Extraction methods and bioautography for evaluation of medicinal plant antimicrobial activity

A comparative study on the antimicrobial properties of extracts from medicinal plants obtained by two different methods was carried out. The screening of the antimicrobial activity of extracts from six plants was conducted by a disc diffusion test against Gram-positive, -negative and fungal organisms. The most active extracts (inhibition diameter >/=12 mm) were assayed for the minimum inhibitory concentration and submitted to phytochemical screening by thin-layer chromatography and bioautography. The results obtained indicate that the diethyl ether extracts were the most efficient antimicrobial compounds. The activity was more pronounced against Gram-positive and fungal organisms than against Gram-negative bacteria. Bioautography showed that the antimicrobial activity was probably due to flavonoids and terpenes.     

Antimicrobial activities of eucalyptus leaf extracts and flavonoids from Eucalyptus maculata

AIMS: We investigated the antimicrobial activities of eucalyptus leaf extracts to find effective antibacterial agents. METHODS AND RESULTS: The antimicrobial activities of leaf extracts from 26 species of eucalyptus were measured. Extracts of Eucalyptus globulus, E. maculata and E. viminalis significantly inhibited the growth of six Gram-positive bacteria (Staphylococcus aureus, MRSA, Bacillus cereus, Enterococcus faecalis, Alicyclobacillus acidoterrestris, Propionibacterium acnes), and of a fungus (Trichophyton mentagrophytes), but they did not show strong antibacterial activity against Gram-negative bacteria (Escherichia coli, Pseudomonas putida). 2',6'-dihydroxy-3'-methyl-4'-methoxy-dihydrochalcone, eucalyptin and 8-desmethyl-eucalyptin, isolated from E. maculata extracts, exhibited potent antimicrobial activities against seven micro-organisms with minimum inhibitory concentrations (MIC) ranging from 1.0 to 31 mg l(-1). CONCLUSIONS: The eucalyptus extracts and three compounds from E. maculata were found to be effective against micro-organisms that cause food poisoning, acne and athlete's foot. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows potential uses of extracts from E. globulus, E. maculata and E. viminalis, and antimicrobial compounds isolated from E. maculata.     

Antimicrobial activity of Calotropis procera Ait. (Asclepiadaceae) and isolation of four flavonoid glycosides as the active constituents

Antimicrobial activity of solvent extracts and flavonoids of Calotropis procera growing wild in Saudi Arabia was evaluated using the agar well-diffusion method. A bioassay-guided fractionation of the crude flavonoid fraction (Cf₃) of MeOH extract which showed the highest antimicrobial activity led to the isolation of four flavonoid glycosides as the bioactive constituents. Structure of compounds have been elucidated using physical and spectroscopic methods including (UV, IR, ¹H, ¹³C-NMR, DEPT, 2D ¹H–¹H COSY, HSQC, HMBC and NOESY). Compounds were found to be the 3-O-rutinosides of quercetin, kaempferol and isorhamnetin, besides the flavonoid 5-hydroxy-3,7-dimethoxyflavone-4′-O-β-glucopyranoside. Most of the isolated extracts showed antimicrobial activity against the test microorganisms, where the crude flavonoid fraction was the most active, diameter of inhibition zones ranged between 15.5 and 28.5 mm against the tested bacterial strains, while reached 30 mm against the fungal Candida albicans. The minimal inhibitory concentrations varied from 0.04 to 0.32 mg/ml against all of the tested microorganisms in case of the crude flavonoid fraction. Quercetin-3-O-rutinoside showed superior activity over the remainder flavonoids. The Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) were more susceptible than the Gram-negative (Pseudomonas aeruginosa and Salmonella enteritidis) and the yeast species were more susceptible than the filamentous fungi. The study recommend the use of such natural products as antimicrobial biorationals.     

Antimicrobial activity of fractions and compounds from Calophyllumbrasiliense (Clusiaceae/Guttiferae)

Calophyllum brasiliense (Clusiaceae/Guttiferae) is a native Brazilian medicinal plant traditionally used against several diseases, including infectious pathologies. Crude methanolic extracts (CME) and two fractions, denoted non-polar (soluble in chloroform) and polar (nonsoluble in chloroform), were prepared from different parts of the plant (roots, stems, leaves, flowers and fruits) and studied. The following compounds were isolated and tested against pathogenic bacteria and yeasts by determination of the minimal inhibitory concentration (MIC): brasiliensic acid (1), gallic acid (2), epicatechin (3), protocatechuic acid (4), friedelin (5) and 1,5-dihydroxyxanthone (6). The results indicated that all the parts of the plant exhibited antimicrobial activity against Gram-positive bacteria, which are selectively inhibited by components of C. brasiliense. No activity was observed against Gram-negative bacteria and yeasts tested. Regarding the isolated compounds, substance 4 showed antimicrobial activity against all the tested microorganisms, whereas compound 6 exhibited antimicrobial activity only against Gram-positive bacteria. The results from the current study confirm and justify the popular use of this plant to treat infectious processes.     

The antimicrobial activity of extracts of the lichen Cetraria aculeata and its protolichesterinic acid constituent

In this study, the antimicrobial activity of the acetone, diethyl ether and ethanol extracts of the lichen Cetraria aculeata has been investigated. The extracts were tested against twelve bacteria and eight fungi and found active against Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, Proteus vulgaris, Streptococcus faecalis, Bacillus cereus, Bacillus subtilis, Pseudomonas aeruginosa, Listeria monocytogenes. No antimicrobial activity against the fungi was detected. It was determined that only one substance in the extracts has antimicrobial activity and it was characterized as protolichesterinic acid. The MICs of the extracts and protolichesterinic acid were also determined.     

In vitro antimicrobial activity of Romanian medicinal plants hydroalcoholic extracts on planktonic and adhered cells

The aim of this study was to assess the antibacterial and antifungal potential of some Romanian medicinal plants, arnica--Arnica montana, wormwood--Artemisia absinthium and nettle--Urtica dioica. In order to perform this antimicrobial screening, we obtained the vegetal extracts and we tested them on a series of Gram-positive and Gram-negative bacteria, and also against two fungal strains. The vegetal extracts showed antimicrobial activity preferentially directed against the planktonic fungal and bacterial growth, while the effect against biofilm formation and development was demonstrated only against S. aureus and C. albicans. Our in vitro assays indicate that the studied plant extracts are a significant source of natural alternatives to antimicrobial therapy, thus avoiding antibiotic therapy, the use of which has become excessive in recent years.     

Antimicrobial activity of Wedelia trilobata crude extracts.

A biological screening of activity against Gram-positive and Gram-negative bacteria, yeasts, and fungi of crude extracts from Wedelia trilobata is reported. The n-hexane extract showed antibacterial activity against Bacillus subtilis, Mycobacterium smegmatis, Staphylococcus aureus, and Staphylococcus epidermidis (Gram-positive bacteria); along with Proteus vulgaris, Pseudomonas aeruginosa, Salmonella group C, Salmonella paratyphi, and Shigella sonnei (Gram-negative bacteria). The ethyl acetate extract was active only against Salmonella group C; and the aqueous extract was inactive against the tested bacteria. None of the tested extracts showed biological activity against the yeasts (Candida albicans, Candida tropicalis, Rhodotorula rubra) or the fungi (Aspergillus flavus, Aspergillus niger, Mucor sp., Trichophyton rubrum).     

Antimicrobial properties of phenolic compounds from berries

AIMS: To investigate the antimicrobial properties of phenolic compounds present in Finnish berries against probiotic bacteria and other intestinal bacteria, including pathogenic species. METHODS AND RESULTS: Antimicrobial activity of pure phenolic compounds representing flavonoids and phenolic acids, and eight extracts from common Finnish berries, was measured against selected Gram-positive and Gram-negative bacterial species, including probiotic bacteria and the intestinal pathogen Salmonella. Antimicrobial activity was screened by an agar diffusion method and bacterial growth was measured in liquid culture as a more accurate assay. Myricetin inhibited the growth of all lactic acid bacteria derived from the human gastrointestinal tract flora but it did not affect the Salmonella strain. In general, berry extracts inhibited the growth of Gram-negative but not Gram-positive bacteria. These variations may reflect differences in cell surface structures between Gram-negative and Gram-positive bacteria. Cloudberry, raspberry and strawberry extracts were strong inhibitors of Salmonella. Sea buckthorn berry and blackcurrant showed the least activity against Gram-negative bacteria. CONCLUSION: Different bacterial species exhibit different sensitivities towards phenolics. SIGNIFICANCE AND IMPACT OF THE STUDY: These properties can be utilized in functional food development and in food preservative purposes.     

Antimicrobial activity of flavonoids extracted from bergamot (Citrus bergamia Risso) peel, a byproduct of the essential oil industry

AIMS: To evaluate the antimicrobial properties of flavonoid-rich fractions derived from bergamot peel, a byproduct from the Citrus fruit processing industry and the influence of enzymatic deglycosylation on their activity against different bacteria and yeast. METHODS AND RESULTS: Bergamot ethanolic fractions were tested against Gram-negative bacteria (Escherichia coli, Pseudomonas putida, Salmonella enterica), Gram-positive bacteria (Listeria innocua, Bacillus subtilis, Staphylococcus aureus, Lactococcus lactis) and the yeast Saccharomyces cerevisiae. Bergamot fractions were found to be active against all the Gram-negative bacteria tested, and their antimicrobial potency increased after enzymatic deglycosylation. The minimum inhibitory concentrations of the fractions and the pure flavonoids, neohesperidin, hesperetin (aglycone), neoeriocitrin, eriodictyol (aglycone), naringin and naringenin (aglycone), were found to be in the range 200 to 800 microg ml(-1). The interactions between three bergamot flavonoids were also evaluated. CONCLUSION: The enzyme preparation Pectinase 62L efficiently converted common glycosides into their aglycones from bergamot extracts, and this deglycosylation increased the antimicrobial potency of Citrus flavonoids. Pairwise combinations of eriodictyol, naringenin and hesperetin showed both synergistic and indifferent interactions that were dependent on the test indicator organism. SIGNIFICANCE AND IMPACT OF THE STUDY: Bergamot peel is a potential source of natural antimicrobials that are active against Gram-negative bacteria.     

Screening of plant extracts for antimicrobial activity against bacteria and yeasts with dermatological relevance

There is cumulative resistance against antibiotics of many bacteria. Therefore, the development of new antiseptics and antimicrobial agents for the treatment of skin infections is of increasing interest. We have screened six plant extracts and isolated compounds for antimicrobial effects on bacteria and yeasts with dermatological relevance. The following plant extracts have been tested: Gentiana lutea, Harpagophytum procumbens, Boswellia serrata (dry extracts), Usnea barbata, Rosmarinus officinalis and Salvia officinalis (supercritical carbon dioxide [CO2] extracts). Additionally, the following characteristic plant substances were tested: usnic acid, carnosol, carnosic acid, ursolic acid, oleanolic acid, harpagoside, boswellic acid and gentiopicroside. The extracts and compounds were tested against 29 aerobic and anaerobic bacteria and yeasts in the agar dilution test. U. barbata-extract and usnic acid were the most active compounds, especially in anaerobic bacteria. Usnea CO2-extract effectively inhibited the growth of several Gram-positive bacteria like Staphylococcus aureus (including methicillin-resistant strains - MRSA), Propionibacterium acnes and Corynebacterium species. Growth of the dimorphic yeast Malassezia furfur was also inhibited by Usnea-extract. Besides the Usnea-extract, Rosmarinus-, Salvia-, Boswellia- and Harpagophytum-extracts proved to be effective against a panel of bacteria. It is concluded that due to their antimicrobial effects some of the plant extracts may be used for the topical treatment of skin disorders like acne vulgaris and seborrhoic eczema.     

Antimicrobial and antioxidant activities with acute toxicity,cytotoxicity and mutagenicity of Cystoseira compressa (Esper) Gerloff & Nizamuddin from the coast of Urla (Izmir,Turkey)

The aim of the study was to evaluate the biological activities with toxic properties of the methanol, hexane, and chloroform extracts of Cystoseira compressa (Esper) Gerloff & Nizamuddin from the Coast of Urla in the Aegean Sea. The extracts of C. compressa were tested for their antimicrobial and antioxidant activities in this study. Cytotoxic and mutagenic potentials of the extracts were also evaluated using cell culture and mutagenicity assays. Hexane extract was found to have higher total flavonoid and phenolic contents than the other extracts and exerted higher antioxidant activity than other extracts. All extracts exhibited moderate antimicrobial activity against tested microorganisms (minimum inhibitory concentration ranges are 32–256 μg/mL). The results indicated that the extracts had no significant cytotoxic activity against human hepatocellular carcinoma Hep 3B cell line in all treated concentrations (5–50 μg/mL) and did not show mutagenicity in the Ames test. Lethality was not observed among mice treated with oral doses of the extracts. In conclusion, results of investigations indicate that brown alga C. compressa is a natural source of antioxidant. It has moderate antimicrobial activities with no toxicity.     

Commensal bacilli inhibitory to mastitis pathogens isolated from the udder microbiota of healthy cows

AIMS: To isolate from the microbiota of the healthy cow udder commensal bacteria having antimicrobial activity against bovine mastitis pathogens, with a long-term view to their potential application as antimastitis probiotics. METHODS AND RESULTS: Bacterial isolates from four healthy cow udders were tested for inhibitory activity against three Gram-positive indicator bacteria. This led to the selection of nine broadly inhibitory strains. All were of the Bacillus genus and their antimicrobial activities, which appeared heterogeneous on the basis of their antibacterial spectra and heat susceptibilities, enabled grouping of the inhibitory bacilli into six different inhibitory profiles. All displayed strong in vitro activity against Gram-positive mastitis pathogens. Inhibitory bacilli were recovered from each of the 11 udder samples collected over 7 months from one of these cows and the isolates included representatives of all six inhibitory profiles. CONCLUSIONS: Bacilli present in the udder microbiota of healthy cows can produce a variety of broadly active inhibitors of Gram-positive bacteria, including potential mastitis pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Inhibitor-producing strains of commensal Bacillus species have been identified, which may have the potential for use as possible antimastitis probiotics.     

Evaluation of antimicrobial activity of the lichens Lasallia pustulata, Parmelia sulcata, Umbilicaria crustulosa, and Umbilicaria cylindrica

The antimicrobial properties of acetone, methanol, and aqueous extracts of the lichens Lasallia pustulata, Parmelia sulcata, Umbilicaria crustulosa, and Umbilicaria cylindrica were studied comparatively in vitro. Antimicrobial activities of the extracts of different lichens were estimated by the disk diffusion test for Gram-positive bacteria, Gram-negative bacteria, and fungal organisms, as well as by determining the MIC (minimal inhibitory concentration). The obtained results showed that the acetone and methanol extracts of Lasallia pustulata, Parmelia sulcata, and Umbilicaria crustulosa manifest antibacterial activity against the majority of species of bacteria tested, in addition to selective antifungal activity. The MIC of lichen extracts was lowest (0.78 mg/ml) for the acetone extract of Lasallia pustulata against Bacillus mycoides. Aqueous extracts of all of the tested lichens were inactive. Extracts of the lichen Umbilicaria cylindrica manifested the weakest activity, inhibiting only three of the tested organisms.     

Eradication of Propionibacterium acnes biofilms by plant extracts and putative identification of icariin, resveratrol and salidroside as active compounds

Propionibacterium acnes is a Gram-positive bacterium that plays an important role in the pathogenesis of acne vulgaris. This organism is capable of biofilm formation and the decreased antimicrobial susceptibility of biofilm-associated cells may hamper efficient treatment. In addition, the prolonged use of systemic antibiotic therapy is likely to lead to the development and spread of antimicrobial resistance. In the present study we investigated whether P. acnes biofilms could be eradicated by plant extracts or their active compounds, and whether other mechanisms besides killing of biofilm cells could be involved. Out of 119 plant extracts investigated, we identified five with potent antibiofilm activity against P. acnes (extracts from Epimedium brevicornum, Malus pumila, Polygonum cuspidatum, Rhodiola crenulata and Dolichos lablab). We subsequently identified icariin, resveratrol and salidroside as active compounds in three of these extracts. Extracts from E. brevicornum and P. cuspidatum, as well as their active compounds (icariin and resveratrol, respectively) showed marked antibiofilm activity when used in subinhibitory concentrations, indicating that killing of microbial cells is not their only mode of action.     

Antimicrobial activity of extracts of the lichen Xanthoparmelia pokornyi and its gyrophoric and stenosporic acid constituents

The antimicrobial activity of the diethyl ether, acetone, chloroform, petroleum ether, and ethanol extracts of the lichen Xanthoparmelia pokornyi and its gyrophoric acid and stenosporic acid constituents has been screened against some foodborne bacteria and fungi. Both the extracts and the acids showed antimicrobial activity against Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Proteus vulgaris, Staphylococcus aureus, Streptococcus faecalis, Yersinia enterocolitica, Candida albicans and Candida glabrata. The extracts were inactive against the tested filamentous fungi. The MIC values of the extracts and the acids for the bacteria have also been determined.     

Antimicrobial activity of ultrasound-assisted solvent-extracted spices

AIMS: The objective of this research was to determine the antimicrobial activity of conventional and high-intensity ultrasound-assisted (HI-US) solvent-extracted Thai spices, including ginger (Zingiber officinale Rose), fingerroot (Bosenbergia pandurata Holtt) and turmeric (Curouma longa Linn). METHODS AND RESULTS: Extracts were obtained using hexane, isopropanol and a 7 : 3 isopropanol : hexane mixture as solvents with and without HI-US. The antimicrobial activity of the extracts was assayed against four strains each of Listeria monocytogenes and Salmonella Typhimurium DT 104 using an agar dilution assay. Application of HI-US did not alter antibacterial activity against S. Typhimurium, but antilisterial activity of some HI-US spice extracts decreased. Solvent type affected antimicrobial efficacy of extracts with hexane producing the least antimicrobial activity. Fingerroot extracted with isopropanol-hexane and without HI-US had the best antilisterial effect while HI-US-isopropanol fingerroot extract had the greatest antimicrobial efficacy against S. Typhimurium. CONCLUSIONS: Application of HI-US reduced time of extraction to 5 min, compared with the 24 h required for conventional extraction and maintained antimicrobial activity against Salmonella but slightly reduced activity against Listeria. SIGNIFICANCE AND IMPACT OF THE STUDY: HI-US in combination with proper solvent selection may offer a new tool to optimize extraction of spice essential oil for use as antimicrobial agents, and reduce processing time and costs.     

Antimicrobial defense mechanisms in the horseshoe crab, Limulus polyphemus: preliminary observations with heat-derived extracts of Limulus amoebocyte lysate.

Heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) were found to contain potent “broad-spectrum” antimicrobial activity. Additional heating of the LAL extracts to 100°C for 30 min completely inactivated the antimicrobial activity and served as a control. Antimicrobial activity was observed over a temperature range of 0° to 37°C (higher temperatures not tested) with greatest activity at 37°C. Antimicrobial activity of LAL extracts was variable when tested against Gram-negative bacteria of the family Enterobacteriaceae. A twofold concentration of the extracts resulted in a significant decrease in antimicrobial effectiveness. Dialysis of single- and double-strength LAL extracts against deionized water produced a marked and significant enhancement of antimicrobial activity against both resistant and sensitive species, confirming the presence of a dialyzable inhibitor(s). Dialyzed LAL extracts were active against 13 of 14 species of Enterobacteriaceae tested. Two strains of Pseudomonas aeruginosa were susceptible as were two of three Gram-positive cocci tested. Highly sensitive bacterial species were rapidly killed with a greater than 90% reduction in viable counts occurring within the first 30 min of reaction time. Dialyzed LAL extracts also possessed considerable antifungal activity. The role of the Limulus polyphemus amoebocyte in defense against microbial invasion and dissemination is discussed.     

Antimicrobial activity of extracts of the lichen Parmelia sulcata and its salazinic acid constituent

The antimicrobial activity of the acetone, chloroform, diethyl ether, methanol, and petroleum ether extracts of the lichen Parmelia sulcata and its salazinic acid constituent have been screened against twenty eight food-borne bacteria and fungi. All of the extracts with the exception of the petroleum ether extract showed antimicrobial activity against Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Listeria monocytogenes, Proteus vulgaris, Yersinia enterocolitica, Staphylococcus aureus, Streptococcus faecalis, Candida albicans, Candida glabrata, Aspergillus niger, Aspergillus fumigatus, and Penicillium notatum. Salazinic acid did not show antimicrobial activity against L. monocytogenes, P. vulgaris, Y. enterocolitica, and S. faecalis but showed activity against Pseudomonas aeruginosa and Salmonella typhimurium as well. The MIC values of the extracts and the acid for the bacteria and fungi have also been determined.     

Influence of hydrogen peroxide foliar applications on in vitro antimicrobial activity in Capsicum chinense Jacq.

The aim of this study was to evaluate the effect of foliar applications of hydrogen peroxide on the antimicrobial activity of Capsicum chinense Jacq. methanolic extracts. The effects of hydrogen peroxide application on metabolites accumulation of C. chinense var. Jaguar and var. Chichen Itza were evaluated. Total flavonoid and phenolic contents, as well as HPLC quantification of capsaicin and dihidrocapsaicin were carried out. Methanolic extracts were microbiologically tested against Staphylococcus aureus, Escherichia coli, Streptococcus mutants, Salmonella thompson, Listeria monocytogenes, Streptococcus faecalis, and Candida albicans. Total phenolics, flavonoids, and capsaicinoids contents in both varieties treated with hydrogen peroxide were found significantly higher as compared to control. The antibacterial activity of chili extracts was observed against Gram-positive and Gram-negative bacteria as well as anti-yeast. The results of in vitro antibacterial activity showed that hydrogen peroxide application increases the inhibitory effect against the pathogenic micro-organisms. Methanolic extracts of var. Jaguar, were the most active against S. aureus, S. Thompson, and C. albicans, while var. Chichen Itza was most potent against E. faecalis and E. coli. Thus, this study confirmed that metabolite-induced factors (MIFs) as hydrogen peroxide, increased secondary metabolites accumulation in C. chinense methanolic extracts and augmented their antimicrobial activity.     

Antimicrobial activity and phytochemical analyses of Polygonum aviculare L. (Polygonaceae), naturally growing in Egypt

Polygonum aviculare (Polygonaceae) is an herb commonly distributed in Mediterranean coastal regions in Egypt and used in folkloric medicine. Organic and aqueous solvent extracts and fractions of P. aviculare were investigated for antimicrobial activities on several microorganisms including bacteria and fungi. Phytochemical constituents of air-dried powered plant parts were extracted using aqueous and organic solvents (acetone, ethanol, chloroform and water). Antimicrobial activity of the concentrated extracts was evaluated by determination of the diameter of inhibition zone against both Gram-negative and Gram-positive bacteria and fungi using paper disc diffusion method.Results of the phytochemical studies revealed the presence of tannins, saponins, flavonoids, alkaloids and sesquiterpenes and the extracts were active against both Gram-negative and Gram-positive bacteria. Chloroform extract gave very good and excellent antimicrobial activity against all tested bacteria and good activity against all tested fungi except Candida albicans. Structural spectroscopic analysis that was carried out on the active substances in the chloroform extract led to the identification of panicudine (6-hydroxy-11-deoxy-13 dehydrohetisane).Evaluation of the antimicrobial activity of panicudine indicated significant activity against all tested Gram-negative and Gram-positive organisms. Panicudine displayed considerable activity against the tested fungi with the exception of C. albicans. Antimicrobial activity of the extracts was unaffected after exposure to different heat treatments, but was reduced at alkaline pH. Studies of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of panicudine on the tested organisms showed that the lowest MIC and the MBC were demonstrated against Salmonella paratyphi, Bacillus subtilis and Salmonella typhi and the highest MIC and MBC were against Staphylococcus aureus.