient broth MHB was employed for the bacterial assays. The growth of the microorganisms was determined by turbidity. Clear wells indicated absence of bacterial growth. For every experiment, a sterility check (50% DMSO and medium), negative control (50% DMSO, medium and inoculum) and positive control
G.Y. Zuo et al. / Journal of Ethnopharmacology 120 (2008) 287–290
289
Table 2 Antibacterial activity of ethanol crude extracts of 19 Chinese medicinal plants No.
Species
Parta
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Anemone rivalry Buch.-Ham. Biota orientalis (L.) Endl. Conyza Blinii Levl. Dendrobenthamia capitata (Wall.) Hutch. Dichrocephala chrysanthemifolia (Bl.) DC. Duchesnea indica (Arulr.) Forke Elsholtzia blanda Benth. Elsholtzia rugulosa Hemsl. Gaultheria yurmaneiisis (Fr.)Rehd. Geranium strictipes K. Kunth. Keineckea carnea (Andr.) Kunth. Physalis alkekengi L. Polygonum multiflorum Thunb. Potentilla fulgens Wall. Rosa laevigata Michx. Rubia cordifolia L. Schizandra spaeraridra Stapf. Senecio scandens Buch.-Ham. Tetrastigma hypoglaucum PI.
Rh Ap Ap Ap Wp Wp Ap Wp Wp R Wp F Rh F Rh Wp Ap Wp R
a b
MIC (mean ± S.E.M., mg/ml)b MSSA
MRSA
2.56 2.17 1.40 1.17 2.32 1.66 1.08 1.13 2.06 0.95 2.06 2.04 1.21 2.11 1.17 1.33 1.90 1.69 1.08
3.07 2.52 1.58 1.25 2.96 2.71 1.32 1.43 2.56 1.34 2.96 2.82 1.34 3.07 1.58 1.50 2.28 1.82 2.32
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.32 0.39 0.21 0.18 0.16 0.54 0.16 0.13 0.48 0.30 0.19 0.64 0.22 0.48 0.17 0.24 0.27 0.30 0.19
Ap, aerial parts; F, fruits; R, roots; Rh, rhizome; Wp, whole plants. A total of nine isolates of MRSA strain were tested. All samples were tested in triplicate.
(50% DMSO, medium, inoculum and vancomycin) were included. The microtitre plates were incubated at 35 ◦ C for 24 h and were examined for growth in daylight. The MIC of the preparations was the lowest concentration in the medium that completely inhibited the visible growth. The solvent value was deducted accordingly to get the final results of activity. All experiments were performed in triplicate. 2.5. Bioassay-guided isolation and the activity of betulinic acid An enlarged 100 g part of the active extract from Dendrobenthamia capitata (600 g) was suspended in 200 ml of sterilized and deionated water. Then the suspension was successively fractionated between water and petroleum ether, chloroform and ethyl acetate to form four fractions. The chloroform fraction (12 g) which showed the most active by the subsequent susceptability test was subjected to vacuum liquid chromatography (VLC, Navarro and Delgado, 1999) over silica gel-H (Qingdao, China) and eluted with gradient systems of chloroform (C)–methanol (M) (C:M = 10:1–1:1) to afford 40 mg of pure betulinic acid (3-Hydroxy-20(29)-lupen28-oic-acid). Amorphous powder; [˛]D + 6◦ (EtOH; c 0.1). IR vmax cm−1 : 3470, 2942, 2865, 1680, 875; 1 H NMR (CDCl3 -pyridine-d5 , 1:1): ı 0.45 (3H, s, H-26), 0.48 (3H, s, H-24), 0.60 (3H, s, H-25), 0.63 (3H, s, H-27), 0.66 (3H, s, H-23), 1.34 (3H, s, H-30), 4.25 and 4.40 (2H, 2 × br s, H-29); EIMS m/z (rel. int.): 456 [M]+ (8), 438 [M−H2 O]+ (5), 248 (100), 219 (15), 207 (30), 203 (80), 191 (7), 190 (12), 189 (15). All the spectral data were identical with the literature (Nick et al., 1995) and co-TLC result was also in agreement with an authentic sample. MICs of the fractions and betulinic acid from Dendrobenthamia capitata were determined by the same procedure as the foregoing serial dilution method (Dickson et al., 2006). The minimal bactericidal concentrations (MBCs) were determined by inoculating the surfaces of MHA plates with 25 l of samples taken from the clear wells of the microbroth susceptibility studies. After the bacterial suspensions had fully absorbed into the agar, the plates were incubated at 35 ◦ C for 24 h and were examined for growth in daylight. The MBC was defined as the concentration of drug that resulted in >99.9% killing of the bacterium relative to the concentration of bacterium that was present in test wells at 0 h (NCCLS, 1999). All experiments were performed in triplicate.
3. Results and discussion The ZD of 36 herbal samples were examined (data not shown), of which 19 ethanolic extracts with ZD > 10 mm and belonging to 17 different plant families were subjected to determination of the MICs. The values are in the range of 0.95–3.07 mg/ml against both MRSA and MSSA strains (Table 1). The phenomena is in accordance with the previous findings we studied on other plant samples and revealed that there might be ascribed to different mechanisms of the action other than inhibition against -lactamase (Zuo et al., 2006), such as the inhibition against bacterial topoisomerase (Cheng et al., 2007) and efflux pump (Kaatz, 2005; Gibbons et al., 2003; Michalet et al., 2007; Schmitz et al., 1998; Stavri et al., 2007). One of the most active extract from Dendrobenthamia capitata was subjected to bioassay-guided fractionation and isolation, and betulinic acid was identified as the principal constitution from the chlorform fraction. Its MIC and MBC were measured as 62.5 and 125.0 g/ml, respectively (Table 2). Although the antimicrobial activities of betulinic acid have been reported for various pathogenic microbes, including anti-HIV activity and induces apoptosis (Kuete et al., 2007; Rzeski et al., 2006; Yogeeswari and Sriram, 2005), its activity against Gram-positive MSSA has been reported contradictorily, and the MIC against MRSA was >128 g/ml (Horiuchi et al., 2007). Here we definitely determined the simliar anti-MRSA activity of the compound. (Table 3) The MIC/MBC values Table 3 Antibacterial activity of fractions and betulinic acid from Dendrobenthamia capitata Fraction and pure compound
Petroleum ether Chloroform Ethyl acetate Water Betulinic acid Vancomycin
MIC/MBC (mean ± S.E.M., g/ml)a MSSA
MRSA
ND/NDb ND/ND ND/ND ND/ND 31.2 ± 3.9/62.5 ± 7.8 1.17 ± 0.20/2.24 ± 0.32
NA/NAc 625 ± 62.5/1250 ± 125 916.7 ± 154.5/1500 ± 216.5 750 ± 108.3/1250 ± 125 62.5 ± 7.8/125.0 ± 15.6 1.23 ± 0.15/2.99 ± 0.44
a A total of nine isolates of MRSA strain were tested. All samples were tested in triplicate. b ND, not determined. c NA, not active.
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for vancomycin are in agreement with the results of Giacometti et al. By comparison, the difference in values are acceptale and our results fall in the range between 1and 4 g/ml of this previous report Giacometti et al. (2007). Apart from the triterpenoids, polyphenol constitutions in the active extracts of Dendrobenthamia capitata were also shown by TLC while spraying with 5% FeCl3 reagent (in 95% aqueous EtOH). From this study we can conclude that Dendrobenthamia capitata, Elsholtzia rugulosa, Elsholtzia blanda, Geranium strictipes, Polygonum multiflorum and Potentilla fulgens were the most active anti-MRSA plants. All of these species are perennial plants widely distributed in Lijiang and other district in the northwest of Yunnan, China. As little was known about their phytochemical constitution and antimicrobial activity prior to our investigation, we are continuing our further investigations of other antibacterial components in these plants. Acknowledgements This research was financially supported by a grant of the National Natural Science Foundation of China (NSFC 30472147). Spectral data were measured in the Department of Analysis, KIB. References Anonymous, 1970. Selected Yunnan Traditional Chinese Herbs. Tianjin People’s Press, Tianjin, China. Barrett, J.F., 2005. MRSA—what is it, and how do we deal with the problem? Expert Opinion on Therapeutic Targets 9, 253–265. Berger, B.B., Rohrer, S., 2002. Factors influencing methicillin resistance in Staphylococci. Archives of Microbiology 178, 165–171. Cheng, B., Liu, I.F., Dinh, Y.C.T., 2007. Compounds with antibacterial activity that enhance DNA cleavage by bacterial DNA topoisomerase I. Journal of Antimicrobial Chemotherapy 59, 640–645. Clinical and Laboratory Standards Institute, 2006a. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, seventh ed, vol. M7-A7. Approved Standard. Document M7. CLSI, Wayne, PA. Clinical and Laboratory Standards Institute, 2006b. Performance standards for antimicrobial disk susceptibility tests, ninth ed. Approved Standard. Document M2-A9. CLSI, Wayne, PA. Clinical and Laboratory Standards Institute, 2007. Performance standards for antimicrobial susceptibility testing—17th informational supplement. Approved standard, CLSI document M100-S17. CLSI, Wayne, PA. Deurenberg, R.H., Kalenic, C., Vink, S., Friedrich, A.W., Bruggeman, C.A., Stobberingh, E.E., 2007. The molecular evolution of methicillin-resistant Staphylococcus aureus. Clinical Microbiology and Infection 13, 222–235.
Dickson, R.A., Houghton, P.J., Hyhinds, P.J., Gibbon, S., 2006. Antimicrobial, resistance-modifying effects, antioxidant and free radical scavenging activities of Mezoneuron benthamianum Bail., Securinega virosa Roxb. and Wlld. and Microglossa pyrifolia Lam. Phytotherapy Research 20, 41–45. Enright, M.C., 2003. The evolution of a resistant pathogen—the case of MRSA. Current Opinion in Pharmacology 3, 474–479. Giacometti, A., Cirioni, O., Riva, A., Kamysz, W., Silvestri, C., Nadolski, P., Vittoria, D.A., Lukasiak, J., Scalise, G., 2007. In vitro activity of aurein 1.2 alone and in combination with antibiotics against Gram-positive nosocomial cocci. Antimicrobial Agents and Chemotherapy 51, 1494–1496. Gibbons, S., Leimkugel, J., Oluwatuyi, M., Heinrich, M., 2003. Activity of Zanthoxylum clava-herculis extracts against multi-drug resistant methicillin-resistant Staphylococcus aureus (mdr-MRSA). Phytotherapy Research 17, 274–275. Goldstein, F.W., 2007. Combating resistance in a challenging, changing environment. Clinical Microbiology and Infection 13, 2–6. Horiuchi, K., Shiot, S., Hatno, T., Yoshida, T., Kuroda, T., Tsuchiya, T., 2007. Antimicrobial Activity of oleanolic acid from Salvia officinalis and related compounds on vancomycin-resistant enterococci (VRE). Biological Pharmaceutical Bulletin 30, 1147–1149. Kaatz, G.W., 2005. Bacterial efflux pump inhibition. Current Opinion in Investigation of Drugs 6, 191–198. Kuete, V., Wabo, G.F., Ngamenib, B., Mbaveng, A.T., Metunob, R., Etoa, F.-X., Ngadjui, B.T., Benga, V.P., Marion Meyer, J.J.M., Lall, N., 2007. Antimicrobial activity of the methanolic extract, fractions and compounds from the stem bark of Irvingia gabonensis (Ixonanthaceae). Journal of Ethnopharmacology 114, 54–60. Michalet, S., Cartier, G., David, B., Mariotte, A.M., franca, M.G.D., Kaatz, G.W., Stavrid, M., Gibbons, S., 2007. N-caffeoylphenalkylamide derivatives as bacterial efflux pump inhibitors. Bioorganic and Medicinal Chemistry Letters 17, 1755–1758. National Committee for Clinical Laboratory Standards, 1999. Methods for determining bactericidal activity antimicrobial agents. Approved guidelines. Document M26-A. CLSI (formerly NCCLS), Wayne, PA. Navarro, V., Delgado, G., 1999. Two antimicrobial alkaloids from Bocconia arborea. Journal of Ethnopharmacology 66, 223–226. Nick, A., Wright, A.D., Topul, R., Sticher, O., 1995. Antibacterial triterpenoids from Dillenia papuana and their structure–activity relationships. Phytochemistry 40, 1691–1695. ´ Rzeski, W., Stepulak, A., Szymanski, M., Sifringer, M., Kaczor, K., Wejksza, K., ´ ´ M., 2006. Betulinic acid decreases expression Zdzisinska, B., Kandefer-Szerszen, of bcl-2 and cyclin D1, inhibits proliferation, migration and induces apoptosis in cancer cells. Naunyn-Schmiedeberg’s Archives of Pharmacology 374, 11–20. Schmitz, F.J., Fluit, A.C., Luckefahr, M., Engler, B., Hofmann, B., Verhoef, J., Heinz, H.P., Hadding, U., Jones, M.E., 1998. The effect of reserpine, an inhibitor of multidrug efflux pumps, on the in-vitro activities of ciprofloxacin, sparfloxacin and moxifloxacin against clinical isolates of Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 42, 807–810. Stavri, M., Piddock, L.J., Gibbons, S., 2007. Bacterial efflux pump inhibitors from natural sources. Journal of Antimicrobial Chemotherapy 59, 1247–1260. Yogeeswari, P., Sriram, D., 2005. Betulinic acid and its derivatives: a review on their biological properties. Current Medicinal Chemistry 12, 657–666. Zeng, Y.L., 1999. Yu-long Pen-ts’ao (Materia Medica of Na-xi Nationality). Chinese Journal of Ethnomedicine and Ethnopharmacy 36, 1–10. Zuo, G.Y., Wang, G.C., Xu, G.L., Yu, W., Peng, H., Kuang, W.X., 2006. Screening of antiMRSA activity from 30 Chinese herbage medicine extracts. Chinese Journal of Modern Applied Pharmacy 23, 293–295.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communications
Screening of Chinese medicinal plants for inhibition against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) G.Y. Zuo a,∗ , G.C. Wang a , Y.B. Zhao a , G.L. Xu a , X.Y. Hao b , J. Han c , Q. Zhao c a
Research Center for Natural Medicines, Kunming General Hospital, PLA, Kunming, China Guiyang Medical College, Guiyang, China c Yunnan Traditional Chinese Medical College, Kunming, China b
a r t i c l e
i n f o
Article history: Received 7 April 2008 Received in revised form 7 August 2008 Accepted 20 August 2008 Available online 28 August 2008 Keywords: Anti-MRSA activity Crude extract Herbal medicine Betulinic acid
a b s t r a c t Ethnopharmacological relevance: Traditional herbs are a valuable source of novel antibacterials in combating pathogenic isolates of methicillin-resistant Staphylococcus aureus (MRSA), a global nosocomial problem. Aim of the study: To assess in vitro anti-MRSA activity of extracts from Chinese herbs. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were determined in the setting of clinical MRSA isolates. Materials and methods: A collection of 19 plant extracts were obtained and bioassay-guided phytochemical analysis performed. Antibacterial susceptibilities were screened for inhibitory zone and MICs/MBCs determined by serial dilution with a standardized microdilution broth methodology. 9 MRSA isolates and a standard control strain (ATCC 25923) were cultured and exposed to the plant extract and isolated compound. Vancomycin was used as a positive control agent. Results: All the presented 19 plants showed anti-MRSA activity with MIC of 1.25–3.07 mg/ml. The most active antimicrobial plants were Dendrobenthamia capitata, Elsholtzia rugulosa, Elsholtzia blanda, Geranium strictipes and Polygonum multiflorum (MIC ≤ 1.43 mg/ml), and betulinic acid isolated from the active ethyl acetate fraction of Dendrobenthamia capitata extract was determined with MIC/MBC values as 62.5/125.0 mg/ml. Conclusions: Dendrobenthamia capitata, Elsholtzia rugulosa, Elsholtzia blanda, Geranium strictipes Polygonum multiflorum and betulinic acid demonstrate promising anti-MRSA potential. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Methicillin-resistant Staphylococcus aureus (MRSA) which is resulted from the selective pressure of antibiotics currently used has increased relentlessly and well recognized as a global
Abbreviations: Ap, aerial parts; ATCC, American type culture collection; CFU, colony forming unit; CLSI, Clinical and Laboratory Standards Institute; DMSO, dimethyl sulfoxide; EtOH, ethanol; F, fruits; HIV, human immunodeficiency virus; ICU, intensive care unit; KIB, Kunming Institut of Botany; KGH, Kunming General Hospital; MBC, minimal bactericidal concentration; MHA, Mueller–Hinton agar; MHB, Mueller–Hinton broth; MIC, minimal inhibitory concentration; MRSa, methicillin resistant Staphylococci aureus; MSSa, methicillin resistant Staphylococcus aureus; NA, not active; NCCLS, National Committee for Clinical Laboratory Standards; ND, not determined; NMR, nuclear magnetic resonance; NSFC, National Natural Science Foundation of China; Rh, rhizomes; R, roots; SEM, standard error of mean; TCM, traditional Chinese medicine; TLC, thin layer chromatography; VLC, vacuum liquid chromatography; Wp, whole plants; ZD, zone diameter. ∗ Corresponding author. Tel.: +86 871 5359434; fax: +86 871 5414186. E-mail address: [email protected] (G.Y. Zuo). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.08.021
nosocomial problem in recent years. It has now emerged as the predominant and serious pathogenic bacterium, leading to high morbidity and mortality. MRSA normally possesses a multidrug-resistant genotype which causes it resistant to lactams, aminoglycosides, fluoroquinolones and macrolides. It is only sensitive to the glycopeptides, i.e. vancomycin and teicoplanin that are considered as the last few effective agents. But resistant isolate has also been reported (Barrett, 2005; Berger and Rohrer, 2002; Deurenberg et al., 2007; Goldstein, 2007). Clinical isolates of MRSA are often encountered, especially in patients of intensive care units (ICU) admission and of those who are elderly and repeatedly hospitalized. There is an urgent need to develop anti-MRSA agents with novel mechanisms of action (Enright, 2003). The importance of Chinese herbal medicines has now been increasingly recognized. These herbal medicines are integrated into the traditional Chinese medicine (TCM), together with multinationalities’ medical treatment. In this study, the 80% ethanol extracts of different parts of 19 Chinese herbal plants which have
288
G.Y. Zuo et al. / Journal of Ethnopharmacology 120 (2008) 287–290
Table 1 Species of 19 Chinese medicinal plants and their folk usage No.
Species
Family
Speciesmen number
Folk usage
1
Anemone rivalry Buch.-Ham.
Ranunculaceae
KUN 0259076
2 3
Biota orientalis (L.) Endl. Conyza Blinii Levl.
Cupressaceae Compositae
KGH 0509006 KUN 0865179
4
Dendrobenthamia capitata (Wall.) Hutch.
Cornaceae
KUN 789239
5 6 7 8 9
Dichrocephala chrysanthemifolia (Bl.) DC. Duchesnea indica (Arulr.) Forke Elsholtzia blanda Benth. Elsholtzia rugulosa Hemsl. Gaultheria yurmaneiisis (Fr.)Rehd.
Compositae Rosaceae Labiatae Labiatae Ericaceae
KUN 0488566 KUN 652421 KUN 0215408 KUN 0090675 KGH 0509001
10 11
Geranium strictipes K.Kunth. Keineckea carnea (Andr.) Kunth.
Geraniaceae Liliaceae
KGH 0509002 KGH 0509003
12
Physalis alkekengi L.
Solanaceae
KUN 0789117
13 14
Polygonum multiflorum Thunb. Potentilla fulgens Wall.
Polygonaceae Rosaceae
KUN 814035 KGH 0509005
15 16
Rosa laevigata Michx. Rubia cordifolia L.
Rosaceae Rubiaceae
KUN 684647 KUN 0328292
17 18 19
Schizandra spaeraridra Stapf. Senecio scandens Buch.-Ham. Tetrastigma hypoglaucum PI.
Shisandraceae Compositae Vitaceae
KGH 0509004 KUN 0724534 KUN 800216
Cholagogue, chronic hepatitis, hepatocirrhosis, laryngitis Haematemesis cough and hemoptysis Tympanitis, stomatitis, diminish inflammation, clearing away heat, faucitis, and poison Ascarid, leprosy, hepatitis, cholagogue, insecticidal Acesodyne, diarrhea, hepatitis, piles, verruca Pharyngitis, acute quinsy enteritis Pyelitis Cold and rheum, fever, detoxification Vas inflammation, invigorate the circulation of blood Enteritis, diarrhea, chronic gastritis Asthma and cough, cholecystitis, cystitis, gastralgia Clearing away heat and poison, rhinitis, tracheitis, rheum, pharyngitis Chronic hepatitis Constringency, diarrhea, enteritis, tuberculosis and emptysis Diarrhea Gastroenteritis, gastric ulcer, hemostasia, acesodyne Rheumatism and lumbago, injuries from falls Sore and furuncle, eczema, acute conjunctivitis Puff, scald, malignant pustule
been described in herbal books and folklore medicine of China and possess the properties of clearing away heat and poison (Here “poison” is a term of TCM generally refer to infectious factor of the body) and anti-inflammation (Anonymous, 1970; Zeng, 1999) were screened for their anti-MRSA activity. The isolation of an active compound betulinic acid from Dendrobenthamia capitata extract was also included.
2.3. Extract preparation The air-dried and ground plant material (30 g) was macerated with 80% ethanol (500 ml) for 5 days, filtered and the mare was further macerated twice with the same solvent overnight and filtered after sonicated for 30 min. The filtrate was combined and the solvent was evaporated at 40 ◦ C in vacuum. The extracts were prepared for each plant part.
2. Materials and methods 2.4. Antibacterial screening 2.1. Plant materials The selected plants were collected in the mountainous area of Lijiang, Yunnan province, the southwest of China, at the altitudes of 2100–3000 m in September 2005. They were identified at the Botany Department, Kunming Institute of Botany (KIB), the Chinese Academy of Sciences. Voucher specimens are preserved at the herbarium of KIB. 2.2. Microorganisms tested MRSA strains were clinical isolates from infectious samples of critically ill patients in Kunming General Hospital (KGH). Pathogen purification and identification (including colonial morphology, Gram staining and coagulase testing) were conducted in our clinical microbiology laboratory and further confirmed by standard cefoxitin disk diffusion test following CLSI (Clinical and Laboratory Standards Institute) standard procedures (CLSI, 2006b; CLSI, 2007). Standard MSSA (Methicillin-resistant Staphylococcus aureus) strain ATCC25923 was used as a control strain. Vancomycin (Eli Lilly Japan K.K., Seishin Laboratories) was used as a control anti-MRSA agent. Standard Mueller–Hinton agar and Mueller–Hinton broth (MHA and MHB, Tianhe Microbial Agents Co., Hangzhou, China) were used as culture media in testing the inhibition zones (inoculums 0.5 McFarland suspension, 1.5 × 108 CFU/ml) and MIC/MBC (inoculums 5 × 105 CFU/ml) for strains, measured as described in CLSI/NCCLS standards (CLSI, 2006a; CLSI, 2007; NCCLS, 1999), respectively. Nine of MRSA strains were tested.
Antimicrobial activities of the crude extracts were firstly screened for their inhibitory zone by the agar diffusion method as described previously (Dickson et al., 2006). Briefly, Crude extracts were prepared at concentrations of 30 mg/ml using 50% aqueous DMSO, sonicated and filter sterilized using a 0.25 m Millipore filter. Wells of 6 mm diameter were made in 20 ml MHA seeded with 20 l of a suspension of test organisms under aseptic conditions. 70 l (2.1 mg) of extracts were fed into wells, allowed to diffuse for 1 h and then incubated at 37 ◦ C for 24 h, after which time they were examined for zones of inhibition. The solvent value was deducted accordingly to get the final results of activity. All experiments were carried out in triplicate. The extracts with zone diameter (ZD) ≥ 10 mm were further subjected to determination of their MICs (minimal inhibitory concentrations) by serial dilution method according to the procedures reported previously (Dickson et al., 2006; CLSI, 2006a). Briefly, stock solutions (30 mg/ml in 50% DMSO) of all extracts were passed through a pyrogenic filter to sterilize the solution and serially diluted to arrive at concentrations between 30 mg/ml and 0.1 mg/ml (100 g/ml and 0.2 g/ml for vancomycin). The 96-well plates were prepared by dispensing into each well 100 l each of an appropriate medium, test extracts and 20 l of the inoculum. A standard nutrient broth MHB was employed for the bacterial assays. The growth of the microorganisms was determined by turbidity. Clear wells indicated absence of bacterial growth. For every experiment, a sterility check (50% DMSO and medium), negative control (50% DMSO, medium and inoculum) and positive control
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Table 2 Antibacterial activity of ethanol crude extracts of 19 Chinese medicinal plants No.
Species
Parta
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Anemone rivalry Buch.-Ham. Biota orientalis (L.) Endl. Conyza Blinii Levl. Dendrobenthamia capitata (Wall.) Hutch. Dichrocephala chrysanthemifolia (Bl.) DC. Duchesnea indica (Arulr.) Forke Elsholtzia blanda Benth. Elsholtzia rugulosa Hemsl. Gaultheria yurmaneiisis (Fr.)Rehd. Geranium strictipes K. Kunth. Keineckea carnea (Andr.) Kunth. Physalis alkekengi L. Polygonum multiflorum Thunb. Potentilla fulgens Wall. Rosa laevigata Michx. Rubia cordifolia L. Schizandra spaeraridra Stapf. Senecio scandens Buch.-Ham. Tetrastigma hypoglaucum PI.
Rh Ap Ap Ap Wp Wp Ap Wp Wp R Wp F Rh F Rh Wp Ap Wp R
a b
MIC (mean ± S.E.M., mg/ml)b MSSA
MRSA
2.56 2.17 1.40 1.17 2.32 1.66 1.08 1.13 2.06 0.95 2.06 2.04 1.21 2.11 1.17 1.33 1.90 1.69 1.08
3.07 2.52 1.58 1.25 2.96 2.71 1.32 1.43 2.56 1.34 2.96 2.82 1.34 3.07 1.58 1.50 2.28 1.82 2.32
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.32 0.39 0.21 0.18 0.16 0.54 0.16 0.13 0.48 0.30 0.19 0.64 0.22 0.48 0.17 0.24 0.27 0.30 0.19
Ap, aerial parts; F, fruits; R, roots; Rh, rhizome; Wp, whole plants. A total of nine isolates of MRSA strain were tested. All samples were tested in triplicate.
(50% DMSO, medium, inoculum and vancomycin) were included. The microtitre plates were incubated at 35 ◦ C for 24 h and were examined for growth in daylight. The MIC of the preparations was the lowest concentration in the medium that completely inhibited the visible growth. The solvent value was deducted accordingly to get the final results of activity. All experiments were performed in triplicate. 2.5. Bioassay-guided isolation and the activity of betulinic acid An enlarged 100 g part of the active extract from Dendrobenthamia capitata (600 g) was suspended in 200 ml of sterilized and deionated water. Then the suspension was successively fractionated between water and petroleum ether, chloroform and ethyl acetate to form four fractions. The chloroform fraction (12 g) which showed the most active by the subsequent susceptability test was subjected to vacuum liquid chromatography (VLC, Navarro and Delgado, 1999) over silica gel-H (Qingdao, China) and eluted with gradient systems of chloroform (C)–methanol (M) (C:M = 10:1–1:1) to afford 40 mg of pure betulinic acid (3-Hydroxy-20(29)-lupen28-oic-acid). Amorphous powder; [˛]D + 6◦ (EtOH; c 0.1). IR vmax cm−1 : 3470, 2942, 2865, 1680, 875; 1 H NMR (CDCl3 -pyridine-d5 , 1:1): ı 0.45 (3H, s, H-26), 0.48 (3H, s, H-24), 0.60 (3H, s, H-25), 0.63 (3H, s, H-27), 0.66 (3H, s, H-23), 1.34 (3H, s, H-30), 4.25 and 4.40 (2H, 2 × br s, H-29); EIMS m/z (rel. int.): 456 [M]+ (8), 438 [M−H2 O]+ (5), 248 (100), 219 (15), 207 (30), 203 (80), 191 (7), 190 (12), 189 (15). All the spectral data were identical with the literature (Nick et al., 1995) and co-TLC result was also in agreement with an authentic sample. MICs of the fractions and betulinic acid from Dendrobenthamia capitata were determined by the same procedure as the foregoing serial dilution method (Dickson et al., 2006). The minimal bactericidal concentrations (MBCs) were determined by inoculating the surfaces of MHA plates with 25 l of samples taken from the clear wells of the microbroth susceptibility studies. After the bacterial suspensions had fully absorbed into the agar, the plates were incubated at 35 ◦ C for 24 h and were examined for growth in daylight. The MBC was defined as the concentration of drug that resulted in >99.9% killing of the bacterium relative to the concentration of bacterium that was present in test wells at 0 h (NCCLS, 1999). All experiments were performed in triplicate.
3. Results and discussion The ZD of 36 herbal samples were examined (data not shown), of which 19 ethanolic extracts with ZD > 10 mm and belonging to 17 different plant families were subjected to determination of the MICs. The values are in the range of 0.95–3.07 mg/ml against both MRSA and MSSA strains (Table 1). The phenomena is in accordance with the previous findings we studied on other plant samples and revealed that there might be ascribed to different mechanisms of the action other than inhibition against -lactamase (Zuo et al., 2006), such as the inhibition against bacterial topoisomerase (Cheng et al., 2007) and efflux pump (Kaatz, 2005; Gibbons et al., 2003; Michalet et al., 2007; Schmitz et al., 1998; Stavri et al., 2007). One of the most active extract from Dendrobenthamia capitata was subjected to bioassay-guided fractionation and isolation, and betulinic acid was identified as the principal constitution from the chlorform fraction. Its MIC and MBC were measured as 62.5 and 125.0 g/ml, respectively (Table 2). Although the antimicrobial activities of betulinic acid have been reported for various pathogenic microbes, including anti-HIV activity and induces apoptosis (Kuete et al., 2007; Rzeski et al., 2006; Yogeeswari and Sriram, 2005), its activity against Gram-positive MSSA has been reported contradictorily, and the MIC against MRSA was >128 g/ml (Horiuchi et al., 2007). Here we definitely determined the simliar anti-MRSA activity of the compound. (Table 3) The MIC/MBC values Table 3 Antibacterial activity of fractions and betulinic acid from Dendrobenthamia capitata Fraction and pure compound
Petroleum ether Chloroform Ethyl acetate Water Betulinic acid Vancomycin
MIC/MBC (mean ± S.E.M., g/ml)a MSSA
MRSA
ND/NDb ND/ND ND/ND ND/ND 31.2 ± 3.9/62.5 ± 7.8 1.17 ± 0.20/2.24 ± 0.32
NA/NAc 625 ± 62.5/1250 ± 125 916.7 ± 154.5/1500 ± 216.5 750 ± 108.3/1250 ± 125 62.5 ± 7.8/125.0 ± 15.6 1.23 ± 0.15/2.99 ± 0.44
a A total of nine isolates of MRSA strain were tested. All samples were tested in triplicate. b ND, not determined. c NA, not active.
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for vancomycin are in agreement with the results of Giacometti et al. By comparison, the difference in values are acceptale and our results fall in the range between 1and 4 g/ml of this previous report Giacometti et al. (2007). Apart from the triterpenoids, polyphenol constitutions in the active extracts of Dendrobenthamia capitata were also shown by TLC while spraying with 5% FeCl3 reagent (in 95% aqueous EtOH). From this study we can conclude that Dendrobenthamia capitata, Elsholtzia rugulosa, Elsholtzia blanda, Geranium strictipes, Polygonum multiflorum and Potentilla fulgens were the most active anti-MRSA plants. All of these species are perennial plants widely distributed in Lijiang and other district in the northwest of Yunnan, China. As little was known about their phytochemical constitution and antimicrobial activity prior to our investigation, we are continuing our further investigations of other antibacterial components in these plants. Acknowledgements This research was financially supported by a grant of the National Natural Science Foundation of China (NSFC 30472147). Spectral data were measured in the Department of Analysis, KIB. References Anonymous, 1970. Selected Yunnan Traditional Chinese Herbs. Tianjin People’s Press, Tianjin, China. Barrett, J.F., 2005. MRSA—what is it, and how do we deal with the problem? Expert Opinion on Therapeutic Targets 9, 253–265. Berger, B.B., Rohrer, S., 2002. Factors influencing methicillin resistance in Staphylococci. Archives of Microbiology 178, 165–171. Cheng, B., Liu, I.F., Dinh, Y.C.T., 2007. Compounds with antibacterial activity that enhance DNA cleavage by bacterial DNA topoisomerase I. Journal of Antimicrobial Chemotherapy 59, 640–645. Clinical and Laboratory Standards Institute, 2006a. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, seventh ed, vol. M7-A7. Approved Standard. Document M7. CLSI, Wayne, PA. Clinical and Laboratory Standards Institute, 2006b. Performance standards for antimicrobial disk susceptibility tests, ninth ed. Approved Standard. Document M2-A9. CLSI, Wayne, PA. Clinical and Laboratory Standards Institute, 2007. Performance standards for antimicrobial susceptibility testing—17th informational supplement. Approved standard, CLSI document M100-S17. CLSI, Wayne, PA. Deurenberg, R.H., Kalenic, C., Vink, S., Friedrich, A.W., Bruggeman, C.A., Stobberingh, E.E., 2007. The molecular evolution of methicillin-resistant Staphylococcus aureus. Clinical Microbiology and Infection 13, 222–235.
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