Synergism of prenylflavonoids from Morus alba root bark against clinical MRSA isolates.

BACKGROUND: Clinical methicillin-resistant Staphylococcus aureus (MRSA) is a thorny problem in current anti-infective therapeutics and a challenge of new drug development. Plant prenylflavonoids possess anti-MRSA activity, but few of the prenylflavonoids have been reported the synergistic anti-MRSA effect when they are used in combination with conventional antibacterial agents. PURPOSE: This study deals with anti-MRSA activity of four prenylflavonoids from the root bark of Morus alba and their synergism with 11 conventional antibacterial agents. METHODS: Chromatographic methods and spectral analysis were used to isolate and identify the prenylflavonoids. The antibacterial activity and synergism were assessed by the broth microdilution method, checkerboard dilution test, and time-kill curve assay, respectively. RESULTS: Four prenylflavonoids, i.e., cyclocommunol (Cy, 1), morusinol (Ml, 2), morusin (Mi, 3) and kuwanon E (Ku, 4), were isolated from Morus alba bark ethanol extract. Compounds 1, 3 and 4 showed high antimicrobial activity on both methicillin-susceptible S. aureus (MSSA) and MRSA strains with MICs/MBCs at 4-16/32-64 and 4-32/16-128 µg/ml, respectively. Ml (2) was not active. Compound 2 showed synergy with amikacin (AK) and streptomycin (SM) against all the ten MRSA isolates. Ml (2) and Ku (4) also showed synergy with ciprofloxacin (CI), etimicin (EM) and vancomycin (VA) against 7-9 isolates. The fractional inhibitory concentration indices (FICIs) ranged 0.09-1.00 and the dose reduction indices (DRIs) of these antibacterial agents ranged 2-128. Cy (1) and Mi (3) showed synergy with the tested antibacterial agents against only 1-3 MRSA isolates except VA. Furthermore, the MRSA resistance could be reversed in the combinations of AK with Cy, Ml, Mi and Ku; EM with Mi and Ku; and SM with Ml by the criteria of MIC interpretive standards for Staphylococcus spp. of CLSI. All the combinations showed only indifference in the 1 × MIC time-killing experiments. The prenylated substitutions play an important role in the activity of the compounds used alone and combined with the tested antibacterials. CONCLUSIONS: The study revealed for the first time the anti-MRSA synergism of prenylflavonoids 1-4 with eleven antibacterial agents and the reversal of MRSA resistance to aminoglycosides, especially amikacin. The results might be valuable for the development of new antibacterial drugs and synergists against MRSA infection.

Synergism of coumarins from the Chinese drug Zanthoxylum nitidum with antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA).

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious therapeutic challenge in current clinic and new drug development. Natural coumarins have diverse bioactivities and the potential of resistance modifying effects. PURPOSE: This study is to present in-depth evaluations of in vitro antimicrobial activities of four natural coumarins 5-geranyloxy-7-methoxycoumarin (Gm, 1), (5,7-dimethoxy-8-prenyloxycoumarin (artanin, Ar, 2)), isopimpinellin (Is, 3) and phellopterin (Ph, 4) from Zanthoxylum nitidum (Roxb.) DC. (Rutaceae) extracts, focusing on their potential restoration the activity of conventional antibacterial agents against clinical MRSA strains. METHODS: Bioactivity-guided fractionation and spectral analyses were used to isolate the coumarins and identify the structures, respectively. The double broth microdilution method was used to assay the coumarins' alone activity. The classic checkerboard microdilution and dynamic time-killing methods were used to evaluate combinatory effects. RESULTS: The four plant coumarins Gm (1), Ar (2), Is (3) and Ph (4) were isolated and identified from Z. nitidum extracts. Coumarins 1-4 displayed promising inhibition against both MSSA and MRSA with minimal inhibitory concentrations (MICs) of 8-64µg/ml, but very weak against Gram-negative pathogen and yeast with MICs of 256 to ≥1024µg/ml. The geranyloxy and prenyloxy substitutions showed to be more active than the methoxy substitution on the coumarin skeletons. 1-4 also showing different extent of synergism with a total of eight conventional antibacterial agents, i.e. chloramphenicol (CL), gentamicin (CN), fosfomycin (FF), levofloxacin (LE), minocycline (MI), piperacillin/tazobactam (P/T), teicoplanin (TE) and vancomycin (VA) against ten clinical MRSA strains. Four to ten of the tested MRSA strains showed bacteriostatic synergy in the eleven combinations. The anti-MRSA modifying effects were related to different arrangement in the combinations with fractional inhibitory concentration indices (FICIs) from 0.187 to 1.125 and the three combinations CN (Is), CL (Ph) and MI (Gm) were the best ones. The enhancement of activity was also shown by 2-64 of dose reduction indices (DRIs) of the combined MICs, with VA (Ph) combination resulted the biggest DRI. The resistance of MRSA to antibacterial agents could be reversed in the combinations of CL (Gm or Ph), LE (Ph) and MI (Is) following the Clinical and Laboratory Standards Institute (CLSI) criteria. Six combinations P/T (Gm), TE (Ar), CN (Is), VA (Ph) and CL (Gm or Ph) also showed bactericidal synergy with Δlog10CFU/ml >2 at 24h incubation. CONCLUSIONS: The coumarins showed high potentiating effects of the antibacterial agents against multi-drug resistant SA. The resistance reversal effect of CL, LE and MI warrants further pharmacological investigation on combinatory therapy for the sake of fighting against MRSA infections.

Potentiation activity of multiple antibacterial agents by Salvianolate from the Chinese medicine Danshen against methicillin-resistant Staphylococcus aureus (MRSA).

Salvianolate (SAL) is a prescribed medicine from the Chinese herb Danshen (Salvia miltiorrhiza Bunge). It has been widely used in treatment of coronary and other diseases with significant effects. The in vitro antimicrobial activities of SAL against infectious pathogens were assayed and its combined effects on 10 clinical isolates of SCCmec III type methicillin-resistant Staphylococcus aureus (MRSA) with ten antibiotics were evaluated. Susceptibility to each agent alone was tested using a broth microdilution method, and the chequerboard and time-kill experiments were used for the combined activities. The results showed MIC was 128-256 mg/L for SAL used alone against MRSA. Significant synergies were observed for SAL/Ampicillin (Fosfomycin, Erythromycin, Piperacillin-tazobactam or Clindamycin) combination against over half of the isolates, with their MICs reduced by times of dilution (TOD) to 4-32 (FICIs 0.375-0.5), respectively. SAL/AMP combination showed the best combined effect of synergy on bacteriostatic and bactericidal activities, while SAL/AMK combination reversed the resistance of MRSA to AMK. The results demonstrated that SAL enhanced widely the in vitro anti-MRSA efficacy of the ten antibacterial agents, which had potential for combinatory therapy of patients infected with MRSA and warrants further investigations.

In vitro synergism of magnolol and honokiol in combination with antibacterial agents against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA).

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a problematic pathogen posing a serious therapeutic challenge in the clinic. It is often multidrug-resistant (MDR) to conventional classes of antibacterial agents and there is an urgent need to develop new agents or strategies for treatment. Magnolol (ML) and honokiol (HL) are two naturally occurring diallylbiphenols which have been reported to show inhibition of MRSA. In this study their synergistic effects with antibacterial agents were further evaluated via checkerboard and time-kill assays. METHODS: The susceptibility spectrum of clinical MRSA strains was tested by the disk diffusion method. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of ML and HL were assayed by broth microdilution. The synergy was evaluated through checkerboard microdilution and time-killing experiments. RESULTS: ML and HL showed similar activity against both MSSA and MRSA with MIC/MBC at 16 ~ 64 mg/L, with potency similar to amikacin (AMK) and gentamicin (GEN). When they were used in combination with conventional antibacterial agents, they showed bacteriostatic synergy with FICIs between 0.25 ~ 0.5, leading to the combined MICs decreasing to as low as 1 ~ 2 and 1 ~ 16 mg/L for ML (HL) and the agents, respectively. MIC50 of the combinations decreased from 16 mg/L to 1 ~ 4 mg/L for ML (HL) and 8 ~ 128 mg/L to 2 ~ 64 mg/L for the antibacterial agents, which exhibited a broad spectrum of synergistic action with aminoglycosides (AMK, etilmicin (ETM) and GEN), floroquinolones (levofloxacin (LEV), ciprofloxacin and norfloxacin), fosfomycin (FOS) and piperacillin. The times of dilution (TOD, the extent of decreasing in MIC value) were determined up to 16 for the combined MIC. A more significant synergy after combining was determined as ML (HL) with AMK, ETM, GEN and FOS. ML (HL) combined with antibacterial agents did not show antagonistic effects on any of the ten MRSA strains. Reversal effects of MRSA resistance to AMK and GEN by ML and HL were also observed, respectively. All the combinations also showed better dynamic bactericidal activity against MRSA than any of single ML (HL) or the agents at 24 h incubation. The more significant synergy of combinations were determined as HL (ML) + ETM, HL + LEV and HL + AMK (GEN or FOS), with △LC24 of 2.02 ~ 2.25. CONCLUSION: ML and HL showed synergistic potentiation of antibacterial agents against clinical isolates of MRSA and warrant further pharmacological investigation.

Antimicrobial activity and synergy of antibiotics with two biphenyl compounds, protosappanins A and B from Sappan Lignum against methicillin-resistant Staphylococcus aureus strains.

OBJECTIVES: This study aims to investigate antimicrobial ingredients from Sappan Lignum and to evaluate their synergy on methicillin-resistant Staphylococcus aureus strains with antibiotics. METHODS: Bioactivity-guided phytochemical procedures were used to screen the active compounds. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were assayed by broth microdilution. The synergy was evaluated through checkerboard microdilution and loss of viability assays. KEY FINDINGS: Protosappanins A (PsA) and B (PsB) were identified from Sappan Lignum extracts. They showed active against both S. aureus and MRSA with MIC or MIC50 at 64 (PsA) and 128 (PsB) mg/L alone. When they were used in combination with antibiotics, they showed best synergy with amikacin and gentamicin with MIC50 (mg/L) of amikacin reduced more significantly from 32 to four (with PsA) and eight (with PsB), and the fractional inhibitory concentration index (FICI) ranged between 0.078 and 0.500 (FICI50 = 0.375). Moreover, the resistance of MRSA towards amikacin and gentamicin could be reversed by the Clinical and Laboratory Standards Institute criteria. The combined bactericidal mode could as well be synergy. PsA and PsB showed very low cytotoxicity in comparison with their promising activity against MRSA. CONCLUSIONS: Protosappanins A and B showed both alone activities and resistance reversal effects of amikacin and gentamicin against MRSA, which warrant further investigations for potential combinatory therapy of MRSA infection.

One new flavonoid from Oroxylum indicum.

One new flavonoid, 5,6,7-trimethoxyflavone-8-O-ß-D-glucopyranoside (1), along with six known compounds 2-7, was isolated from Oroxylum indicum. Their structures were determined on the basis of spectral data. The antibacterial activities of compounds 1-4 were studied. Compounds 1 and 3 showed medium antibacterial activity against Staphylococcus aureus with MIC/MBC at 32-128 µg/ml.

Limonoids from the fruits of Cipadessa cinerascens.

A new limonoid, 3-de(2-methylbutanoyl)-3-propanoylcipadesin (1), along with 10 known limonoids and 1 known triterpenoid, was isolated from the fruits of Cipadessa cinerascens. Their structures were elucidated on the basis of spectroscopic analysis. All compounds were evaluated for their antimicrobial activities, and compounds 6 and 12 showed weak antimicrobial activities against MRSA 82(#) and MRSA 92(#).

Myrifabine, the first dimeric Myrioneuron alkaloid from Myrioneuron faberi.

One Myrioneuron alkaloid, myrifabine (1), the first example of a dimer with 12 chiral centers embraced in a decacyclic novel skeleton, was isolated from Myrioneuron faberi . Its structure was elucidated by spectroscopic data and single-crystal X-ray diffraction. The antimicrobial and cytotoxic activities of 1 were evaluated in vitro.

Chemical constituents from Aphanamixis grandifolia.

Four new terpenoids, nemoralisins D-G (1-4), were isolated from the leaves and stems of Aphanamixis grandifolia, along with two known diterpenoids, nemoralisin C and nemoralisin. Among them, compound 1 is the first example of norsesquiterpenoid with δ-lactone moiety, and nemoralisins E-G (2-4), are a class of acyclic diterpenoids, which are structurally related nemoralisin C and nemoralisin. These structures were established on the basis of spectroscopic methods and the absolute configuration of 1 was determined by comparison of quantum chemical TDDFT calculated and experimental ECD spectra. Nemoralisins D-G (1-4) were tested for their cytotoxicities on HL-60, SMMC-7721, A-549, MCF-7, and SW480 human tumor cell lines (IC50>40 µM), as well as the antimicrobial activities on Staphylococcus aureus, Pseudomonas aeruginosa, MRSA92(#) and MRSA98(#) (MIC>50 µg/mL).

Two new compounds from Khaya senegalensis.

Two new compounds, khayseneganin I (1) and 2α,3α,16ß-trihydroxy-20-acetoxy-20(R)-pregnane (2), along with six known compounds, 2α,3α,20-trihydroxy-16ß-acetoxy-20(R)-pregnane (3), 2α,3ß-dihydroxypregnan-16-one-2ß,19-hemiketal (4), (+)-catechin (5), ivorenolide A (6), luteolin-7-O-α-l-rhamnoside (7), and ( - )-5'-methoxy-isolariciresinol-2a-O-ß-d-xylopyranoside (8), were isolated from the leaves and twigs of Khaya senegalensis. The structures of new compounds were elucidated by 2D NMR spectroscopy and MS. Selected compounds (2-8) were evaluated for their antimicrobial activities and compounds 5 and 7 showed weak antimicrobial activities against MRSA 92(#) and MRSA 98(#).

Khayseneganins A-H, limonoids from Khaya senegalensis.

Eight new limonoids, khayseneganins A-H (1-8), and 31 known limonoids were isolated from the leaves and twigs of Khaya senegalensis. The structures of the new compounds were elucidated by 2D-NMR spectroscopy and mass spectrometry, and the absolute configuration of 1 was determined by the CD exciton chirality method. Compounds 9, 10, 12, and 15 showed antimicrobial activities against Pseudomonas aeruginosa, MRSA 92(#), and MRSA 98(#), all with a MIC value of 12.5 µg/mL.

Trigohowilols A-G, degraded diterpenoids from the stems of Trigonostemon howii.

Two new degraded diterpenoids, trigohowilols A (1) and B (2), four new heterodimers, trigohowilols C-F (3-6), one new homodimer, trigohowilol G (7), and three known degraded diterpenoids (8-10) were isolated from the methanol extract of the stems of Trigonostemon howii. Compounds 1-7 were evaluated for their cytotoxic activity against five human tumor cell lines by an MTT assay, and trigohowilols E (5) and F (6) exhibited inhibitory activity with IC50 values ranging from 2.33 to 12.57 µM. Moreover, compounds 1-6 showed weak antimicrobial activities (MIC values: 6.25-25 µg/mL) against Staphylococcus aureus, Pseudomonas aeruginosa, MRSA 92(#), and MRSA 98(#) using a 2-fold dilution method.

Isojacareubin from the Chinese herb Hypericum japonicum: potent antibacterial and synergistic effects on clinical methicillin-resistant Staphylococcus aureus (MRSA).

Through bioassay-guided fractionation of the extracts from the aerial parts of the Chinese herb Hypericum japonicum Thunb. Murray, Isojacareubin (ISJ) was characterized as a potent antibacterial compound against the clinical methicillin-resistant Staphylococcus aureus (MRSA). The broth microdilution assay was used to determine the minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of ISJ alone. The results showed that its MICs/MBCs ranged from 4/16 to 16/64 µg/mL, with the concentrations required to inhibit or kill 50% of the strains (MIC(50)/MBC(50)) at 8/16 µg/mL. Synergistic evaluations of this compound with four conventional antibacterial agents representing different types were performed by the chequerboard and time-kill tests. The chequerboard method showed significant synergy effects when ISJ was combined with Ceftazidime (CAZ), Levofloxacin (LEV) and Ampicillin (AMP), with the values of 50% of the fractional inhibitory concentration indices (FICI(50)) at 0.25, 0.37 and 0.37, respectively. Combined bactericidal activities were also observed in the time-kill dynamic assay. The results showed the ability of ISJ to reduce MRSA viable counts by log(10)CFU/mL at 24 h of incubation at a concentration of 1 × MIC were 1.5 (LEV, additivity), 0.92 (CAZ, indifference) and 0.82 (AMP, indifference), respectively. These in vitro anti-MRSA activities of ISJ alone and its synergy with conventional antibacterial agents demonstrated that ISJ enhanced their efficacy, which is of potential use for single and combinatory therapy of patients infected with MRSA.

Antibacterial and synergy of berberines with antibacterial agents against clinical multi-drug resistant isolates of methicillin-resistant Staphylococcus aureus (MRSA).

Antibacterial activity of berberine (Ber) and 8-acetonyl-dihydroberberine (A-Ber) alone and combined uses with antibacterial agents ampicillin (AMP), azithromycin (AZM), cefazolin (CFZ) and levofloxacin (LEV) was studied on 10 clinical isolates of SCCmec III type methicillin-resistant Staphylococcus aureus (MRSA). Susceptibility to each agent alone was tested using a broth microdilution method and the chequerboard and time-kill tests for the combined evaluations, respectively. The alone MICs/MBCs (µg/mL) ranges were 32-128/64-256 (Ber) and 32-128/128-512 (A-Ber). Significant synergies were observed for the Ber (A-Ber)/AZM and Ber (A-Ber)/LEV combinations against 90% of the tested MRSA strains, with fractional inhibitory concentration indices (FICIs) values ranged from 0.188 to 0.500. An additivity result was also observed for the Ber/AZM combination by time-kill curves. These results demonstrated for the first time that Ber and A-Ber enhanced the in vitro inhibitory efficacy of AZM and LEV to a same extent, which had potential for further investigation in combinatory therapeutic applications of patients infected with MRSA.

Trigoflavidols A-C, degraded diterpenoids with antimicrobial activity, from Trigonostemon flavidus.

Trigoflavidols A (1) and B (2), tetranorditerpenoid dimers possessing a rearrangement skeleton with a spiroketal core moiety, and trigoflavidol C (3), a hexanorditerpenoid, have been isolated from Trigonostemon flavidus along with two known compounds. Compounds 1 and 2 showed moderate antimicrobial activities (MIC values: 3.12-6.25 µg/mL) against Staphylococcus aureus, 8(#)MRSA, and 82(#)MRSA, and 1, 2, and 5 showed weak activities (IC(50) values: 3.75-28.99 µM) against various human tumor cell lines.

Evaluation of traditional Chinese medicinal plants for anti-MRSA activity with reference to the treatment record of infectious diseases.

The in vitro antimicrobial activities of 30 Chinese medicinal plants were evaluated with reference to the treatment record of infectious diseases in the Traditional Chinese Medicine (TCM) literature. The plant materials were extracted with 80% ethanol and the extracts were primarily screened against conventional clinical pathogens like Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans by the agar diffusion method. Their inhibition zone diameters (IZDs, mm, 50 mg/mL) ranged from 2,048 by the standard broth microdilution method. The seven extracts from M. yunnanensis, S. sinensis, G. morella, E. daneillii, M. squamulata, S. arborescens and B. hancei were determined as the most active extracts, with MICs of 8-64 µg/mL. The results were in good agreement with their traditional applications in skin and other infections.

Synergistic antibacterial and antibiotic effects of bisbenzylisoquinoline alkaloids on clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA).

The antibacterial activity of two bisbenzylisoquinoline alkaloids, tetrandrine (Tet) and demethyltetrandrine (d-Tet), alone and in combination with the antibiotics ampicillin (AMP), azithromycin (AZM), cefazolin (CFZ) and levofloxacin (LEV) against 10 clinical isolates of staphylococcal chromosomal cassette mec (SCCmec) III type methicillin-resistant Staphylococcus aureus (MRSA) was studied. Susceptibility to each agent alone was tested using a broth microdilution method. The chequerboard and time-kill tests were used for the combined evaluations. The minimal inhibitory concentrations/minimal bactericidal concentrations (MICs/MBCs, µg/mL) ranges alone were 64-128/256-1,024 for both Tet and d-Tet. Significant synergies against 90% of the isolates were observed for the Tet/CFZ combination, with their MICs being reduced by 75-94% [fractional inhibitory concentration indices (FICIs) ranged from 0.188 to 0.625], respectively. An additive bactericidal result was also observed for the Tet (d-Tet)/CFZ combination in the time-kill experiments. These results demonstrated that Tet and d-Tet enhanced the in vitro inhibitory efficacy of CFZ. Their potential for combinatory therapy of patients infected with MRSA warrants further pharmacological investigation.

In vitro activity of plant extracts and alkaloids against clinical isolates of extended-spectrum ß-lactamase (ESBL)-producing strains.

The antibacterial activity of 80% ethanol extracts of 10 medicinal plants collected in Yunnan (Southwest China), was tested against clinical isolates of extended-spectrum ß-lactamase (ESBL)-producing strains. Their MIC values ranged between 1.56-12.50 mg/mL. The most active plant extract was Chelidonium majus L. (MIC = 1.56 mg/mL). Two potent isoquinoline alkaloids, 8-hydroxydihydrosanguinarine and 8-hydroxydihydrochelerythrine, were identified as the major active principles through bioassay-guided fractionation and identification of the active ethyl acetate fraction from C. majus, with minimum MIC/MBC values of 15.63/62.50 mg/mL.

Cytotoxicity of labdane-type diterpenoids from Hedychium forrestii.

Two new labdane-type diterpenoids, hedyforrestin D (1) and 15-ethoxy-hedyforrestin D (2), and three known compounds, yunnancoronarin A (4), B (3) and C (5) were isolated from the rhizomes of Hedychium forrestii. The structure of the new diterpenoids was established as 6beta,15xi-dihydroxylabda-8(17),11,13-trien-15,16-olide (1), and 6beta-hydroxy-15xi-ethoxylabda-8(17),11,13-trien-15,16-olide (2) on the basis of spectroscopic analyses. In addition, the isolated compounds were evaluated for their cytotoxicity against the lung adenocarcinoma cells A549 and leukemia cells K562 through 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. Of these, compounds 3 and 4 exhibited the most activity with IC(50) values of 0.92 and 2.20 microM, respectively, whereas 5 was inactive against A549 cells and 1 was inactive against both cell lines up to a concentration of 300.81 microM. This shows that both the hydroxy substitution and orientation of unsaturated lactone group in the five-membered ring of C-13 to C-16 seem to play an important role in the anti-tumor activities of human lung adenocarcinoma and leukemia.

Antibacterial alkaloids from chelidonium majus linn (papaveraceae) against clinical isolates of methicillin-resistant Staphylococcus aureus.

PURPOSE: This study describes the antibacterial effect of extracts and compounds isolated from the aerial part of Chelidonium majus Linn. (Papaveraceae) acting against clinical strains of methicillin-resistant Staphylococcus aureus (MRSA). METHODS: The activities were evaluated by using the macrobroth dilution method and reported as the MICs/MBCs. RESULTS: Bioassay-guided fractionation of the most active extract from the aerial parts (EtOAc) led to the isolation of benzo[c]phenanthridine-type alkaloids 8-hydroxydihydrosanguinarine (hhS), 8-hydroxydihydrochelerythrine (hhC), which were potently active against MRSA strains. CONCLUSIONS: The selective antibacterial activity reported in this paper for 8-hydroxylated benzo[c]phenanthridine-type alkaloids isolated from C.majus opens the possibility that they could be helpful for the developing of new antibacterial agents for treating the infection of MRSA which has created nosocomial problem worldwide.