Yong K. Park

In vitro antimicrobial activity of propolis and Arnica montana against oral pathogens

Arnica and propolis have been used for thousands of years in folk medicine for several purposes. They possess several biological activities such as anti-inflammatory, antifungal, antiviral and tissue regenerative, among others. Although the antibacterial activity of propolis has already been demonstrated, very few studies have been done on bacteria of clinical relevance in dentistry. Also, the antimicrobial activity of Arnica has not been extensively investigated. Therefore the aim here was to evaluate in vitro the antimicrobial activity, inhibition of adherence of mutans streptococci and inhibition of formation of water-insoluble glucan by Arnica and propolis extracts. Arnica montana (10%, w/v) and propolis (10%, w/v) extracts from Minas Gerais State were compared with controls. Fifteen microorganisms were used as follows: Candida albicans--NTCC 3736, F72; Staphylococcus aureus--ATCC 25923; Enterococcus faecalis--ATCC 29212; Streptococcus sobrinus 6715; Strep. sanguis--ATCC 10556; Strep. cricetus--HS-6; Strep. mutans--Ingbritt 1600; Strep. mutans--OMZ 175; Actinomyces naeslundii--ATCC 12104, W 1053; Act. viscosus OMZ 105; Porphyromonas gingivalis; Porph. endodontalis and Prevotella denticola (the last three were clinical isolates). Antimicrobial activity was determined by the agar diffusion method and the zones of growth inhibition were measured. To assess cell adherence to a glass surface, the organisms were grown for 18 h at 37 degrees C in test-tubes at a 30 degree angle. To assay water-insoluble glucan formation, a mixture of crude glucosyltransferase and 0.125 M sucrose was incubated for 18 h at 37 degrees C in test-tubes at a 30 degree angle. Arnica and propolis extracts (20 microl) were added to these tubes to evaluate the % of inhibition of cell adherence and water-insoluble glucan formation. The propolis extract significantly inhibited all the microorganisms tested (p < 0.05), showing the largest inhibitory zone for Actinomyces spp. The Arnica extract did not demonstrate significant antimicrobial activity. Cell adherence and water-insoluble glucan formation were almost completely inhibited by the propolis extract at a final concentration of 400 microg/ml and 500 microg/ml, respectively. The Arnica extract showed slight inhibition of the adherence of the growing cells (19% for Strep. mutans and 15% for Strep. sobrinus) and of water-insoluble glucan formation (29%) at these same concentrations. Thus, the propolis extract showed in vitro antibacterial activity, inhibition of cell adherence and inhibition of water-insoluble glucan formation, while the Arnica extract was only slightly active in those three conditions.

Effects of compounds found in propolis on Streptococcus mutans growth and on glucosyltransferase activity.

ABSTRACT Propolis, a resinous bee product, has been shown to inhibit the growth of oral microorganisms and the activity of bacterium-derived glucosyltransferases (GTFs). Several compounds, mainly polyphenolics, have been identified in this natural product. The present study evaluated the effects of distinct chemical groups found in propolis on the activity of GTF enzymes in solution and on the surface of saliva-coated hydroxyapatite (sHA) beads. Thirty compounds, including flavonoids, cinnamic acid derivatives, and terpenoids, were tested for the ability to inhibit GTFs B, C, and D from Streptococcus mutans and GTF from S. sanguinis (GTF Ss). Flavones and flavonols were potent inhibitors of GTF activity in solution; lesser effects were noted on insolubilized enzymes. Apigenin, a 4′,5,7-trihydroxyflavone, was the most effective inhibitor of GTFs, both in solution (90.5 to 95% inhibition at a concentration of 135 μg/ml) and on the surface of sHA beads (30 to 60% at 135 μg/ml). Antibacterial activity was determined by using MICs, minimum bactericidal concentrations (MBCs), and time-kill studies. Flavanones and some dihydroflavonols, as well as the sesquiterpene tt-farnesol, inhibited the growth of S. mutans and S. sobrinus; tt-farnesol was the most effective antibacterial compound (MICs of 14 to 28 μg/ml and MBCs of 56 to 112 μg/ml). tt-Farnesol (56 to 112 μg/ml) produced a 3-log-fold reduction in the bacterial population after 4 h of incubation. Cinnamic acid derivatives had negligible biological activities. Several of the compounds identified in propolis inhibit GTF activities and bacterial growth. Apigenin is a novel and potent inhibitor of GTF activity, and tt-farnesol was found to be an effective antibacterial agent.

Antimicrobial activity of propolis on oral microorganisms.

Abstract. Formation of dental caries is caused by the colonization and accumulation of oral microorganisms and extracellular polysaccharides that are synthesized from sucrose by glucosyltransferase of Streptococcus mutans. The production of glucosyltransferase from oral microorganisms was attempted, and it was found that Streptococcus mutans produced highest activity of the enzyme. Ethanolic extracts of propolis (EEP) were examined whether EEP inhibit the enzyme activity and growth of the bacteria or not. All EEP from various regions in Brazil inhibited both glucosyltransferase activity and growth of S. mutans, but one of the propolis from Rio Grande do Sul (RS2) demonstrated the highest inhibition of the enzyme activity and growth of the bacteria. It was also found that propolis (RS2) contained the highest concentrations of pinocembrin and galangin.

Brazilian Red Propolis—Chemical Composition and Botanical Origin

Propolis contains resinous substances collected by honey bees from various plant sources and has been used as a traditional folk medicine since ca 300 BC. Nowadays, the use of evidence-based complementary and alternative medicine (CAM) is increasing rapidly and so is the use of propolis in order to treat or support the treatment of various diseases. Much attention has been focused on propolis from Populus sp. (Salicaceae) and Baccharis dracunculifolia (Asteracea), but scientific information about the numerous other types of propolis is still sparse. We gathered six samples of red propolis in five states of Northeastern Brazil. The beehives were located near woody perennial shrubs along the sea and river shores. The bees were observed to collect red resinous exudates on Dalbergia ecastophyllum (L) Taub. (Leguminosae) to make propolis. The flavonoids of propolis and red resinous exudates were investigated using reversed-phase high-performance liquid chromatography and reversed-phase high-performance thin-layer chromatography. We conclude that the botanical origin of the reddish propolis is D. ecastophyllum. In areas where this source (D. ecastophyllum) was scarce or missing, bees were collecting resinous material from other plants. Propolis, which contained the chemical constituents from the main botanical origin, showed higher antimicrobial activity.

Apigenin and tt-Farnesol with Fluoride Effects on S. mutans Biofilms and Dental Caries:

Apigenin (Api) and tt-farnesol (Far) are two naturally occurring agents that affect the development of cariogenic biofilms. Fluoride (F) interferes physicochemically with caries development and also exhibits antibacterial activity. We examined whether the association of Api and Far enhance the anti-caries properties of F by acting cooperatively on the expression of virulence of Streptococcus mutans. The biological effects of each of the agents were greatly enhanced when used in combination with F. In general, biofilms treated with Api and/or Far in combination with F displayed less biomass and fewer insoluble glucans and iodophilic polysaccharides than did those treated with the test agents alone (P < 0.05). The combination of the test agents with F was highly effective in preventing caries development in rats, especially Api+Far+F, and results were comparable with those observed with chlorhexidine + F (positive control). Results from these studies showed that apigenin and tt-farnesol may enhance the cariostatic effectiveness of fluoride.

Effect of Apis mellifera Propolis from Two Brazilian Regions on Caries Development in Desalivated Rats

The purpose of the present study was to evaluate the effect of Apis mellifera propolis collected from two regions of Brazil on caries development in desalivated rats. Ethanolic extracts of propolis (EEP) were prepared from crude propolis samples collected in Minas Gerais state (MG), southeastern Brazil, and Rio Grande do Sul state (RS), southern Brazil. The flavonoid composition of EEP was analyzed by high–performance thin–layer chromatography (HPTLC) and reversed–phase high–performance liquid chromatography (HPLC). For the animal study, 30 specific pathogen–free Wistar rats were infected with Streptococcus sobrinus 6715 and surgically desalivated. The rats were randomly divided into three groups which were treated with 80% ethanol (control), EEP from MG and EEP from RS. The animals were placed in a König–Höfer programmed feeder and received 17 meals of diet 2000 daily at hourly intervals. The solutions were applied on the rat molars (25 μl on molars of each quadrant) twice a day, by using graduate syringes. After 3 weeks, the animals were killed by CO2 asphyxiation. For microbial assessment, the left jaw was removed and sonicated in 154 mM NaCl solution. Dental caries was evaluated according to Larson’s modification of Keyes’ system. The HPTLC patterns and HPLC profiles demonstrated that both quality and quantity of flavonoid aglycones of EEP from MG were different compared to EEP from RS. In general, it is apparent that EEP from RS contained the highest concentrations of pinocembrin, chrysin, acacetin and galangin. The group of animals treated with EEP from RS showed the lowest smooth–surface and sulcal caries scores as well as less caries severity in smooth–surface and sulcal lesions, and these data were statistically different when compared with the control group. The group treated with EEP from MG only demonstrated a significant difference in the severity of sulcal lesions when compared to the control group. The percentage of S. sobrinus was lower in the groups treated with EEP, but did not differ statistically from the control group. The results showed that the cariostatic effect of propolis depends on its composition, and consequently the region of collection of propolis samples.

Effect of a Mouthrinse Containing Selected Propolis on 3-Day Dental Plaque Accumulation and Polysaccharide Formation

The aim of this study was to evaluate the effect of a mouthrinse containing propolis SNB-RS on 3-day dental plaque accumulation. Six volunteers took part in a double-blind crossover study performed in two phases of 3 days. During each phase the volunteers refrained from all oral hygiene and rinsed with 20% sucrose solution 5 times a day to enhance dental plaque formation and with mouthrinse (placebo or experimental) twice a day. On the 4th day, the plaque index (PI) of the volunteers was scored and the supragingival dental plaque was analyzed for insoluble polysaccharide (IP). The PI (SD) for the experimental group was 0.78 (0.17), significantly less than for the placebo group, 1.41 (0.14). The experimental mouthrinse reduced the IP concentration in dental plaque by 61.7% compared to placebo (p < 0.05). An experimental mouthrinse containing propolis SNB-RS was thus efficient in reducing supragingival plaque formation and IP formation under conditions of high plaque accumulation.

Effects of Apis mellifera propolis on the activities of Streptococcal glucosyltransferases in solution and adsorbed onto saliva-coated hydroxyapatite

Propolis, a resinous hive product collected by Apis mellifera bees, has been used for thousands of years in folk medicine. Ethanolic extracts of propolis (EEP) have been shown to inhibit the activity of a mixture of crude glucosyltransferase (Gtf) enzymes in solution. These enzymes synthesize glucans from sucrose, which are important for the formation of pathogenic dental plaque. In the present study, the effects of propolis from two different regions of Brazil on the activity of separate, purified Gtf enzymes in solution and on the surface of saliva–coated hydroxyapatite (sHA) beads were evaluated. The EEP from Minas Gerais (MG; Southeastern Brazil) and Rio Grande do Sul (RS; Southern Brazil) were tested for their ability to inhibit the enzymes GtfB (synthesis of insoluble glucan), GtfC (insoluble/soluble glucan) and GtfD (soluble glucan). The effects of propolis on Gtf from Streptococcus sanguis (soluble glucan synthesis) was also explored. The EEP from both regions effectively inhibited the activity of all Gtfs in solution (75–95%) and on the surface of sHA beads (45–95%) at concentrations between 0.75 and 3.0 mg of propolis/ml. However, the two samples of propolis showed different levels of inhibition on each of the enzymes tested. In general, EEP RS demonstrated a significantly higher inhibitory activity on GtfB and C activities (both solution and surface assays) than EEP MG at concentrations between 0.047 and 0.187 mg/ml (p<0.05). EEP MG, on the other hand, exhibited a greater inhibitory effect on the activities of surface GtfD (at 0.375, 0.75 and 1.5 mg/ml) and S. sanguis Gtf (at 1.5 and 3.0 mg/ml; p<0.05). These data indicate that EEP is a potent inhibitor of Gtf enzymes in solution and adsorbed on an experimental pellicle; however, its effect on Gtf activity is variable depending on the geographical origin of the propolis samples. There is a need to identify the active compounds of propolis.

Effect of a new variety of Apis mellifera propolis on mutans streptococci.

The effects of a new variety of propolis, from Northeastern Brazil (BA), on growth of mutans streptococci, cell adherence, and water-insoluble glucan (WIG) synthesis were evaluated. Propolis from Southeastern (MG) and Southern (RS) Brazil were also tested as an extension of our previous work. Ethanolic extracts of propolis (EEP) were prepared and analyzed by reversed-phase HPLC. For the antibacterial activity assays, minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of EEPs against Streptococcus mutans, S. sobrinus, and S. cricetus were determined. Cell adherence of S. mutans and S. sobrinus to a glass surface was measured spectrophotometrically at 550 nm. WIG synthesized from sucrose by glucosyltransferase (Gtf) was extracted and quantified by the phenol-sulfuric method. The HPLC profile of the new variety of propolis was entirely different from Southeastern and Southern propolis. Neither flavonoid aglycones nor p-coumaric acid were detected in EEP BA. All EEPs demonstrated biological activities against mutans streptococci; EEP BA showed the highest potency in all in vitro parameters evaluated in this study. The ranges of MIC values were 50 (EEP BA)–400 μg/ml (MG), for S. mutans; and 25 (BA)–400 μg/ml (MG), for S. sobrinus and S. cricetus. The bactericidal concentration of EEPs was four to eight times the MIC values. The adherence of S. mutans and S. sobrinus cells and WIG synthesis were markedly inhibited by EEPs, demonstrating significant inhibition at all concentrations compared with the control (80% ethanol) (p < 0.05). EEP BA showed 80% inhibition of cell adherence and WIG synthesis at concentrations as low as 12.5 and 7.8 μg/ml, respectively. The results show that the new variety of propolis was exceptionally effective in all in vitro parameters tested against mutans streptococci; biological effects of propolis are likely not to be due solely to flavonoids and (hydroxy)cinnamic acid derivatives.

Comparison of Effects of the Ethanolic Extracts of Brazilian Propolis on Human Leukemic Cells As Assessed with the MTT Assay

Propolis is a resinous product collected by honey bees. It was also reported that propolis has a wide variety of biological actions, including antimicrobial activity and antioxidant, anti-inflammatory, and suppressive effects of dioxin toxicity activities. The aim of this study was to compare the in vitro cytotoxic activities of green propolis (G12) and red propolis (G13) in human leukemia cells. These cells were incubated with different concentrations of propolis and 48 hours after the IC50 was calculated for each cell. The results showed that the red propolis has cytotoxic effect in vitro higher than green propolis. Red propolis was showed to be cytostatic in K562 cells and caused the same amount of apoptosis as its control Gleevec. In conclusion, these results showed that red propolis is more cytotoxic than the green propolis in a variety of human cell lines of leukemia. Red propolis may contain drugs capable of inhibiting cancer cell growth. Therefore, further isolation of respective chemical ingredients from the red propolis (G13) for identification of the activities is necessary.