Technical Field

The present disclosure relates to the evaluation of the in-vitro antimicrobial effects of a triple essential oil mixture of standardized Mentha piperita, Thymus serpyllum, and Pelargonium graveolens or specifically designed formulations containing combinations of Juniperus communis or Zingiber officinalis or Pistacia lentiscus essential oils with chlorhexidine against dental caries pathogens.

State of the Art (Background)

Dental infections, such as dental caries and periodontal diseases, are among the most common bacterial infections in humans. As the main etiological agent of human dental caries, Streptococcus mutans is found in biofilms, which are formed on tooth surfaces and are also known as dental plaque. They live in the oral cavity and form dental plaque. S. mutans increases the acidity of the oral environment by producing organic acids from food residues, and as a result, it has a mineralizing effect on tooth structure. Many antimicrobial agents are available to inhibit biofilm formation and bacterial tooth mineralization, but these agents may exhibit side effects such as fluorosis, chromatism, taste disturbance, burning sensation, ulcerative lesions, and enamel erosion.

Since dental caries is seen as a serious concern for global public health, therapeutic alternatives to improve oral health seem to be very important. Numerous plant essential oils are known to act as antibiofilm agents against mouth-dwelling bacteria, particularly S. mutans. The activities of Mentha and Thymus species against S. mutans and various other microorganisms are stated, but no S. mutans activity is specified. For the P. graveolens essential oil, there is no study on the synergistic effect that may occur as a result of combinations of essential oils of these species. J. communis (common juniper) is a genus that spreads naturally in Tiirkiye and has different uses ethnobotanically such as urine enhancer, diaphoretic, gastric, antiseptic, emmenagogue, anti-inflammatory, hemorrhagic inhibitor.

Z. officinalis (ginger) is a tuber-rooted plant that grows under the ground. It is used among people to help digestion, relieve nausea, regulate bleeding, reduce rheumatism, regulate respiration, to prevent clotting and cholesterol.

P. lentiscus (gum tree) grows in various places in Tiirkiye. It has been used ethnobotanically for many years as a treatment for rabies, stomach, intestinal, lung, and various dental conditions.

These plants have been used for anti-inflammatory and antimicrobial purposes ethnobotanically since ancient times. It is known that the extracts of Zingiber officinalis and Pistacia lentiscus have used in oral care and to protect the gums.

Brief Description and Objects of the Invention

In the present invention, a triple essential oil mixture consisting of Mentha piperita, Thymus serpyllum and Pelargonium graveolens or any of the essential oils Juniperus communis, Zingiber officinalis, Pistacia lentiscus were combined with different concentrations of the chlorhexidine substance frequently used in oral care on the market, and as a result, antimicrobial essential oil-chlorhexidine combinations were obtained. Accordingly, it is aimed to prepare a safe, innovative, and naturally sourced oral care preparation that requires less chemical use by the inventors.

Detailed Description of the Invention

The displayed invention intends to standardize the essential oils selected in line with their use among the public and experimental study results by gas chromatography over major components/markers, to examine the antimicrobial activities and synergistic effects of the selected essential oils both in-house and with chlorhexidine by in vitro checkerboard method, and to design the pharmaceutical formulation suitable for oral use from the effective combined doses determined as a result of the activity studies of the standardized oils. Moreover, based on the main object of the invention, which is to prepare a safe, innovative and naturally sourced oral care preparation that requires less chemical use, the antimicrobial activities of Juniperus communis, Zingiber officinalis, Pistacia lentiscus essential oils, which have not previously had a specific antibacterial study for bacteria in the oral flora, are compared in themselves and in combination with chlorhexidine, which is frequently used in dentistry, are examined by in vitro experiments.

One of the primary objectives of the study conducted in comparison with chlorhexidine aims to minimize daily chemical use and to include natural products in human life in a controlled, evidence-based manner. According to our findings,, the preparation of natural oral care preparation is the main object of the present invention. The fact that the essential oils used in the study have been studied in different combinations and doses on bacteria that cause problems especially in the oral flora clearly presents the innovative aspect of the invention. This invention, in which the synergistic effect of three different essential oils as well as their combinations and synergism with chlorhexidine are investigated by the Checkerboard method, aims to reveal an evidence-based traditional medicinal product.

The standardization process of the supplied oils is based on the given literature and the major substances given in the European Pharmacopoeia. The standardization process is presented in detail with methods such as diode array detector, mass spectrometer, etc. The analysis of the oils is illuminated by simultaneous analyses of the GC and GC/MS systems. GC analysis is performed with the Agilent 6890N GC system. The analysis is performed using the Innowax FSC column (60 m x 0.25 mm, film thickness 0.25 pm) and Helium (0.8 mL/min) as carrier gas. The GC oven temperature is increased to 220°C with an increase of 4°C/min at 60°C for 10 minutes, kept for 10 minutes, and then increased to 240°C with an increase of l°C/min. The FID detector temperature is set to 300°C. The relative ratios of the compounds are calculated using the FID chromatogram computer. The volatile compounds separated in the GC column were then identified by taking individual mass spectra of each that act as detectors. Volatile compounds are separated into GC columns and then identified by taking individual mass spectra in mass spectrometry. Here, the mass spectrum is used in the detector function. GC/MS analysis is performed by using the Agilent 5975 GC/MSD system. Innowax FSC column (60 m x 0.25 mm, film thickness 0.25 pm) is used for separation of components. Helium (0.8 mL/min) is used as the carrier gas. The GC oven temperature is increased to 220°C with an increase of 4°C/min at 60°C for 10 minutes, kept for 10 minutes, and then increased to 240°C with an increase of l°C/min. Split applied at a rate of 1 in 40 and the energy of the mass spectra is taken as 70 eV. Mass spectra are taken with an energy of 70 eV. Mass values are recorded between m/z 35 and 450. The adhesion indices (RRI) of the substances in the column are calculated with reference to n-Alkanes. Component analyses were carried out using "Ba§er Essential Oil Components Library", Wiley, and MassFinder 3.1 Library Scanning Software.

The formulations contain 1-5% volatile oils by weight. The essential oils were weighed and dissolved in ethanol; sodium chloride and sodium bicarbonate were gradually added using a mechanical stirrer (500 rpm, 30 minutes), respectively. The formulation was filtered, and the volume of filtrate was made up to 10 mL using distilled water.

Essential oils were found to be effective against dental caries microorganisms in the concentration range of 0.01-0.5 mg/mL.

In the preferred embodiment of the invention, chlorhexidine: essential oil was combined separately and the chlorhexidine: essential oil ratios were determined in the range of 0.001- 0.004: 1-0.25 mg/mL.

In single combinations with chlorhexidine, combinations such as Pistacic . chlorhexidine (0.25:0.001 mg/ml), Juniperus communis', chlorhexidine (0.0125:0.001 mg/ml), Zingiber officinalis', chlorhexidine (0.5:0.001 mg/ml) found to be effective.

The aim is to evaluate the efficacy, toxicity, and stability of the designed formulations and to achieve a combination of essential oils with high antimicrobial activity and a synergistic effect.