Antibacterial activity of phytochemicals isolated from Erythrina zeyheri against vancomycin-resistant enterococci and their combinations with vancomycin

Six phytochemicals were isolated from the roots of Erythrina zeyheri (Leguminosae) by repeated silica gel column chromatography using various eluting solvents. Extensive spectroscopic studies revealed that all were isoflavonoids. The antibacterial activity of the six compounds against vancomycin-resistant enterococci (VRE) was estimated by determining the minimum inhibitory concentration (MIC). Of the six isoflavonoids, erybraedin A ((6aR, 11aR)-3,9-dihydroxy-4,10-di(gamma,gamma-dimethylallyl)pterocarpan) exhibited the highest growth inhibitory potency against VRE with an MIC value of 1.56-3.13 microg/mL, followed by eryzerin C ((3R)-7,2',4'-trihydroxy-6,8-di(gamma,gamma-dimethylallyl)isoflavan) (MIC 6.25 microg/mL). These compounds also inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA) at 3.13-6.25 microg/mL. The antibacterial effects of the two compounds against VRE and MRSA were based on bacteriostatic action. When erybraedin A or eryzerin C was combined with vancomycin, the fractional inhibitory concentration (FIC) index against VRE ranged from 0.5306 to 1.0 and from 0.5153 to 0.75, respectively. The combinations also showed FIC indices of 0.6125-1.0 against MRSA. The results indicate that, depending on the case, both compounds act either synergistically or additively with vancomycin against VRE and MRSA. Erybraedin A and eryzerin C show evidence of being potent phytotherapeutic agents against infections caused by VRE and MRSA.     

Evaluation of the flora of northern Mexico for in vitro antimicrobial and antituberculosis activity

The aim of the present study was to evaluate the potential antimicrobial activity of 14 plants used in northeast México for the treatment of respiratory diseases, against drug-sensitive and drug-resistant strains of Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae type b and Mycobacterium tuberculosis. Forty-eight organic and aqueous extracts were tested against these bacterial strains using a broth microdilution test. No aqueous extracts showed antimicrobial activity, whereas most of the organic extracts presented antimicrobial activity against at least one of the drug-resistant microorganisms tested. Methanol-based extracts from the roots and leaves of Leucophyllum frutescens and ethyl ether extract from the roots of Chrysanctinia mexicana showed the greatest antimicrobial activity against the drug-resistant strain of Mycobacterium tuberculosis; the minimal inhibitory concentration (MIC) were 62.5, 125 and 62.5 microg/mL, respectively; methanol-based extract from the leaves of Cordia boissieri showed the best antimicrobial activity against the drug-resistant strain of Staphylococcus aureus (MIC 250 microg/mL); the hexane-based extract from the fruits of Schinus molle showed considerable antimicrobial activity against the drug-resistant strain of Streptococcus pneumoniae (MIC 62.5 microg/mL). This study supports that selecting plants by ethnobotanical criteria enhances the possibility of finding species with activity against resistant microorganisms.     

Bioactive carbazole alkaloids from Clausena wallichii roots

Four new carbazole alkaloids, clausenawallines C-F (1-4), along with 18 known compounds (5-22) were isolated from the roots of Clausena wallichii. Compounds 3, 9, and 22 exhibited significant antibacterial activity against methicillin-resistant Staphylococcus aureus SK1 (MRSA SK1) and Staph. aureus TISTR 1466 with MIC values in the range 4-16 μg/mL, whereas compound 4 showed the highest cytotoxicity against oral cavity cancer (KB) and small-cell lung cancer (NCI-H187) with IC(50) values of 10.2 and 4.5 μM, respectively.     

Antimicrobial and anti-inflammatory activity of four known and one new triterpenoid from Combretum imberbe (Combretaceae)

Combretum imberbe is used widely in Africa inter alia for treating bacterial infections. In addition to four known triterpenoids, 1alpha,3beta-dihydroxy-12-oleanen-29-oic (1), 1-hydroxy-12-olean-30-oic acid (2), 3,30-dihydroxyl-12-oleanen-22-one (3), and 1,3,24-trihydroxyl-12-olean-29-oic acid (4), a new pentacyclic triterpenoid (1alpha,23-dihydroxy-12-oleanen-29-oic acid-3beta-O-2,4-di-acetyl-L-rhamnopyranoside) 5 has been isolated through a bioassay-guided procedure from the leaves of Combretum imberbe. The structures of the compounds were elucidated on the basis of 1D and 2D NMR experiments, as well as mass spectrometric data. All compounds isolated had moderate (62 microg/ml) to strong (16 microg/ml) antibacterial activity (MIC values) against Staphylococcus aureus and Escherichia coli, with 1 and 5 being most active. Compounds 1 and 5 also showed very strong inhibition of 3alpha-hydroxysteroid dehydrogenase with an IC(50) of 0.3 microg/ml. Compound 5 indicated a moderate anti-proliferative (GI(50)=16.5 and 13.2 microg/ml) and cytotoxic activity (CC(50)=17.6 micro/ml) against K-562, L-929 and HeLa cell lines, respectively. The results of this study give credence to the ethnomedicinal use of Combretum imberbe and expand our knowledge on the biological activity of its metabolites.     

Antibacterial steroid alkaloids from the stem bark of Holarrhena pubescens

Holarrhena pubescens (syn. H. antidysenterica) (L.) WALL. stem bark was tested for antibacterial efficacy against Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus faecalis, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa using the microdilution broth method as well as the disc diffusion method. The crude methanolic extract was active against all tested bacteria. Further chemical fractionation indicated that the antibacterial activity was mainly associated with the alkaloids. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for the crude extract, the total alkaloids and the neutral fraction using microdilution broth method. The results were compared with reference antibiotics. The total alkaloids showed remarkable activity against S. aureus (MIC = 95 microg/ml).     

ent-rosane and labdane diterpenoids from Sagittaria sagittifolia and their antibacterial activity against three oral pathogens

Seven new ent-rosane diterpenoids, sagittines A-G (1-7), together with one new labdane diterpene, 13-epi-manoyl oxide-19-O-alpha-l-2',5'-diacetoxyarabinofuranoside (8), were isolated from the whole plant of Sagittaria sagittifolia. The structures and relative configurations of 1-8 were characterized using spectroscopic means, chemical methods, and X-ray crystallography. Compounds 1-4 exhibited antibacterial activity against the oral pathogens Streptococcus mutans ATCC 25175 and Actinomyces naeslundiis ATCC 12104, with MIC values between 62.5 and 125 microg/mL. Compound 5 was active against only A. naeslundiis ATCC 12104, with an MIC value of 62.5 microg/mL.     

Antibacterial activities and cytotoxicity of terpenoids isolated from Spirostachys africana

Spirostachys africana Sond. stem bark is used traditionally for the treatment of diarrhoea and dysentery in Limpopo Province of South Africa. Bioassay-guided fractionation of ethanolic extract from bark of Spirostachys africana led to the isolation of four known compounds, two triterpenoids, compound 1 [d-Friedoolean-14-en-oic acid (3-acetyl aleuritolic acid)] and compound 2 (Lupeol), and two diterpenes, compound 3 [ent-2,6alpha-dihydroxy-norbeyer-1,4,15-trien-3-one (diosphenol 2)] and compound 4 (ent-3beta-hydroxy-beyer-15-ene-2-one). Isolated compounds were tested for antibacterial activity using micro-dilution method. Compound 1, exhibited minimum inhibitory concentration (MIC) of 50 microg/ml against Staphylococcus aureus, Salmonella typhy, Vibrio cholera, Escherichia coli and Shigella dysentery. Compound 2 was not active against all tested microorganisms at 200 microg/ml, which was the highest concentration tested. At this concentration, all four compounds were not active against Shigella sonnei. Cytotoxicity of ethanol crude extracts and isolated compounds from Spirostachys africana was determined using the sodium-2,3-bis-[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) assay on Vero cells. Compounds 2 and 3, isolated from Spirostachys africana, had up to three times higher [50% inhibitory concentration (IC(50) values; 300.9 and 308.9 microg/ml)] than the ethanol crude extracts (102.8 microg/ml) suggesting higher toxicity of the crude extract as compared to these two compounds. In contrast, compounds 1 and 4 were not cytotoxic to Vero cell lines (African green monkey) in vitro at the concentrations tested (IC(50)>400 microg/ml). This is the first report on the antibacterial activity and cytotoxicity of purified compounds from Spirostachys africana.     

Antimicrobial activity of xanthones from Calophyllum species, against methicillin-resistant Staphylococcus aureus (MRSA).

During the past 5 years, a considerable number of known and new xanthones from the Calophyllum species of Sri Lanka have been isolated and characterized. We have investigated the antimicrobial activity of Calophyllum xanthones, with a special reference to methicillin-resistant Staphylococcus aureus (MRSA). These activity studies were carried out using the agar plate method. Calozeloxanthone, a xanthone which has been isolated from C. moonii and C. lankensis, showed the highest activity against methicillin-resistant S. aureus (MRSA) strains at a concentration of 8.3 microg/ml. Hence, calozeyloxanthone appears to hold promise as an antimicrobial agent in the treatment of infections with S. aureus, including methicillin-sensitive S. aureus (MRSA), and should be investigated further.     

Antimicrobial activities of naphthazarins from Arnebia euchroma

Bioassay-directed fractionation of extract of Arnebia euchroma led to the isolation of alkannin (1), shikonin (2), and their derivatives (3-8) as the active principles against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). The stereochemistry of alpha-methylbutyryl alkannin (8) is revealed for the first time, and the antimicrobial activity of 8 was compared with its corresponding diastereomer (9). The derivatives 3-9 showed stronger anti-MRSA activity [minimum inhibitory concentrations (MICs) ranged from 1.56 to 3.13 microg/mL] than alkannin or shikonin (MIC = 6.25 microg/mL). Anti-MRSA activity of derivatives was bactericidal with minimum bactericidal concentration (MBC)/MIC < or = 2. In a time-kill assay, the bactericidal activity against MRSA was achieved as rapidly as 2 h. The derivatives 3-9 were also active against vancomycin-resistant Enterococcus faecium (F935) and vancomycin-resistant Enterococcus faecalis (CKU-17) with MICs similar to those with MRSA. Aromatic ester derivatives were also synthesized for antimicrobial activity comparison. None of these compounds were active against Gram-negative bacteria tested. Their cytotoxicity was also evaluated on selected cancer cell lines, and they expressed their activity in the range 0.6-5.4 microg/mL (CD(50)). Our results indicate that the ester derivatives of alkannin are potential candidates of anti-MRSA and anti-VRE agents with antitumor activity.     

Isopimaric acid from Pinus nigra shows activity against multidrug-resistant and EMRSA strains of Staphylococcus aureus

The diterpene isopimaric acid was extracted from the immature cones of Pinus nigra (Arnold) using bioassay-guided fractionation of a crude hexane extract. Isopimaric acid was assayed against multidrug-resistant (MDR) and methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentrations (MIC) were 32-64 microg/mL and compared with a commercially obtained resin acid, abietic acid, with MICs of 64 microg/mL. Resin acids are known to have antibacterial activity and are valued in traditional medicine for their antiseptic properties. These results show that isopimaric acid is active against MDR and MRSA strains of S. aureus which are becoming increasingly resistant to antibiotics. Both compounds were evaluated for modulation activity in combination with antibiotics, but did not potentiate the activity of the antibiotics tested. However, the compounds were also assayed in combination with the efflux pump inhibitor reserpine, to see if inhibition of the TetK or NorA efflux pump increased their activity. Interestingly, rather than a potentiation of activity by a reduction in MIC, a two to four-fold increase in MIC was seen. It may be that isopimaric acid and abietic acid are not substrates for these efflux pumps, but it is also possible that an antagonistic interaction with reserpine may render the antibiotics inactive. 1H-NMR of abietic acid and reserpine taken individually and in combination, revealed a shift in resonance of some peaks for both compounds when mixed together compared with the spectra of the compounds on their own. It is proposed that this may be due to complex formation between abietic acid and reserpine and that this complex formation is responsible for a reduction in activity and elevation of MIC.     

Screening of some Tanzanian medicinal plants from Bunda district for antibacterial, antifungal and antiviral activities

Extracts from 50 plant parts obtained from 39 different plants belonging to 22 families used to treat infectious diseases in Bunda district, Tanzania, were screened against twelve microorganisms, including the bacteria Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhimurium, the fungi Aspergillus niger, Candida albicans, and the viruses Herpes Simplex Virus type 1, Vesicular Stomatitis Virus T2, Coxsackie B2 and Semliki Forest A7. The highest activity was obtained for the n-hexane extract of Elaeodendron schlechteranum root bark against the Gram-positive bacteria Bacillus cereus (MIC 0.97 microg/ml and MBC 1.95 microg/ml) and Staphylococcus aureus (MIC 3.90 microg/ml and MBC 31.25 microg/ml). Gram-negative bacteria were less sensitive. Only Balanites aegyptiaca stem bark exhibited a high antifungal activity against Candida albicans (MIC 125 microg/ml and MFC 250 microg/ml). Extracts from four plants; Lannea schweinfurthii, Combretum adenogonium, Ficus sycomorus and Terminalia mollis showed strong antiviral activity with RF values of 10(3) and 10(4) against Herpes Simplex Virus type 1 at various concentrations. Our results support, at least in part, the use of most plants as claimed by traditional healers/informants especially against the Gram-positive bacteria Bacillus cereus and Staphylococcus aureus.     

Antibacterial terpenes from the oleo-resin of Commiphora molmol (Engl.)

Two octanordammaranes, mansumbinone (1) and 3,4-seco-mansumbinoic acid (2), and two sesquiterpenes, beta-elemene (3) and T-cadinol (4) have been isolated from the oleo-resin of Commiphora molmol (Engl.). The structures of these compounds were established unambiguously by a series of 1D and 2D-NMR analyses. We have also unambiguously assigned all (1)H and (13)C NMR resonances for 2 and revised its (13)C data. The crude extract of the oleo-resin of C. molmol displayed potentiation of ciprofloxacin and tetracycline against S. aureus, several Salmonella enterica serovar Typhimurium strains and two K. pneumoniae strains. The antibacterial activity of terpenes 1-4 was determined against a number of Staphylococcus aureus strains: SA1199B, ATCC25923, XU212, RN4220 and EMRSA15 and minimum inhibitory concentration (MIC) values were found to be in the range of 4-256 microg/ml. The highest activity was observed by the seco-A-ring octanordammarane 2 with an MIC of 4 microg/ml against SA1199B, a multidrug-resistant strain which over-expresses the NorA efflux transporter, the major characterized antibiotic pump in this species. This activity compared favorably to the antibiotic norfloxacin with an MIC of 32 microg/ml. Compound 2 also displayed weak potentiation of ciprofloxacin and tetracycline activity against strains of Salmonella enterica serovar Typhimurium SL1344 and L10.     

In Vitro Antibacterial Activity and Synergistic Antibiotic Effects of Phlorotannins Isolated from Eisenia bicyclis Against Methicillin‐Resistant Staphylococcus aureus

Six phlorotannins, isolated from Eisenia bicyclis, were evaluated for antibacterial activity against methicillin‐resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentrations (MIC) of the compounds were in the range 32 to 64 µg/mL. Phlorofucofuroeckol‐A (PFF) exhibited the highest anti‐MRSA activity, with an MIC of 32 µg/mL. An investigation of the interaction between these compounds and the β‐lactam antibiotics ampicillin, penicillin, and oxacillin revealed synergistic action against MRSA in combination with compound PFF. To our knowledge, this is the first report on the anti‐MRSA activity of phlorotannins from E. bicyclis. The results obtained in this study suggest that the compounds derived from E. bicyclis can be a good source of natural antibacterial agents against MRSA. Copyright © 2012 John Wiley & Sons, Ltd.     

Erythrina alkaloids and a pterocarpan from the bark of Erythrina subumbrans

Three new erythrina alkaloids, (+)-10,11-dioxoerythratine (1), (+)-10,11-dioxoepierythratidine (2), and (+)-10,11-dioxoerythratidinone (3), and a new pterocarpan, 1-methoxyerythrabyssin II (4), were isolated from the bark of Erythrina subumbrans, together with seven known pterocarpans, erythrabyssin II, erybraedin A, erystagallin A, erycristagallin, erythrabissin-1, eryvarin A, and hydroxycristacarpone, three flavanones, 5-hydroxysophoranone, abyssinone V, and lespedezaflavanone B, three triterpenes, sophoradiol, soyasapogenol B, and lupeol, and one isoflavanone, vogelin C. Their structures were elucidated on the basis of spectroscopic data. Some isolates were tested for antiplasmodial, antimycobacterial, and cytotoxic activities.     

Antimicrobial activity of Alstonia macrophylla: a folklore of bay islands

The methanolic crude and methanol-aqueous extract of Alstonia macrophylla leaves and n-butanol part of the crude extract showed antimicrobial activity against various strains of Staphylococcus aureus, Staphylococcus saprophyticus, Streptococcus faecalis, Escherichia coli, Proteus mirabilis, Trichophyton rubrum, Trichophyton mentagrophytes var. mentagrophytes and Microsporum gypseum. The minimum inhibitory concentration (MIC) values ranges from 64 to 1000 microg/ml for bacteria and 32-128 mg/ml for dermatophytes. However, the strains of Pseudomonas aeruginosa, Klebsiella sp. and Vibrio cholerae showed resistance against in vitro treatment of the extracts up to 2000 microg/ml concentration, while the two yeast species were resistant even at 128 mg/ml concentration. The stem bark extract prepared similarly was found to be less active compared to the leaves. Phytochemical study indicates that the crude extract contains tannins, flavonoids, saponins, sterols, triterpene and reducing sugars. Further fractionation and purification of n-butanol part of the extract showed the presence of beta-sitosterol, ursolic acid, beta-sitosterol glucoside and a mixture of minor compounds only detected in TLC.     

Lunacridine from Lunasia amara is a DNA intercalating topoisomerase II inhibitor

An ethnobotanical survey of plants used to treat tropical ulcers in Papua New Guinea identified Lunasia amara as possessing anti-Staphylococcus aureus activity. Activity-guided fractionation of the aqueous bark extract resulted in the identification of the quinoline alkaloid lunacridine as the active principle. Lunacridine tends to cyslise at room temperature but the 2'-O-trifluoroacetyl derivative was found to be stable and therefore more suitable for biological assays. The compound exhibited a minimal inhibitory concentration (MIC) of 64 micro g/ml against Staphylococcus aureus NCTC 6571 and activity in the low micromolar range against HeLa and H226 cells; the latter showing signs of caspase-3/7 mediated apoptotic cell death. Experiments with drug resistant strains of Streptococcus pneumoniae suggested topoisomerase as a likely target for the drug in bacteria whilst decatenation assays with human topoisomerase II showed the compound to be a potent inhibitor of this isoform (IC(50)<5 micro M) thus explaining the drug's activity against human cell lines. Both lunacridine and 2'-O-trifluoroacetyl lunacridine exhibited mild DNA intercalation activity giving 50% decrease in ethidium DNA fluorescence at 0.22 and 0.6 mM, respectively, placing the drug amongst the DNA intercalating class of topoisomerase II inhibitors.     

Antibacterial activity of totarol and its potentiation.

Antimicrobial activity of six diterpenoids isolated from the bark of Podocarpus nagi (Podocarpaceae) has been tested against twelve selected microorganisms. Totarol [1], the most abundant compound among the six, exhibited potent bactericidal activity only against Gram-positive bacteria, among which Propionibacterium acnes was the most sensitive bacterium. Totarol also showed strong activity against four other Gram-positive bacteria tested: Streptococcus mutans, Bacillus subtilis, Brevibacterium ammoniagenes, and Staphylococcus aureus (both penicillin-resistant and penicillin-susceptible strains). The bactericidal activity of totarol was enhanced when it was tested in combination with several other natural products. Noticeably, the activity of totarol against Sta. aureus was increased eightfold when tested in combination with 1/2MIC of anacardic acid [9]. The synergistic activity of anacardic acid caused the minimum bactericidal concentration (MBC) of totarol to be lowered from 1.56 to 0.2 micrograms/ml.     

Antimicrobial activity of the methanolic extract, fractions and compounds from the stem bark of Irvingia gabonensis (Ixonanthaceae)

The antimicrobial activity of the methanolic extract from the stem bark of Irvingia gabonensis (IGM), fractions and compounds isolated from IGM [3-friedelanone (1), betulinic acid (2), oleanolic acid (3), 3,3',4'-tri-O-methylellagic acid (4), 3,4-di-O-methylellagic acid (5) and hardwickiic acid (6)] was evaluated against Gram-positive bacteria (6 species), Gram-negative bacteria (13 species) and three Candida species using dilution methods for the determination of the minimal inhibition concentration (MIC) and the minimal microbicidal concentration (MMC). From the obtained results, IGM prevented the growth of all the species of microorganisms tested at a concentration limit of 312.50 microg/ml. Compounds 4-6 also inhibited the growth of all the tested microbial species while compounds 1-3 showed selective activities. The lowest MIC values (78.12 microg/ml) were obtained with IGM on 13 of the 22 microorganisms tested. The corresponding value of 1.22 microg/ml (4.26 microM) for compounds was recorded with compound 6 on Neisseria gonorrhoeae. The obtained results confirmed the use of Irvingia gabonensis in the treatment of bacterial and fungal infections.     

Traditional herbal drugs of southern Uganda. Part III: isolation and methods for physical characterization of bioactive alkanols from Rubus apetalus

The East African plant Rubus apetalus Poir. was collected as a component of an ethnobotanical survey in southern Uganda. No phytochemical investigations of this plant have been found in the literature. Preliminary antimicrobial susceptibility tests performed in Uganda indicated biological activity against several bacterial and one fungal human pathogen. Bulk re-collection of Rubus apetalus was accomplished and crude extraction performed in preparation for further testing. Two chemical fractions of the crude extract were active in the antimicrobial susceptibility assay. Fractionation of one of the active crude fractions led to the isolation and elucidation of a mixture of related compounds that exhibit antimicrobial activity against Staphylococcus aureus (MIC=62 microg/ml), Streptococcus faecalis (16 microg/ml) and Candida albicans (32 microg/ml).     

Synergistic effects of baicalein with ciprofloxacin against NorA over-expressed methicillin-resistant Staphylococcus aureus (MRSA) and inhibition of MRSA pyruvate kinase

Ethnopharmacological relevance

Baicalein, the active constituent derived from Scutellaria baicalensis Georgi., has previously been shown to significantly restore the effectiveness of β-lactam antibiotics and tetracycline against methicillin-resistant Staphylococcus aureus (MRSA). With multiple therapeutic benefits, the antibacterial actions of baicalein may also be involved in overcoming other bacterial resistance mechanisms. The aim of the present study was to further investigate antibacterial activities of baicalein in association with various antibiotics against selected Staphylococcus aureus strains with known specific drug resistance mechanisms.

Material and methods

A panel of clinical MRSA strains was used for further confirmation of the antibacterial activities of baicalein. The effect of baicalein on inhibiting the enzymatic activity of a newly discovered MRSA-specific pyruvate kinase (PK), which is essential for Staphylococcus aureus growth and survival was also examined.

Results

In the checkerboard dilution test and time-kill assay, baicalein at 16 μg/ml could synergistically restore the antibacterial actions of ciprofloxacin against the NorA efflux pump overexpressed SA-1199B, but not with the poor NorA substrate, pefloxacin. Moreover, synergistic effects were observed when baicalein was combined with ciprofloxacin against 12 out of 20 clinical ciprofloxacin resistant strains. For MRSA PK studies, baicalein alone could inhibit the enzymatic activity of MRSA PK in a dose-dependent manner.

Conclusion

Our results demonstrated that baicalein could significantly reverse the ciprofloxacin resistance of MRSA possibly by inhibiting the NorA efflux pump in vitro. The inhibition of MRSA PK by baicalein could lead to a deficiency of ATP which might further contribute to the antibacterial actions of baicalein against MRSA.