INTRODUCTION:

In recent years, the urgency of treatment with medicinal plants and preparations based on them has increased. This is primarily due to the growth of toxic-allergic diseases, side effects from the use of synthetic drugs. The centuries-old traditions and experience of traditional medicine convincingly prove the advisability of using medicinal plants in the prevention, maintenance or course therapy of a number of diseases. It should be noted that the flora of Kazakhstan, including essential oil plants, has not been sufficiently studied.

Plants of the families Asteraceae, Apiaceae, Lamiaceae, etc. are richest in essential oils. Essential oils have proven to be a promising source of bioactive molecules with potential use in the treatment of dental caries ^1-4. In the oral cavity, the antimicrobial properties of essential oils have shown promising health benefits including reducing gum inflammation and bad breath and controlling biofilm formation⁵. In addition to native essential oils their main components monoterpenoids are active against cariogenic bacteria, for example in ⁶ limonene, linalool and β-ocymene reduced the growth of Streptococcus mutans (S. mutans) and reduced the expression of genes comC, comD, comE, gtfB, gtfC and gbpB. The results in publication⁷ demonstrated that kaffir lime leaf oil and lemongrass oil showed a potent anti- S. mutans activity and inhibited biofilm formation with the possible mechanism targeted on the cell membrane. Biofilm formation by S. mutans was also inhibited in the presence of the tested essential oils (Eucalyptus globulus and Eucalyptus urograndis), which yielded more effective results when compared to 0.1% commercial NaF (sodium fluoride)⁸. In publication ⁹ the substances (Cymbopogon citratus essential oil and the citral and myrcene) evaluated showed significant antimicrobial effects; hence, these should be studied further as potential co-adjuvants to prevent dental caries that cause minor adverse effects.

Thus, essential oils and their components can be used as alternative antimicrobial drugs that can reduce or inhibit biofilm formation. If essential oils are effective against biofilm formation, especially in bacteria that develop antibiotic resistance, they can be incorporated into new antimicrobial drugs. Therefore, there is a need to explore the potential of the chemical diversity of essential oils and their enormous potential in antimicrobial therapy. In this regard, we have studied the component composition of 4 essential oils (Hyssopus ambiguus (Trautv.) Iljin ex Prochorov. & Lebel, Nepeta cataria L., Origanum vulgare L., Ziziphora clinopodioides Lam.) And their effect on the biofilm formation of Streptococcus mutans.

Hyssopus ambiguus Hjin is a mountain-steppe species inhabiting Central Asia; in Siberia. According to M.I. Goryaeva ¹⁰ the herb of the plant contains 0.21-0.6% of essential oil, and the amount of the latter increases by the time of full flowering. Light yellow oil with a strong pungent odor. The oil contains up to 8.21% cineole, 22% pinene, 12.6% l-pinocamphone. According to E. Suleimen the study of the component composition of the essential oil of Hyssopus ambiguus made it possible to reveal that it contains terpene hydrocarbons and their oxygen-containing derivatives, as well as aromatic compounds of predominantly phenolic nature. The investigated essential oil contains 48 components, the content of which varies from 0.1 to 26.4%. The main components of H. ambiguus essential oil are: 1,8-cineole (26%), cis-verbenol (18.2%), pinocarvone (14%), β-pinene (10.5%), p-menta-1,8- diene (8.7%) ¹¹.

Nepeta cataria L. is a perennial plant with an erect, branched stem and heart-shaped leaves. Flowers on short pedicels, collected in significant numbers on short common peduncles. Cultivated in the wild found in the CIS, the Caucasus, Western Siberia, and Central Asia: in Kyrgyzstan and Turkmenistan and in all regions of Kazakhstan - a steppe and mountain-steppe species. Nepetolactone, an iriroid monoterpenoid, was first identified in the essential oil of Nepeta cataria and has a relatively high analgesic activity. In the essential oil of catnip, nepetolactone is the main component ^12-17. The essential oil of Nepeta cataria has antimicrobial, antifungal, antioxidant, nootropic, repellent and anti-inflammatory activities ^18-20.

Origanum vulgare L. is a steppe and meadow-steppe species widespread in all regions of Kazakhstan with the exception of deserts. The chemical composition of the essential oil depends on the weather and geographical conditions of the environment, the chemotype, the storage conditions of the raw materials and the method of extracting essential oils. For example, research shows that the plant performed well in terms of body weight, oil yield, antioxidant potential and essential oil composition when grown at high altitudes ²¹. The characteristic components of oregano essential oil are: α-pinene, β-pinene, myrcene, selenene, camphene, sabinene, ocymene, limonene, α-terpinene, β-caryophyllene, borneol, 1,8-cineole, α-terpineol, thymol, carvacrol, methyl esters of thymol and carvacrol ^22,23. Earlier studies have reported anti-mutagenic, anti-oxidant, anti-hyperglycemic, antifungal, anti-viral, anti-inflammatory and potent antibacterial effects of this plant ^24-26. Work was carried out to study the inhibitory and antibiofilm action of Origanum vulgare essential oil in vitro and in vivo on Streptococcus mutans isolates obtained from primary school students. Limonene and myrcene were the most effective constituents of the essential oil. It has been revealed that the essential oil and its main components have powerful antibiotic-film and antibacterial properties and can be used for the production of new plant-based mouthwashes ²⁷.

Ziziphora clinopodioides Lam. is a perennial herb with a strong odor. Grows in Central Asia, Altai and Western Siberia. Grows on rocky slopes and river banks. In the essential oil Ziziphora clinopodioides which grows in Kazakhstan, Russia, Turkey and Iran, the main component is pulegon ^28-31. Studies have shown that the essential oil from Ziziphora clinopodioides can be considered as a potential potent antimicrobial agent that can be used to inhibit the growth of Salmonella typhimurium and Staphylococcus aureus bacteria in foods adding Ziziphora clinopodioides essential oil as a natural antibacterial agent to foods increases the shelf life storage of these products ^32-38.

As the analysis of the range of external dosage forms of phytopreparations shows new developments in the field of drug technology using plant essential oils are promising for the treatment of various wounds ³⁹. Creation of new dosage forms of dental products for external use in the form of gels, ointments, pastes, etc. will allow it to remain on the mucous membrane for a long time and maintain a constant concentration of the drug ^40-42. In this regard, we have selected 4 types of essential oils of plants of the Lamiaceae family, studied their component compositions and screened these oils for anti-caries activity to create new effective, environmentally friendly, low-toxic anti-caries agents.

MATERIALS AND METHODS:

The object of research is essential oils of 4 species of plants of the Lamiaceae family growing in the territory of the Republic of Kazakhstan.

Essential oil raw materials were collected in June-August 2020 in the vicinity of the Karaganda and East Kazakhstan regions. Essential oils of leaves and inflorescences were obtained by hydrodistillation from air-dry raw materials for 3 hours using a Clevenger laboratory setup.

Study of the component composition of essential oils:

The component composition of the assayed samples of the essential oils was investigated by Gas chromatography-mass spectroscopy (GC/MS). GC/MS analysis of the essential oil samples was performed on an Agilent 7890A GC System coupled to an Agilent 5975C Mass Selective Detector. The HP-5MS capillary column was 30 m x 0.25 mm (film thickness 0.25 μm). The analysis was performed using the temperature program such as oven isotherm at 70 °C for 2 min, then 70°C to 270 °C at 20°C/min and 270 °C for 30 min. Helium was used as a carrier gas at a flow rate of 2 ml/min, without separation. The temperatures of the nozzle 250°C and the detector were 230 °C. Mass spectra were recorded using the ionization energy of 70 eV and the separation temperature of 280 °C, acquisition mass range m/z 10–650. MSD ChemStation software supplied by Agilent Technologies combined with AMDIS 32 and NIST 2017 were used to process data.

Biofilm formation, treatment and analysis by colorimetric assay:

S. mutans (strain UA159) stock in skim milk was thawed, and 10 μl of its suspension was inoculated to the starter culture vials containing 990 µl of Todd Hewitt (TH) broth. Then, the starter culture vials were incubated anaerobically (95% N₂ + 5% CO₂) at 37 ºC for 18 h. For purity check, the loopful of S. mutans stock suspension was inoculated to Columbia agar plates with 7% sheep blood. Then, the plates were incubated anaerobically (95% N₂ + 5% CO₂) at 37º C for 48 h. After 18 h, optical density (OD) adjustment of starter cultures was performed in 1:5 dilutions in 96-well microplate using the Dynex MRX™ microplate-reader spectrophotometer at 630 nm (Figure 1). TH broth without and with 1% sucrose was dispensed into 24-well, flat-bottomed, polystyrene tissue culture plates (the final volume of liquid per well was 1 ml). The stock concentrations of essential oils (100 mg/ml) were prepared in the pure dimethyl sulfoxide (DMSO). The essential oils were added to the plates at the final concentrations of 2, 4, 6, 8 and 10 mg/ml, and DMSO was added to the plates at the final concentrations of 2, 4, 6, 8 and 10%. The plate wells were inoculated with S. mutans starter culture at the final dilution of 1:100, and the plates were incubated anaerobically (95% N2 + 5% CO2) at 37 ºC for 24 h. The plate wells were rinsed with distilled water in order to remove loosely bound bacterial cells, and then the biofilm in bottom of wells was fixed with 95% ethanol. The biofilm was stained with 0.01% crystal violet solution, and then the bound dye was extracted using 33% acetic acid solution. Afterwards, the OD of samples was measured using Dynex MRX™ microplate-reader spectrophotometer at 595 nm. The data were analyzed with Statistical Package for Social Science (SPSS) program (version 23.0) using One-Way ANOVA Least Significant Difference (LSD) Post-Hoc test for comparison of means. A p value less than 0.05 was considered statistically significant.

Figure 1 - Сolorimetric assay

All of the tested essential oils showed inhibitory activity on S. mutans biofilm formation in the medium containing 1% sucrose (which is the main inducer of biofilm formation for S. mutans bacteria).

RESULTS:

In the course of this work, an essential oil of yellow-green color, light and fluid, with a tart sweet aroma, was isolated from the aboveground part of Hyssopus ambiguus. The analysis of essential oil obtained from flowers and leaves of plants growing in Kazakhstan showed a high content of eucalyptol (61.7%), moderate content of α-pinene (14.1%), β-fellandrene (5.4%) and 1-octen-3-ol (8.8%) (Table 1).

The essential oil extracted by us from Nepeta cataria is a light yellow mobile liquid with a pleasant smell. The yield of essential oil is 0.45%. Table 1 shows the chemical composition of Nepeta cataria essential oil collected in Central Kazakhstan. About 50 components were identified in the essential oil isolated from the aboveground part of the Nepeta cataria by the GC-MS method, the main ones are 1,8-cineol (15.8%) and (4aR,7S,7As)-nepetolactone (29.3) (Table 1). The main sesquiterpenoids are represented by caryophyllene (3.5 %) and germacrene D (6.3%).

The essential oil from Origanum Vulgare L. is a yellow transparent liquid with a strong aromatic smell. Chromatographic analysis of the essential oil of Origanum vulgare L., growing in Eastern Kazakhstan, showed that the main components are carvacrol (65.4%) and o-Cymene (13.2%), also present in significant concentrations of α-thuyene (2.7%), β-myrcene (2.3), γ-terpinene (6.7%). α -pinene (1.7%), α-terpinene (1.5%) and α-fenchene (0.5%) were found in small amounts (Table 1).

The yield of Ziziphora clinopodioides essential oil was 0.3%. The essential oil is liquid, yellow in color with a pleasant smell. A total of 24 components were identified. The major components of the essential oil of Ziziphora clinopodioides are pulegone 42.7%, isomenthone 15.3%.

Table 1- Component composition of essential oils of Hyssopus ambiguus, Nepeta cataria, Origanum vulgare and Ziziphora clinopodioides.

HT, min.	Component name	Content of components in essential oil, %
HT, min.	Component name	Nepeta cataria	Hyssopus ambiguus	Origanum vulgare	Ziziphora clinopodioides
1	2	4	5	6	7
10.167	Camphene	0.1	0.1	0.1	2.1
10.715	α-thuyene	-	-	1.7	0.1
10.922	b-Phellandrene	-	5.4	-	0.1
10.931	a-Pinene	3.3	8.6	1.7	2.3
10.965	Sabinene	0.9	4.1	-	0.8
10.986	b-Pinene	0.4	-	-	2.1
11.135	1-Octen-3-ol	0.2	8.8		-
11.453	b- Myrcene	0.1	2.9	2.3	0.5
11.465	a-fenchene	-	-	0.5	-
11.570	3- Octanol	0.9	06	0.1	-
12.102	a-Terpinene	2.3	-	2.3	-
12.314	о- Cymene	-	2.5	13.2	-
12.421	D- Limonene	-	-	0.6	1.1
12.474	1,8-Cineol	15.8	61.7	-	2.7
12.398	g- Terpinene	2.9	1.9	6.7	0.1
12.686	Trans-b- Ocimene	-	0.5	-	0.1
12.890	b- Terpinene	0.2	0.1	0.2	-
12.699	1-dodecene-3-ol	-	-	0.1	-
13.547	Trans-Linalool oxide	0.2	0.1	0.1	-
13.709	g-carene	-	-	0.1	-
14.206	Linalool	0.1	0.1	0.1	-
14.493	1-Octen-3-yl-acetate	-	-	-	0.1
14.786	(+)-menthone	-	-	-	2.5
15.001	(-)-isomenthone	-	-	-	16.3
15.365	isoterpinolene	-	-	0.7	-
15.835	isopulegone	-	-	-	1.2
16.045	isomenthol	-	-	-	6.5
16.267	pulegone	-	-	-	42.7
17.076	2,6-Octadien-1-ol, 3,7-dimethyl-(Z)-	0.1	-	-	-
17.767	2,6-Octadien-1-ol, 3,7-dimethyl-(E)-	-	-	-	0.1
18.320	Bornyl acetate	-	-	-	0.1
18.810	endoborneol			0.4
18.585	o-Cymen-5-ol	-	-	0.1	-
19.164	Terpinen-4-ol			0.6
19.453	(4aS,7S,7aS)-nepetolactone	9.6	-	-	-
19.691	eugenol	-	-	tr	-
20.286	(-)-b-Bourbonene	-	-	-	0.2
20.360	Germacrene D	6.3	-	-	-
20.785	α-Cedrene	0.1	-	-	-
20.886	Thymol methyl	-	-	0.1	-
20.950	carvacrol	-	-	65.4	-
21.079	(4aS,7S,7aS)-nepetolactone	29.3
21.115	Caryophyllene	0.1	0.1	0.9	2.3
21.179	trans-a-Bergamotene	-	-	-	-
21.296	Aromandendrene	0.1	-	-	-
21.508	cis-b-Farnesene	0.5	-	-	0.1
21.561	Humulene	0.4	-	-
21.699	Alloaromadendrene	0.7	-	-	-
21.944	g-Muurolene	0.1	-	-	-
22.337	Bicyclogermacrene	0.2	-	-	-
22.465	b-bisabolene	0.7	-	-	0.2
22.752	Cadina-1(6),4-diene	0.1	-	-	-
23.039	Cis-a-Bisabolene	0.2	-	-	-
23.730	Spatulenol	0.2	-	-	0.1
24.707	(-)-Spathulenol	0.1	-	-	-
24.760	Muurolol	0.3	-	-	-
24.973	γ-Eudesmol	0.1	-	-	-
25.494	γ-Cadinene	0.4	-	-	-
25.679	Caryophyllene oxide	2.8	-	-	-

Note: tr - trace amount

the main components are highlighted in bold

Evaluation of the effectiveness of essential oils Hyssopus ambiguus, Nepeta cataria, Origanum vulgare and Ziziphora clinopodioides for inhibiting S. mutans biofilm formation using colorimetric analysis showed its ability to significantly inhibit the development of biofilms on the surface of polystyrene in 24-well cell culture plates.

The assessment of the effectiveness of the Nepeta cataria, Origanum vulgare essential oils for inhibition of S. mutans biofilm formation using colorimetric assay revealed its capacity to considerably suppress biofilm development on the polystyrene surface in the 24-well cell culture plates. Treatment with the Nepeta cataria, Origanum vulgare essential oils at a concentration of 2 mg/ml resulted in only a slight reduction in biofilm production at the bottom of the wells in the 24-well cell culture plates. However, concentrations of 4 mg/ml, 6 mg/ml, 8 mg/ml, and 10 mg/ml of the Nepeta cataria and Origanum vulgare essential oil almost completely inhibited S. mutans biofilm accumulation at the bottom of the wells in the 24-well cell culture plates.

As shown in Figure 2, the Origanum vulgare essential oil at a concentration of 2 mg/ml caused an insignificant reduction of 9% in S. mutans biofilm biomass versus the untreated control bacteria (p > 0.05, as determined quantitatively using a one-way ANOVA, followed by a post hoc leastsignificant difference test). However, the Origanum vulgare essential oil at concentrations of 4 mg/ml, 6 mg/ml, 8 mg/ml, and 10 mg/ml significantly decreased S. mutans biofilm biomass by 98% compared to the untreated control bacteria (p < 0.05, as determined quantitatively using a one-way ANOVA, followed by a post hoc least-significant difference test).

Figure 2 - Quantities of Streptococcus mutans biofilm biomass following 24 h of incubation within Todd Hewitt broth in the presence of 1% sucrose and various concentrations of Origanum vulgare essential oil and dimethyl sulfoxide (DMSO). Values are shown as the mean + standard error obtained from a single experiment (n =2-26).

*p < 0.05 versus the control; **p < 0.05 versus the DMSO.

Figure 3 shows that Nepeta cataria essential oil concentrations of 4, 6, 8 and 10 mg/ml decreased S. mutans biofilm formation by 97% in comparison to the untreated bacteria (p < 0.05) in the TH broth with 1% sucrose.

Figure 3 - Quantities of Streptococcus mutans biofilm biomass following 24 h of incubation within Todd Hewitt broth in the presence of 1% sucrose and various concentrations of Nepeta cataria essential oil and dimethyl sulfoxide (DMSO). Values are shown as the mean + standard error obtained from a single experiment (n = 2-26).

*p < 0.05 versus the control; **p < 0.05 versus the DMSO.

The inhibitory activity of Nepeta cataria, Origanum vulgare essential oils did not occur in a dose-dependent manner. Of note, the DMSO concentration of 2% did not significantly decrease the biofilm biomass in comparison with the untreated bacteria (p > 0.05, as determined quantitatively using a one-way ANOVA, followed by a post hoc least-significant difference test). On the other hand, treatment of bacterial cells with DMSO alone caused a significant reduction in S. mutans biofilm biomass, versus the untreated bacteria, at DMSO concentrations of 4%, 6%, 8%, and 10% (p < 0.05, as determined quantitatively using a one-way ANOVA, followed by a post hoc least-significant difference test). This DMSO activity was induced in a dose-dependent manner.Importantly, the suppressive effect of Nepeta cataria, Origanum vulgare essential oils at concentrations of 4 mg/ml, 6 mg/ml, 8 mg/ml, and 10 mg/ ml was significant compared to the corresponding concentrations of DMSO (p < 0.05, as determined quantitatively using a one-way ANOVA, followed by a post hoc leastsignificant difference test). This indicates that Nepeta cataria, Origanum vulgare essential oils exhibited inhibitory activity against S. mutans biofilm formation independently from DMSO, causing a considerably greater effect than DMSO alone.

Treatment with Hyssopus ambiguus essential oil at 2 mg / ml and 4 mg / ml resulted in only a slight reduction in biofilm formation at the bottom of the wells in 24-well cell culture plates. However, concentrations of 6 mg / ml, 8 mg / ml, and 10 mg / ml of Hyssopus ambiguus essential oil almost completely suppressed the accumulation of S. mutans biofilm at the bottom of the wells in a 24-well well. As shown in Figure 4, Hyssopus ambiguus essential oil concentrations of 6, 8 and 10 mg/ml decreased S. mutans biofilm formation by 73, 98 and 98%, respectively, in comparison to the untreated bacteria (p < 0.05) in the TH broth with 1% sucrose (Figure 4).

Figure 4 - Quantities of Streptococcus mutans biofilm biomass following 24 h of incubation within Todd Hewitt broth in the presence of 1% sucrose and various concentrations of Hyssopus ambiguus essential oil and dimethyl sulfoxide (DMSO). Values are shown as the mean + standard error obtained from a single experiment

(n = 2-26), *p < 0.05 versus the control; **p < 0.05 versus the DMSO.

Figure 5 shows that Ziziphora clinopodioides essential oil concentrations of 6, 8 and 10 mg/ml decreased S. mutans biofilm formation by 96% in comparison to the untreated bacteria (p < 0.05) in the TH broth with 1% sucrose.

Figure 5 - Quantities of Streptococcus mutans biofilm biomass following 24 h of incubation within Todd Hewitt broth in the presence of 1% sucrose and various concentrations of Ziziphora clinopodioides essential oil and dimethyl sulfoxide (DMSO). Values are shown as the mean + standard error obtained from a single experiment

(n = 2-26), *p < 0.05 versus the control; **p < 0.05 versus the DMSO.

Dimethyl sulfoxide concentrations of 2, 4, 6, 8 and 10% decreased S. mutans biofilm formation by 5, 16, 44, 53 and 65% respectively, in comparison to the untreated in the TH broth with 1% sucrose. This effect was statistically significant (p < 0.05) for the DMSO concentrations of 4, 6, 8 and 10%.

Thus, аll of the tested essential oils and borneol showed inhibitory activity on S. mutans biofilm formation in the medium containing 1% sucrose (which is the main inducer of biofilm formation for S. mutans bacteria). Origanum vulgare, Nepeta cataria essential oils and borneol demonstrated the the highest suppressive effect on S. mutans biofilm formation in the medium containing 1% sucrose. Hyssopus ambiguus, Ziziphora clinopodioides essential oils exhibited the inhibitory activity on S. mutans biofilm formation in the concentration-dependent manner. The solvent – dimethyl sulfoxide reduced S. mutans biofilm formation in the concentration-dependent manner in the medium containing 1% sucrose, however the inhibitory activity of essential oils on S. mutans biofilm formation is considerably greater in comparison with the DMSO.

CONCLUSION:

The chemical composition of 4 plant species of the Lamiaceae family was established using GC-MS analysis. 1.8-cineole is the main ingredient for the essential oils of Hyssopus ambiguus and Nepeta cataria, carvacrol for Origanum vulgare, pullegon for Ziziphora clinopodioides, and nepetolactone for Nepeta cataria.

As a result of the experiment, it was revealed that Origanum vulgare, Nepeta cataria essential oils demonstrated the the highest suppressive effect on S. mutans biofilm formation in the medium containing 1% sucrose. As a result of the experiment, it was revealed that Origanum vulgare, Nepeta cataria essential oils demonstrated the highest suppressive effect on S. mutans biofilm formation in the medium containing 1% sucrose.

Hyssopus ambiguus, Ziziphora clinopodioides essential oils exhibited the inhibitory activity on S. mutans biofilm formation in the concentration-dependent manner

The solvent – dimethyl sulfoxide (DMSO) reduced S. mutans biofilm formation in the concentration-dependent manner in the medium containing 1% sucrose, however the inhibitory activity of essential oils on S. mutans biofilm formation is considerably greater in comparison with the DMSO.

The results of the research can be used to develop new therapeutic and prophylactic dental products.

SOURCE OF FUNDING:

This research has been funded by the Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan (Grant No. AP14971364).

REFERENCES

1. Lapinska B, Szram A, Zarzycka B, Grzegorczyk J, Hardan L, Sokolowski J, Lukomska-Szymanska M, An In Vitro Study on the Antimicrobial Properties of Essential Oil Modified Resin Composite against Oral Pathogens // Materials 2020, 13, 4383; doi:10.3390/ma13194383

2. Moghadam P, Dadelahi S, Hajizadeh YS, Matin M, Amini M, Hajazimian S. Chemical Composition and Antibacterial Activities of Sumac Fruit (Rhus coriaria) Essential Oil on Dental Caries Pathogens// The Open Microbiology Journal 14(1):142-146. - DOI: 10.2174/1874285802014010142

3. Dagli N, Dagli R. Possible Use of Essential Oils in Dentistry// Journal of International Oral Health 6(3): i-ii

4. Subasree S, Geetha RV. Essential oils in prevention of Dental Caries - An In-Vitro study// Research J. Pharm. and Tech. 8(7): July, 2015; Page 909-911. doi: 10.5958/0974-360X.2015.00148.1

5. Freires IA, Denny C, Benso B, Alencar SM, Rosalen PL. Antibacterial Activity of Essential Oils and Their Isolated Constituents against Cariogenic Bacteria: A Systematic Review// Molecules 2015, 20, 7329-7358; doi:10.3390/molecules20047329

6. Benzaid С, Belmadani А, Tichati L, Djeribi R, Rouabhia M.Effect of Citrus aurantium L. Essential Oil on Streptococcus mutans Growth, Biofilm Formation and Virulent Genes Expression // Antibiotics (Basel). 2021 Jan 8;10(1):54. doi: 10.3390/antibiotics10010054.

7. Jiamboonsri P, Kanchanadumkerng P. Influence of Gallic Acid and Thai Culinary Essential Oils on Antibacterial Activity of Nisin against Streptococcus mutans//Adv Pharmacol Pharm Sci. 2021 Apr 24;2021:5539459. doi: 10.1155/2021/5539459.

8. Goldbecka JC, Nascimento JE, Jacob RG, Fiorentini AM, Silva WH Bioactivity of essential oils from Eucalyptus globulus and Eucalyptus urograndis against planktonic cells and biofilms of Streptococcus mutans// Industrial Crops and Products. Volume 60, September 2014, Pages 304-309

9. Ortega-Cuadros M, Tofiño-Rivera AP, Merini LS, Martínez-Pabón MC. Antimicrobial activity of Cymbopogon citratus (Poaceae) on Streptococcus mutans biofilm and its cytotoxic effects//Revista de Biología Tropical. Oct./Dec. 2018 http://dx.doi.org/10.15517/rbt.v66i4.33140

10. Goryaev M.I., Pliva I. Methods for the study of essential oils. - Alma-Ata: Publishing House of the Academy of Sciences of the Kazakh SSR, 1969. - 752 p.

11. Sampietro D.A., Gomez A.L., Jimenez C.M., Lizarraga E.F., Ibatayev Z.A, Suleimen Y.M., Catalán C.A. Chemical composition and antifungal activity of essential oils from medicinal plants of Kazakhstan. // Nat Prod Res.- 2017.- N.31(12). – P.1464-1467.

12. Malizia R.A., Molli S.C., Cardell D.A., Retamar J.A.Volatile constituents of the essential oil of Nepeta cataria L. grown in Cordoba Province (Argentina) // Journal of Essential Oil Research. – 1996. - Vol. 8., N. 5. – P. 565–567, 1996.

13. Zenasni L., Bouidida L., Hancali A. The essentials oils and antimicrobial activity of four Nepeta species from Morocco//Journal of Medicinal Plants Research. – 2008. - Vol. 2, N. 5. – P. 111–114.

14. Handjieva N. V., Popov S. S., Evstatieva L. N. Constituents of essential oils from Nepeta cataria L., N. grandiflora M.B. and N. nuda L// Journal of Essential Oil Research. -1996. - Vol. 8, N. 6.- P. 639–643.

15. Bourrel C., Perineau F., Michel G., Bessiere J.M. Catnip (Nepeta cataria L.) essential oil: analysis of chemical constituents, bacteriostatic and fungistatic properties. // Journal of Essential Oil Research. – 1993. - Vol. 5. - N. 2. - P. 159–167, 1993.

16. Safaei-Ghomi J., Djafari-Bidgoli Z., Batooli H. Volatile constituents analysis of Nepeta cataria from central Iran// Chemistry of Natural Compounds. -2009. - Vol. 45, N. 6.-P. 913–915

17. Zomorodian K., Mohammad J. S., Shariati S., Pakshir K., Rahimi M., Khashei R. Chemical composition and antimicrobial activities of essential oils from Nepeta cataria L. against common causes of food-borne infections// International Scholarly Research Network ISRN Pharmaceutics. - Volume 2012, Article ID 591953, 6 p.

18. Adiguzel A., Ozer H., Sokmen M. Antimicrobial and antioxidant activity of the essential oil and methanol extract of Nepeta cataria. // Polish Journal of Microbiology. – 2009. - Vol. 58, N. 1. - P. 69–76.

19. Satish S. Studies on therapeutic potential of essential oils of Nepeta cataria in treatment of Alzheimer’s disease // Asian Journal of Biomedical and Pharmaceutical Sciences. – 2013.- Vol. 3(18). - P. 42-48.

20. Ricci E., Toyama D., Lago J., Romoff P., Kirsten T., Reis-Silva T., Bernardi M. Anti-nociceptive and anti-inflammatory actions of Nepeta cataria L. var. citriodora (Becker) Balb. essential oil in mice// J Health Sci Inst. 2010. –N 28(3). – P.289-293

21. Shweta Goyal , Geeta Tewari , H. K Pandey, Anjali Kumari Exploration of Productivity, Chemical Composition, and Antioxidant Potential of Origanum vulgare L. Grown at Different Geographical Locations of Western Himalaya, India// Journal of Chemistry Volume 2021, Article ID 6683300, 12 pages https://doi.org/10.1155/2021/6683300

22. Bokov D.O., Nizamova L.A., Morokhina S.L., Marakhova A.I., Bobkova N.V., Sergunova E.V., Kovaleva T.Yu., Balobanova N.P., Prostodusheva T.V., Klyukina E.S., Chevidaev V.V., Samylina I.A., Lazareva N.B., Krasnyuk I.I. Pharmacognostic studies of Origanum L. species Medicinal plant raw materials. Research J. Pharm. and Tech. 2020; 13(9):4365-4372. doi: 10.5958/0974-360X.2020.00772.6

23. Fikry, S.; Khalil, N.; Salama, O. Chemical Profiling, Biostatic and Biocidal Dynamics of Origanum vulgare L. Essential Oil. AMB Express 2019, 9, 41.

24. Lukas, B.; Schmiderer, C.; Novak, J. Essential Oil Diversity of European Origanum vulgare L. (Lamiaceae). Phytochemistry 2015, 119, 32–40

25. Adelina Lombrea, Diana Antal, Florina Ardelean, Stefana Avram, Ioana Zinuca Pavel, Lavinia Vlaia, Ana-Maria Mut, Zorita Diaconeasa, Cristina Adriana Dehelean, Codruta Soica and Corina Danciu. A Recent Insight Regarding the Phytochemistry and Bioactivity of Origanum vulgare L. Essential Oil//Int. J. Mol. Sci. 2020, 21, 9653; doi:10.3390/ijms21249653

26. Amal A. Mohamed, Sami I. Ali, Manal Y. Sameeh, Tamer M. Abd El-Razik. Effect of solvents extraction on HPLC profile of phenolic compounds, antioxidant and anticoagulant properties of Origanum vulgare. Research J. Pharm. and Tech 2016; 9(11):2009-2016. doi: 10.5958/0974-360X.2016.00410.8

27. Fatemeh Hejazinia 1, Leila Fozouni, Nasrin Sadat Azami, Seyedgholamreza Mousavi The Anti-Biofilm Activity of Oregano Essential Oil Against Dental Plaque-Forming Streptococcus mutans In Vitro and In Vivo //J Kermanshah Univ Med Sci. 2020 September; 24(3):e107680. doi: 10.5812/jkums.107680

28. Korolyuk E.A., Koenig V., Tkachev A.V. The composition of the essential oil of Ziziphora clinopodioides lam. From the Altai Territory and the Altai Republic // Chemistry of Vegetable Raw Materials. - 2002. - No. 1. - P. 49–52.

29. Belyaev N.F., Demeubaeva A.M. Chromatographic study of the composition of the oil Ziziphora clinopodioides Lam., A vicarious species Origanum vulgare // Chemistry of Natural Compounds. 1999.-T. 1.- S. 66–68.

30. Attila A., De Pooter Herman L., De Buyck, Lauren F. The essential oils of Calamintha Nepeta subsp. Glandulosa and Ziziphora clinopodioides from Turkey // J. Essent. Oil. - 1991. -Vol. 3 (1). - P. 7–10.

31. Hamidreza Khodaverdi-Samani, Abdollah G. P., Hamzeh-Ali Shirmardi, Fatemeh M. Chemical composition of essential oils of Ziziphora clinopodioides Lam. (endemic Iranian herb) collected from different natural habitats. // Indian Journal of Traditional Knowledge. - 2015.- Vol. 14 (1). - P. 57-62.

32. Shahbazi Y. The antibacterial effect of Ziziphora clinopodioides essential oil and nisin against Salmonella typhimurium and Staphylococcus aureus in doogh, a yoghurt-based Iranian drink// Veterinary Research Forum. -2016. - № 7 (3). – Р. 213 – 219.

33. Shahbazi Y, Shavisi N., Mohebi E. Аntibacterial activity of Ziziphora clinopodioides essential oil and nisin against Bacillus subtilis and Salmonella typhimurium in commercial barley soup // Bulgarian Journal of Veterinary Medicine. - 2016.- P.1311-1477

34. Y. Effects of Ziziphora clinopodioides essential oil and nisin on the microbiological properties of milk. Pharm Sci. – 2016. - №22(4). – Р. 272-278.

35. Sharifan А., Beikmohammadi L. Antimicrobial efficiency of Iranian Ziziphora clinopodiodes essential oil on Preservation of Hamburger //J. Med. Microbiol. Infec. Dis. 2014. – N 2 (4). – P. 138-142.

36. Sinaeyan S., Sani A.M. Antimicrobial activity of Ziziphora clinopodioides essential oil and extract on Salmonella enterica, Staphylococcus aureus and Saccharomyces cerevisiae in Low Fat Mayonnaise// BioTechnology: An Indian Journal Research. – 2012. - Vol 12, Iss 8. – P. 34-38.

37. Kheirkhah M., Ghasemi V., Yazdi A., Rahban A. Chemical composition and insecticidal activity of essential oil from Ziziphora clinopodioides Lam. used against the Mediterranean flour moth, Ephestia kuehniella Zeller. Journal of Plant Protection Research. – 2015.- Vol. 55, No. 3.-P.35-41

38. Masrournia M., Shams A. Elemental determination and essential oil composition of Ziziphora clinopodioides and consideration of its antibacterial effects. Asian J. Chem. 2013. - N 25. –P. 6553–6556.

39. Andreia Aires, António Salvador Barreto, Teresa Semedo-Lemsaddek. Antimicrobial Effects of Essential Oils on Oral Microbiota Biofilms: The Toothbrush in Vitro Model // Antibiotics (Basel). 2021 Jan; 10(1): 21. Published online 2020 Dec 29. doi: 10.3390/antibiotics10010021

40. S. Subasree, Geetha R. V. Essential oils in prevention of Dental Caries - An In-Vitro study. Research J. Pharm. and Tech. 8(7): July, 2015; Page 909-911. doi: 10.5958/0974-360X.2015.00148.1

41. Suchithra. A. B, S. Jeganath, E. Jeevitha. Pharmaceutical Gels and Recent Trends –A Review. Research J. Pharm. And Tech. 2019; 12(12): 6181-6186. doi: 10.5958/0974-360X.2019.01073.

42. Nazia Zareen. I, Gopinath Prakasam. Oral Biofilms. Research J. Pharm. and Tech 2016; 9(10):1812-1814. doi: 10.5958/0974-360X.2016.00368.1

Received on 02.09.2021 Modified on 09.03.2022

Research J. Pharm. and Tech 2022; 15(11):4959-4966.

DOI: 10.52711/0974-360X.2022.00834