J Nat Med (2006) 60: 93–95 DOI 10.1007/s11418-005-0011-5 NOTE Phan Minh Giang Ỉ Phan Tong Son Katsuyoshi Matsunami Ỉ Hideaki Otsuka Anti-staphylococcal activity of ent-kaurane-type diterpenoids from Croton tonkinensis Received: 12 July 2005 / Accepted: 19 July 2005 / Published online: 11 October 2005 Ó The Japanese Society of Pharmacognosy and Springer-Verlag 2005 Abstract Ent-kaurane-type diterpenpoids 1–11, isolated from the dried leaves of the endemic Vietnamese medicinal plant Croton tonkinensis Gagnep (Euphorbiaceae), were evaluated for inhibitory activity against Staphylococcus aureus and methicillin-resistant S aureus (MRSA) strains The most active diterpenoids, 2, 3, and 8, exhibited minimum inhibitory concentrations (MICs) of 32, 500, and 125 lg/ml, respectively, against MRSA strains Keywords Ent-kaurane Ỉ Diterpenoid Ỉ Croton tonkinensis Ỉ Euphorbiaceae Ỉ Staphylococcus aureus Ỉ MRSA Staphylococcus aureus is a virulent pathogen that is currently the most common cause of infections in hospitalized patients [6] The increase in the resistance of S aureus to antibacterial agents, coupled with its increasing prevalence as a nosocomial pathogen, is of major concern The core resistance phenotype that seems to be most associated with the persistence of S aureus in the hospital is methicillin resistance In the development of new antimicrobials from plant natural products, research has been conducted on anti-staphylococcal activity of very few ent-kaurane-type diterpenoids [7, 8] Moreover, a recent review did not report any interesting anti-staphylococcal ent-kaurane-type diterpenoids [9] In this study, the evaluation on the antistaphylococcal activity of 11 ent-kaurane-type diterpenoids, 1–11 (Fig 1) from C tonkinensis was conducted Introduction Croton tonkinensis Gagnep (Euphorbiaceae) is an endemic Vietnamese medicinal plant and is used to treat burns, abscesses, impetigo, abdominal pain, dyspepsia, and gastric and duodenal ulcers Recipes containing C tonkinensis are applied to cure urticaria, leprosy, psoriasis, vaginitis due to trichomonas and genital organ prolapse [1, 2] Various ent-kaurane-type diterpenoids were isolated and their structures were determined in our previous phytochemical studies on the plant [3, 4, 5] P M Giang Ỉ P T Son Faculty of Chemistry, College of Natural Science, Vietnam National University, 19 Le Thanh Tong, Hanoi, Vietnam P M Giang Ỉ K Matsunami Ỉ H Otsuka (&) Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan E-mail: hotsuka@hiroshima-u.ac.jp Tel.: +81-82-2575335 Fax: +81-82-2575335 Results and discussion The disk diffusion method was used as a screening test for anti-staphylococcal activity Compounds showing noticeable inhibitory activity against S aureus and methicillin-resistant S aureus (MRSA) strains were tested by the 96-multiwell plate microdillution method for determination of minimum inhibitory concentrations (MICs) against MRSA The results are summarized in Table This is the first report on anti-staphylococcal activity of ent-kaurane-type diterpenoids 1–11 from C tonkinensis The most potent anti-MRSA agents are compounds 2, 3, and with MIC values of 32, 500, and 125 lg/ml, respectively Only compound can be classified as an antibacterial compound, for which a MIC less than 64 lg/ml is often required [9] Interestingly, the MIC of is much higher than that of rosthornins A–D (50, 100, 100, and 400 lg/ml), reported by Kubo group [8] All the active diterpenoids possess the a,b-unsaturated ketone group in the D-ring of kaurane skeleton and the functional groups R2 and R5 may be important in modulating the activity 94 -dihydroxykaur-16-en-15-one (8), ent-11a-acetoxykaur16-en-18-oic acid (9), ent-15a,18-dihydroxykaur-16-ene (10), ent-(16S)-18-acetoxy-7b-hydroxykaur-15-one (11), were performed in our previous phytochemical studies on C tonkinensis [3–5] Experimental Plant material The dried leaves of C tonkinensis Gagnep (Euphorbiaceae) were collected in the suburbs of Hanoi, Vietnam, and identified by Professor Vu Van Chuyen at Hanoi College of Pharmacy (Hanoi, Vietnam) in September 2003 Used microorganisms S aureus (SA) FDA209F strain and methicillin-resistant S aureus (MRSA) B-26 strain were clinically isolated at Hiroshima University Hospital and kindly provided by Professor Sugiyama M of the Graduate School of Biomedical Sciences, Hiroshima University (Hiroshima, Japan) Extraction of plant material and isolation of ent-kaurane-type diterpenoids 1–11 Extraction of the dried leaves of C tonkinensis and isolation of the diterpenoids ent-7b-acetoxy-11a-hy droxykaur-16-en-15-one (1), ent-18-acetoxy-11a-hy droxykaur-16-en-15-one (2), ent-18-acetoxy-7b-hydroxykaur-16-en-15-one (3), ent-1a,14a-diacetoxy-7b-hy droxykaur-16-en-15-one (4), ent-1a,7b-diacetoxy-14ahydroxykaur-16-en-15-one (5), ent-18-acetoxy-14a-hy droxykaur-16-en-15-one (6), ent-1a-acetoxy-7b,14a-dihydroxykaur-16-en-15-one (7), ent-18-acetoxy-7b,14a Fig Chemical structures of ent-kaurane-type diterepnoids 1–11 Disk diffusion method The anti-staphylococcal activity of compounds 1–11 was screened using the disk diffusion method [10] Petri dishes containing 20 ml of Mueller-Hinton agar medium (Difco) were seeded with a 24 h culture of the bacterial strains The inoculum size was adjusted to AcO R5 R1 H R4 H O R3 H H HOOC R2 R1 H H H OAc OAc H OAc H R2 H OAc OAc H H OAc H OAc R3 R4 R5 OAc H OH H H OH OH H H OH OAc H OAc OH H H OH H OH OH H OH OH H H OH H HO 10 H O OH H AcO 11 95 Table Anti-staphylococcal activity of compounds 1–11 against S aureus (SA) FDA209F and methicillinresistant S aureus (MRSA) B-26 strains Positive controls included streptomycin: 13 mm (0.5 lg/ disk), 10 mm (0.2 lg/disk); tetracyclin: 14 mm (0.5 lg/disk), 11 mm (0.2 lg/disk); penicillin G: 16 mm (0.01 lg/disk), 11 mm (0.005 lg/disk) a mm refers to no visible zone of inhibition Compound Zone of inhibition (mm)a 100 (lg/disk) 10 11 MIC (lg/ml) 10 (lg/disk) SA FDA 209F MRSA B-26 SA FDA 209F MRSA B-26 19 20 16 22 22 10 16 23 11 8.5 16 15 18 14 18 11 8.5 12 22 8.5 11 16 12 12 9 11 12 0 0 16 13 8.5 0 11 12 0 approximately 108 colony-forming units (CFU)/ml The solutions of compounds 1–11 were applied to sterile filter paper disks (ADVANTEC, mm in diameter) to give the final amounts of 10 and 100 lg and placed on the surface of the inoculated medium The plates were incubated at 37°C for 24 h Anti-staphylococcal activity was determined by measuring the diameter of the inhibition zone formed around the disk Minimum inhibitory concentration (MIC) determination Assays were performed in a final volume of 100 ll using Mueller–Hinton broth (Difco) [11] The bacterial inocula were adjusted to yield a density of 5·105 CFU/ml Compounds were prepared at a concentration equivalent to twice the highest desired final concentration in culture medium Compounds were then diluted directly in the 96-multiwell plates by serial twofold dilution using a multichannel pipette Microtiter plates were incubated for 24 h at 35°C and were read using a microtiter-plate reader (Molecular Devices) at 650 nm as well as by visual observation The MIC is defined as the lowest concentration of a compound at which the visible growth of the organism is completely inhibited Acknowledgements This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) P.M.G is grateful to acknowledge the International Foundation for Science (Stockholm, Sweden) for the financial support We thank Professor Sugiyama M (Graduate School of Biomedical Sciences, Hiroshima University) for the generous gift of methicillinresistant Staphylococcus aureus B-26 strain MRSA B-26 32 500 125 References Vo VC (1997) Dictionary of Vietnamese medicinal plants Medicine, Ho Chi Minh City Le Van Truyen, Nguyen Gia Tran (eds) (1999) Selected medicinal plants in Vietnam Science and Technology, Hanoi Phan MG, Jin HZ, Phan TS, Lee JH, Hong YS, Lee 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Japan) Extraction of plant material and isolation of ent-kaurane-type diterpenoids 1–11 Extraction of the dried leaves of C tonkinensis and isolation of the diterpenoids ent-7b-acetoxy-11a-hy droxykaur-16-en-15-one... and placed on the surface of the inoculated medium The plates were incubated at 37°C for 24 h Anti-staphylococcal activity was determined by measuring the diameter of the inhibition zone formed... Ent-kaurane diterpenoids from Croton tonkinensis inhibit LPSinduced NF-jB activation and NO production J Nat Prod 66:1217–1220 Phan MG, Phan TS, Lee JJ, Otsuka H (2004) Four ent-kaurane-type diterpenoids