Synergistic antibacterial combination of Scutellaria lateriflora extract and Cistus x Incanus extract

Abstract

To provide a synergistic combination of Scutellaria lateriflora L. extract titrated with baicalin and Cistus x incanus L. extract with high polyphenol content for oral health.

Description

[Technical field]

[0001] Summary of the Invention The object of the present invention is a synergistic combination of Scutellaria lateriflora L. extract titrated with baicalin and Cistus x incanus L. extract with high polyphenol content for oral health. [Background technology]

[0002] prior art The human body constantly lives in symbiosis with a variety of microorganisms that inhabit every organ and surface. Since 2007, the scientific community has investigated the relationship between the microbiota, human physiology and disease onset and chronicity, demonstrating that bacteria positively influence human physiology at a systemic level and regulate overall human health.

[0003] The oral microbiota is an integral part of the human microbiome and is represented by more than 6 billion bacteria belonging to 700 different species. It is highly dynamic due to many factors such as genetics, oral hygiene and lifestyle, including smoking and diet. Diet plays an important role as microbes feed on food debris remaining in the oral cavity after a meal, different types of microbes can more easily colonize the oral cavity because they use preferential metabolic and fermentation pathways depending on the main macronutrients (carbohydrates and proteins), and pH fluctuations affect the growth of certain bacterial species and are unfavorable for others, inducing eubacteriosis / dysbiosis.

[0004] It is therefore clear that diet, although in part affecting oral health, plays an important role in health at a systemic level, as was first demonstrated in 1980. Periodontal disease (infection of the supporting structures around the teeth, such as gums and bone) is known to be associated with cardio- and cerebrovascular disease, asthma, pneumonia, preterm birth and pre-eclampsia, epilepsy and osteoporosis. The association of periodontal disease with secondary systemic disease may be due to the spread of infection from the oral cavity to other organs via the blood circulation of microbial toxins and the systemic inflammatory state caused by cheek inflammation.

[0005] In detail, periodontitis is a multifactorial polymicrobial infection characterized by a destructive inflammatory process. The Gram-negative black-pigmented anaerobic bacterium Porphyromonas gingivalis is the main pathogen involved in the initiation and progression of periodontitis, which can produce a number of virulence factors that stimulate human gingival epithelial (HIGh) cells and human periodontal ligament (HPL) cells to produce a variety of inflammatory mediators.

[0006] To reduce oral infections, chlorhexidine, a broad-spectrum bactericide active against gram-positive bacteria, some species of Pseudomonas and Proteus, as well as some fungi and viruses, is widely used. Chlorhexidine is used in mouthwashes and is effective in reducing inflammation of bacterial origin, but it can induce resistance and has an indiscriminate bactericidal effect, so it acts not only against pathogens but also against eubacteria, causing dysbiosis.

[0007] In order to reduce the side effects of chlorhexidine, the use of mouthwashes based on plant extracts has recently been proposed as a preferred alternative to the use of said antiseptic.

[0008] Medicinal plants have traditionally been used as antimicrobial agents because they contain bioactive substances such as polyphenols and essential oils.

[0009] The literature provides data on the use of plants and extracts to obtain a positive synergistic effect on the oral flora. Bilberry helps to control and reduce the risk of oral inflammation (Widen et al., Acta Odontol Scand, 2015;73(7):539-43), magnolia, cinnamon and aloe vera extracts help to prevent the development of plaque and gingivitis (clinically tested, Hellstrom et al., Int J Dent Hyg, May 2014;12(2):96-102; Gupta et al., J Int Acad Periodontol. July 2015;17(3):91-8; Yeturu et al., J Oral Biol Craniofac Res. January-April 2016;6(1):54-8), and products containing multiple extracts (ginger, rosemary and calendula) have been proven effective against gingivitis (Mahyari et al., Complement Ther Clin Pract, February 2016;22:93-8).

[0010] Scutellaria lateriflora L. is a typical neurosedative plant and is widely used as a sedative-hypnotic, especially in North America, on the basis that the flavonoids characteristic of the plant, baicalein and wogonin, exert their tranquilizing effects through allosteric modulation of the GABA receptor (γ-aminobutyric acid) at the benzodiazepine site (Hui et al., Biochem Pharmacol, 2002 Nov. 1;64(9):1415-24; Wang et al., Neuropharmacology, 2008 Dec.;55(7):1231-7). Additionally, many components and extracts of the Scutellaria genus have shown antibacterial activity in vitro (Shen et al., J Ethnopharmacol, 2021 Jan 30;265:113-198), although only limited testing has been done on Scutellaria lateriflora L. extracts and only against some pathogens (Bergeron et al., International Journal of Pharmacognosy, 34:4, 233-242). More recently, baicalin has shown synergistic activity against Staphylococcus aureus when combined with the β-lactam antibiotic benzylpenicillin (Liu et al., J Pharm Pharmacol, 2000 Mar;52(3):361-6). In a very recent study, aglycone baicalein showed significant inhibitory properties against biofilm formation by Streptococcus mutans, one of the main cariogenic species considered to be a pioneer bacterium in the colonization of tooth surfaces (Vijayakumar et al., Antibiotics 2021, 10, 215).

[0011] Infusions obtained from various species of Cistus have traditionally been used to alleviate gastrointestinal and respiratory disorders, as well as some skin disorders. The antioxidant properties of the plant are also known (Gori et al., Int J Mol Sci, 2016 Aug. 17;17(8):1344). The effectiveness of the plant against gram-positive bacteria is also known for some time (Kuchta et al., Cardiol J, 2019 Mar. 26). Another in vitro study demonstrated the antibacterial and anti-adhesive activity of Cistus x incanus L. extract against Streptococcus mutans (Wittpahl et al., Planta Med, 2015 Dec;81(18):1727-35). [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of the example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Description of the invention It has now been found that a combination of Scutellaria lateriflora L. and Cistus x incanus L. extracts exhibits synergistic antibacterial activity against Porphyromonas gingivalis, a bacterium that causes periodontitis.

[0014] This combination can be advantageously used as an ingredient in currently or functionally used foods (candy, chewing gum, fruit juices and beverages) and also as a food supplement or medical device in view of their daily consumption / use, directing the composition of the oral microbiota towards eubacteriosis, reducing pathogenic bacteria and exerting a protective effect against oral diseases and their associated systemic diseases.

[0015] Thus, a first aspect of the present invention relates to a synergistic antibacterial combination consisting of an extract of Scutellaria lateriflora L. and an extract of Cistus x incanus L.

[0016] The tested combinations are characterized by weight / volume concentration ratios of Scutellaria lateriflora L. extract and Cistus x incanus L. extract ranging from 3:1 to 1:1, respectively, in the form of a solution or suspension.

[0017] The Scutellaria lateriflora L. and Cistus x incanus L. extracts are preferably hydro-alcoholic extracts.

[0018] Said extracts are known or can be obtained by known methods, see for example DE 20 2005 014 459, US 8,691,294, EP 1 446 135 and US 7,771,758.

[0019] The Scutellaria lateriflora L. extract preferably has a baicalin content of 10% by weight or more, while the Cistus x incanus L. extract preferably has a polyphenol content in the range of 18-25% by weight.

[0020] Scutellaria lateriflora L. extracts with a minimum baicalin content of 10% to be used in synergistic combinations can be prepared, after identification by DNA barcode testing (to exclude contamination with other morphologically similar species of the same genus and, above all, with the dangerous Teucrium chamaedrys), by a process including the following steps: chopping the previously dried aerial parts, preferably to a length of 1-2 cm, treating them with an extraction solvent containing water, ethanol and / or their mixtures, maintaining the mixture in a state of percolation under heating, and at the end of the first recycle, repeating the extraction 3-5 times under the same conditions, using fresh solvent each time; combining all the aqueous-alcoholic extracts, filtering and concentrating; removing the extraction solvent by concentration under vacuum, heating and subsequent spray drying on maltodextrin, until a homogeneous green-brown powder is obtained.

[0021] Cistus x incanus L. extracts with polyphenol contents ranging from 18 to 25% can be prepared by a method comprising the following steps: chopping the previously dried aerial parts, preferably to lengths of 1 to 2 cm, then treating them with an extraction solvent comprising water, ethanol and / or a mixture thereof, maintaining the mixture in a state of percolation under heating, and at the end of the first recycle, repeating the extraction 3 to 5 times under the same conditions, using fresh solvent each time; combining all the aqueous-alcoholic extracts, filtering and concentrating; removing the extraction solvent by concentration under vacuum, heating and subsequent spray drying over gum arabic, until a homogeneous brown powder is obtained.

[0022] The synergistic combination according to the invention is suitably formulated in the form of a composition suitable for application in the oral hygiene field, such as a mouthwash, gel, paste, chewing gum, chewable tablet or orodispersible film. The concentration of the extract in the composition may vary over a wide range, generally ranging from 1 to 100 mg / ml of extract.

[0023] The present invention will now be described in detail with reference to the following examples. EXAMPLES

[0024] Evaluation of antibacterial activity against Porphyromonas gingivalis Porphyromonas gingivalis was cultured in trypticase soy broth (TSB) under anaerobic conditions, inoculated into 96-well multiwell plates at an optical density of 0.1 OD and treated with the extracts for 24 h.

[0025] Materials and Reagents: Porphyromonas gingivalis: Strains were obtained from the American Type Culture Collection (ATCC 33277) and grown in TSB broth according to the supplier's instructions.

[0026] Tryptic Soy Broth / Agar Supplement (ATCC® Medium 2722) DI Water - 1000ml Autoclave at 121 °C. Final pH7.4+ / -0.2 Store at 2~8°C. Microbial growth conditions: Temperature: 37℃ Atmosphere: Anaerobic

[0027] Anaerobic conditions were obtained using anaerobic bottles and AnaeroGen® Compact paper packets (Thermo Scientific-AN0025A).

[0028] The lyophilized strains were rehydrated in 1 ml of TSB under sterile conditions. Approximately 100 μl of the bacterial suspension was used to inoculate TSA agar plates. The bacterial suspension and plates were incubated at 37°C under anaerobic conditions for 24–48 h until turbidity increased and colonies appeared.

[0029] To evaluate the antibacterial activity of the extracts against Porphyromonas gingivalis, the microorganisms were inoculated in TSB in 96-well plates at an optical density of 0.1 OD and then treated with the extracts for 24 h. The concentrations tested were 60, 50, 40, 30, 20, 10 and 5 mg / mL, with each test performed in triplicate. The extracts were diluted in sterile broth. The antibiotic amoxicillin 10 μg / ml was used as a positive control. Incubation was performed anaerobically at 37°C for 24 h.

[0030] The absorbance was read at a wavelength of 595 nm. The absorbance value is directly proportional to the bacterial growth. The absorbance data are shown in a graph. Significant differences between groups were evaluated by ANOVA test using GraphPad Prism 6.0. Data are presented as the mean ± standard deviation (SD) of three independent experiments.

[0031] Aqueous-alcoholic extracts of Scutellaria lateriflora L. and Cistus x incanus L. were tested against P. gingivalis first individually and then in a 1:1 ratio with the following results: 30mg / ml of Scutellaria results in 16% growth inhibition 30mg / ml of Cistus results in 4% growth inhibition · 30mg / ml Scutellaria + 30mg / ml Cistus (1:1 ratio) results in 48% growth inhibition, i.e. double (almost 2.5 times) the original combined value (about 20%).

[0032] Considering the observed synergistic effect, the following combinations of both extracts were tested using ampicillin as a positive control and broth medium without extracts as a negative control. Scutellaria+Cistus 55mg / mL+5mg / mL 45mg / mL+15mg / mL 40mg / mL+20mg / mL 30mg / mL+30mg / mL 20mg / mL+40mg / mL 10mg / mL+50mg / mL 5mg / mL+55mg / mL Ampicillin 20μM / mL

[0033] The results obtained, as shown in the attached figures, confirm the synergistic effect of the Scutellaria-Cistus combination (ratio 1:1), with the mixture: 45mg / mL+15mg / mL 40mg / mL+20mg / mL 30mg / mL+30mg / mL Only the extracts from Scutellaria and Cistus are shown to have synergistic effects with respect to the individual extracts, and synergy occurs when the extract from Scutellaria is present in greater amounts relative to the extract from Cistus (up to a ratio of 3:1). Conversely, no synergy occurs when the extract from Cistus is quantitatively more abundant than the extract from Scutellaria.

Claims

Claim 1 A composition comprising an extract of Scutellaria lateriflora L. and an extract of Cistus x incanus L. Claim 2 The composition according to claim 1, which is in the form of a solution or a suspension. Claim 3 The composition according to claim 2, wherein the weight / volume concentration ratio of the extract of Scutellaria lateriflora L. to the extract of Cistus x incanus L. is in the range of 3:1 to 1:1, respectively. Claim 4 The composition according to claim 1, wherein the extracts of Scutellaria lateriflora L. and Cistus x incanus L. are water-alcohol extracts. Claim 5 The composition according to claim 4, wherein the extract of Scutellaria lateriflora L. has a baicalin content of 10% by weight or more. Claim 6 The composition according to claim 4, wherein the extract of Cistus x incanus L. has a polyphenol content in the range of 18-25% by weight. Claim 7 The composition according to any one of claims 1 to 6, for oral hygiene. Claim 8 The composition according to claim 7, which is in the form of a mouthwash, gel, paste, chewing gum, chewable tablet, or orally dispersible film.

Images