Compounds from Olea europaea and Pistacia lentiscus inhibit oral microbial growth

Oleuropein and maslinic acid used in this study were isolated from an extract deriving from O. europaea L. (Oleaceae) leaves that were collected in 2009 at the region of Attica and were identified by Dr. E. Kalpoutzakis. A voucher specimen is deposited at the herbarium of the Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, University of Athens, Greece under the number PROK 006. The preparation of the extract has been described in a previous report [33]. The extract (360 g) was separated, dried and subjected to medium pressure Liquid Chromatography (MPLC) with silica (Si) gel 60 Merck (15–40 mm), using the dichloromethane (CH₂Cl₂) / methanol (MeOH) gradient as the eluent to extract pure oleuropein (≥ 95%) and maslinic acid (≥ 95%). Hydroxytyrosol was isolated from an extract produced from table olive processing wastewater. The preparation of the extract and the isolation of pure hydroxytyrosol (≥ 95%) have been previously described [34]. For the procurement of oleacein and oleocanthal the total polyphenol fraction (TPF) of extra virgin olive oil (EVOO) was used as starting material [35], while for their isolation column chromatography (CC) and preparative Thin Layer Chromatography (TLC) were employed, as previously described [36]. Briefly, 170 L of EVOO was subjected to 25 kg of the adsorbent XAD-7HP resin. The resin was activated with water (H₂O) and ethanol (EtOH), and EVOO remained in the XAD-7HP resin for 2 days with controlled and smoothed shaking and filtered. The resin was washed with 30 L cyclohexane (cHex) for the removal of the lipophilic constituents, and then the polyphenol-enriched extract was obtained by the extraction of the resin with approx. 40 L of ethanol. Further purification was achieved by liquid–liquid extraction using cHex and EtOH, and the obtained ethanolic fraction was filtered through paper and evaporated until dry affording 150 g of TPF. In continuation, 250 mg of TPF were subjected to a Si gel (0.015–0.04 mm) column (25 × 2.7 cm) and mixtures of CH₂Cl₂ and MeOH in increasing polarity (0–10% MeOH) were used for the elution. From the fractions obtained using 98:2 CH₂Cl₂ / MeOH, oleocanthal was isolated, while from the fractions obtained using 97:3 CH₂Cl₂ / MeOH, oleacein was attained. For further purification, preparative TLC was used. Specifically, precoated TLC silica 60 F254 plates, 2 mm layer thickness (purchased from Aldrich), were used while 94:6 CH₂Cl₂ / MeOH was used as the mobile phase. Spots were visualized using ultraviolet (UV) light and vanillin-sulfuric acid reagent. Finally, oleocanthal (≥ 95%) and oleacein (≥ 95%) were purified.

24Z-isomasticadienolic acid, oleanolic acid and oleanonic aldehyde were isolated from mastic gum [37]. Commercially available mastic gum was supplied by Chios Mastic Growers Association which is the exclusive worldwide producer of the resin. Conventional extraction of mastic gum for the preparation of total mastic extract without polymer (TMEWP) (extraction A) has been described in a previous report (Karygianni et al., 2014a). TMEWP partitioned between aqueous 5% Na₂CO₃ and ether as already described (extraction B) [38]. The organic phase was reextracted three times with 5% Na₂CO₃ (extraction C) and afforded the neutral fraction of mastic (135 g) as the organic phase. The aqueous phase was added to that of extraction B and acidified with 1 N HCl. The acidic solution was reextracted with ether (extraction D), and the organic phase afforded the acid fraction of mastic (190 g).

The acidic fraction (20 g) was submitted to MPLC over normal-phase silica gel first with a cHex / CH₂Cl₂ gradient and then with a CH₂Cl₂/MeOH gradient affording 23 fractions. In continuation, oleanolic acid was separated by MPLC over normal-phase silica gel eluted with a CH₂Cl₂ / MeOH gradient, while 24Z-isomasticadienolic acid by column chromatography over silica gel eluted with a CH₂Cl₂ / MeOH gradient. A part of the neutral fraction (17.2 g) was submitted to column liquid chromatography over normal-phase silica gel with a cHex / CH₂Cl₂ gradient to afford 22 fractions. Oleanonic aldehyde was separated in continuation by MPLC over normal-phase silica gel eluted with a cHex / CH₂Cl₂ gradient.

The chemical structures of the tested compounds are demonstrated on Fig. 1. The identity and purity (≥95%) of the isolated compounds were confirmed by nuclear magnetic resonance spectroscopy (NMR), mass spectrometry (MS) and high performance liquid chromatography (HPLC) experiments and by comparison with literature data. All solvents (ethanol, methanol, dichloromethane and cyclohexane) were of p.A. quality and came from Merck (Darmstadt, Germany). Water was purified by double distillation.

A total of eleven microbial strains from ten different bacterial strains and one Candida albicans strain were tested. Among these eight bacterial strains and C. albicans represent typical oral inhabitants, while the reference bacterial strains Escherichia coli and Staphylococcus aureus are members of the intestinal and skin flora, respectively. Their use facilitated the comparison of the antimicrobial activity of the natural compounds within the oral cavity against their general inhibitory impact. Facultative anaerobic Gram-positive species such as Streptococcus mutans DSM 20523, Streptococcus sobrinus DSM 20381, Streptococcus oralis ATCC 35037, Enterococcus faecalis ATCC 29212 and S. aureus ATCC 25923 were tested. Escherichia coli ATCC 25922 served also as a facultative anaerobic bacterium but with a Gram-negative cell wall. The tested obligate anaerobes included Porphyromonas gingivalis W381, Prevotella intermedia ATCC 25611, Fusobacterium nucleatum ATCC 25586 and Parvimonas micra ATCC 23195. All bacterial strains and C. albicans were kindly provided by the Division of Infectious Diseases and the Institute of Medical Microbiology and Hygiene of the Albert-Ludwigs-University, Freiburg. The microorganisms were kept at − 80 °C in basic growth medium containing 15% (v/v) glycerol prior to use.

As described in the Clinical and Laboratory Standards Institute (CLSI) guidelines an overnight culture of each bacterial strain and C. albicans was prepared and each dilution was placed on Columbia blood agar plates (CBA, Becton Dickinson GmbH, Heidelberg, Germany) or yeast-cysteine blood agar plates (HCB, Becton Dickinson GmbH, Heidelberg, Germany) [39, 40]. CBA agar plates were used for the incubation of facultative anaerobic bacteria and C. albicans at 37 °C and 5 -10% CO₂ atmosphere for 24 h. HCB agar plates were used for the incubation of anaerobic bacteria at 37 °C for 48 h (anaerobic chamber, Genbox BioMérieux SA, Marcy/Etoile, France). For the microdilution assay at 10⁶ colony forming units (CFU) mL^− 1 for each strain, Mueller-Hinton Broth (MHB) was utilized for the inoculation of all facultative anaerobic strains, Wilkins–Chalgren broth (WCB) for anaerobic bacteria and Sabouraud Dextrose Broth (SDB) for C. albicans. Then, with the aid of a multi-channel pipette appropriate volumes of the MHB/WCB/SDB microbial cultures were transferred into a 96-well microtiter-plate. Each well of the 96-well microtiter-plate had a total volume of 200 μL. Afterwards the prepared compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma, Steinheim, Germany) and diluted in distilled water. A concentration series ranging from 2500 μg mL-¹ to 2.4 μg mL^− 1 at dilution levels starting from 2-fold to 512-fold was used to screen all compound solutions in DMSO. The experiments were conducted in duplicate. For bacteria and fungi, a 0.5/1A McFarland standard suspension was diluted in normal saline. A dilution series of DMSO was tested in parallel in order to exclude potential inhibitory effects of the DMSO residuals. In case of bacterial growth in the co-tested DMSO dilution series the inhibitory impact of DMSO was taken into account. Wells containing only sterile MHB/WCB/SDB to minimize the possibility of contamination or 0.2% chlorhexidine (CHX) served as positive and negative controls, respectively. Thereafter, facultative anaerobic bacteria and C. albicans were incubated at 37 °C and 5 -10% CO₂ atmosphere for 24 h, while anaerobic bacteria were kept at 37 °C for 48 h (anaerobic chamber, Genbox BioMérieux SA, Marcy / Etoile, France). All assays for each bacterial strain and C. albicans were conducted in duplicate. If the MIC values of a specific strain were not identical, the highest minimum inhibitory concentration (MIC) values were taken into account. MIC was determined as the lowest concentration of each compound at which visible inhibition of bacterial growth occurred, and was expressed by the percentage of bacterial growth at that particular concentration.

As described in the CLSI guidelines the minimum bactericidal concentration (MBC) could also be determined [39, 40]. After the MIC testing, 10 μL from each well of the 96-well microtiter-plates containing the tested compound concentration series were incubated on agar plates. In particular, Columbia blood agar (CBA) agar plates were used for facultative anaerobic bacteria and C. albicans at 37 °C and 5 -10% CO₂ atmosphere for 2 days. Strictly anaerobic bacteria were incubated on yeast-cysteine blood agar (HCB) plates at 37 °C for 5 days (anaerobic chamber, Genbox BioMérieux SA, Marcy/Etoile, France). The colony forming units (CFU) were determined visually. The MBC was determined as the concentration at which a three-log decrease in bacterial growth (= 99.9%) was detected compared to the positive control. In the presence of variations within the yielded MIC/MBC values after the repetition of the experiments, the highest MIC/MBC values were listed to eliminate false positive results.

Five compounds (oleuropein, maslinic acid, hydroxytyrosol, oleocanthal and, oleacein) isolated from O. europaea by-products (leaves, table olives processing wastewater) and products (olive oil) were screened. Table 1 demonstrates the mean MIC and MBC values for each of the aforementioned O. europaea compounds as well as the tested bacterial and fungal strains.

Overall, maslinic acid was more effective than oleuropein, hydroxytyrosol, oleocanthal and oleacein. Maslinic acid was active against almost all anaerobic bacterial strains, with a mean concentration range of 4.9 μg mL^− 1 (Porphyromonas gingivalis) to 312 μg mL^− 1 (Fusobacterium nucleatum). The obligate anaerobe Parvimonas micra (9.8 μg mL^− 1) were efficiently inhibited, whereas maslinic acid showed no inhibitory effect against Prevotella intermedia. For streptococci (Streptococcus mutans, Streptococcus sobrinus, Streptococcus oralis) the MIC value for maslinic acid was estimated at 19.5 μg mL^− 1, for Enterococcus faecalis at 39 μg mL^− 1, for Staphylococcous aureus at 78 μg mL^− 1. The highest MIC value at 1.25 mg mL^− 1 was detected for Escherichia coli and Candida albicans. For obligate anaerobes, maslinic acid showed low MBC values, which ranged from 9.8 μg mL^− 1 (P gingivalis, P. micra) to 25 μg mL^− 1 (F. nucleatum). Streptococci such as S. sobrinus and S. oralis (19.5 μg mL^− 1) as well as S. mutans (156 μg mL^− 1) were more easily eradicated when compared to facultative anaerobic E. faecalis (312 μg mL^− 1) and S. aureus (625 μg mL^− 1). The highest MBC value at 1.25 mg mL^− 1 was detected for E. coli and C. albicans, while P. intermedia was not affected at all by maslinic acid.

Oleacein exhibited a milder inhibitory activity against oral microorganisms. The lowest MIC values of oleacein were observed for obligate anaerobes and were between 156 μg mL^− 1 (F. nucleatum) and 1.25 mg mL^− 1 (P. micra). The anaerobic P. gingivalis showed also a satisfactory MIC value of 312 μg mL^− 1 and P. intermedia could be inhibited only by oleacein (625 μg mL^− 1) after 5 days of culture. All other bacterial strains (streptococci, E. faecalis, E. coli) and C. albicans had MIC and MBC values of 1.25 mg mL^− 1. The MBC values of oleacein for obligate anaerobia were substantially lower ranging from 156 μg mL^− 1 (F. nucleatum), 625 μg mL^− 1 (P. gingivalis) to 1.25 mg mL^− 1 (P. micra).

Oleocanthal also showed an inhibitory effect on oral bacteria. The lowest MIC values of oleocanthal were detected for S. sobrinus (312 μg mL^− 1) as well as obligate anaerobes and varied between 156 μg mL^− 1 (P. gingivalis) and 312 μg mL^− 1 (F. nucleatum, P. micra). C. albicans was eradicated at 625 μg mL^− 1, whereas the streptococci and reference strains had MIC and MBC values of 1.25 mg mL^− 1. Obligate anaerobes (P. gingivalis, F. nucleatum, P. micra) showed the lowest MBC value (312 μg mL^− 1), while P. intermedia did not respond to the treatment with oleocanthal.

Concerning hydroxytyrosol, the lowest compound concentrations of 156 μg mL^− 1 (P. gingivalis), 312 μg mL^− 1 after 5 days of culture (P. intermedia, F. nucleatum) exerted bactericidal effect mainly on strict anaerobic, Gram-negative bacteria. From the streptococci, S. mutans and S. sobrinus presented also satisfactory inhibitory values of 312 μg mL^− 1 and 625 μg mL^− 1, respectively. The highest MIC value of hydroxytyrosol (1.25 mg mL^− 1) was observed for the reference strains, C. albicans. E. faecalis and P. micra. The lowest MBC value of hydroxytyrosol was estimated at 312 μg mL^− 1 (P. gingivalis, F. nucleatum, S. mutans), while 99.9% of E. faecalis, S. aureus and C. albicans were eradicated by 2.5 mg mL^− 1 of hydroxytyrosol.

Finally, oleuropein had the mildest antimicrobial impact on the oral pathogens. The MIC and MBC values of the eradicated microbial strains for oleuropein were between 625 μg mL^− 1 (S. mutans, S. aureus, P. gingivalis) to 1.25 mg mL^− 1 (S. oralis. E. faecalis, E. coli, P. micra, C. albicans). The lowest MBC value of oleuropein (312 μg mL^− 1) was observed for F. nucleatum, whereas P. intermedia was not inhibited by this compound.

Table 2 summarizes the MIC and MBC values of the three compounds (24Z-isomasticadienolic acid, oleanolic acid, and oleanonic aldehyde) isolated from P. lentiscus for all screened microbial strains.

Among all mastic gum compounds, oleanolic acid was the most effective against almost all microorganisms with MIC values ranging from 9.8 μg mL^− 1 (P. gingivalis) to 625 μg mL^− 1 (F. nucleatum, P. micra) for obligate anaerobes. The MIC value for maslinic acid was estimated at 19.5 μg mL^− 1 for streptococci (S. mutans, S. sobrinus, S. oralis), at 78 μg mL^− 1 for E. faecalis and S. aureus. The highest MIC and MBC value of oleanolic acid (1.25 mg mL^− 1) was detected for E. coli and C. albicans, whereas P. intermedia was not affected at all by oleanolic acid. The mean MBC values for strict anaerobic bacteria were 9.8 μg mL^− 1 (P. gingivalis), 625 μg mL^− 1 (F. nucleatum) and 1.25 mg mL^− 1 (P. micra), whereas higher MBC values were estimated for streptococci at 39 μg mL^− 1 (S. mutans, S. sobrinus) and 78 μg mL^− 1 (S. oralis).

Another compound, the 24Z-isomasticadienolic acid also presented a substantial antimicrobial effect against the screened microorganisms. In its presence, a mean inhibitory concentration range of 2.4 μg mL^− 1 (P. gingivalis, P. micra) to 625 μg mL^− 1 (F. nucleatum) was observed for strict anaerobia. The MIC value for 24Z-isomasticadienolic acid was estimated between 39 μg mL^− 1 (S. sobrinus, S. oralis) and 78 μg mL^− 1 (S. mutans) for streptococci, while E. faecalis had a MIC value of 156 μg mL^− 1. The highest MIC and MBC value of 24Z-isomasticadienolic acid (1.25 mg mL^− 1) was detected for E. coli, S. aureus and C. albicans, whereas P. intermedia did not respond to the treatment. The lowest MBC value (9.8 μg mL^− 1) were determined for the obligate anaerobia P. gingivalis and P. micra, while 78 μg mL^− 1 and 156 μg mL^− 1 of the compound killed 99.9% of S. oralis and P. micra and S. mutans, respectively.

Oleanonic aldehyde presented the lowest antimicrobial activity compared to the other two mastic gum compounds. The lowest MIC value of 625 μg mL^− 1 was found for P. gingivalis, whereas all other tested bacterial and fungal strains presented MIC and MBC value of 1.25 mg mL^− 1. Oleanonic aldehyde proved to be ineffective against P. intermedia.