Journal of Pharmaceutical Sciences & Emerging Drugs ISSN: 2380-9477

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Research Article, J Pharm Sci Emerg Drugs Vol: 4 Issue: 2

Antibacterial Activity of Selected Essential oils against Streptococcus sobrinus and Porphyromonas gingivalis

Xiaoyu Wang1,2, Tohru Mitsunaga1*, and Kosei Yamauch
1Institute of Pesticide & Environmental Toxicology, Guangxi University, Nanning 530005, China
2Faculty of Applied Biological Science, Gifu University, Gifu, Japan
Corresponding author : Tohru Mitsunaga
Faculty of Applied Biological Science, Department of Applied Life Science, Gifu University, Japan
Tel: +81-058-293-2920
Fax: +81-058-293-2920
E-mail: [email protected]
Received: March 04, 2016 Accepted: May 02, 2016 Published: May 15, 2016
Citation: Wang X, Mitsunaga T, Yamauchi K (2016) Antibacterial Activity of Selected Essential oils against Streptococcus sobrinus and Porphyromonas gingivalis. J Pharm Sci Emerg Drugs 4:2. doi: 10.4172/2380-9477.1000113

Abstract

Plant essential oils have been used medicinally in history. The antibacterial screening of selected plant essential oils against Porphyromonas gingivalis and Streptococcus sobrinus was evaluated by MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) assay. Among them, thujopsis dolabrata (Asunaro) essential oil was selected as a fairly effective sample against P. gingivalis (MIC=1 mg/ml) and S. sobrinus (MIC=1 mg/ml) respectively. Moreover, Callitrisintratropica (Australian blue cypress; cypress) was also screened as a fairly effective sample only against S. sobrinus (MIC=0.5 mg/ml). Furthermore, five effective compounds, (+)-cuparene, (-)-thujopsene, (+)-cuparenol, (-)-guaiol and a novel sesquiterpenoid were isolated by a performance of a series of chromatography, and identified by nuclear magnetic resonance (NMR) and matrix assisted laser desorption/ionizationtime of flight mass spectrometry (MALDI-TOFMS). Among them, (+)-cuparenol was the most effective compound with a MIC of 0.125 mg/ml against P. gingivais. Additionally, the novel sesquiterpenoid (MIC=0.25 mg/ml) was identified as the first time isolation from C. intratropica.

Keywords: Thujopsis dolabrata; Callitris intratropica; Porphyromonas gingivalis; Streptococcus sobrinus;(+)-cuparene; (-)-thujopsene;(+)-cuparenol;(-)-guaiol; novel sesquiterpenoid

Keywords

Thujopsis dolabrata; Callitris intratropica; Porphyromonas gingivalis; Streptococcus sobrinus;(+)-cuparene; (-)-thujopsene; (+)-cuparenol; (-)-guaiol; novel sesquiterpenoid

Introduction

Plant essential oils and extracts have been used for a wide variety of purposes for thousands of years [1]. In particular, the antibacterial activity of plant oils and extracts has formed the basis of many applications, including raw and processed food preservation, pharmaceuticals, alternative medicine and natural therapies [2,3]. In this study, forty-eight (listed in Table 1) different commercial plant essential oils were screened for inhibitory activity against S. sobrinus and P. gingivalis by MIC and MBC assay.
Table 1: Forty-eight selected plant essential oil samples.
Oral diseases such as dental caries, periodontal disease and tooth loss are major public health problems in all over the world, especially on the disadvantaged and poor population groups [4]. Dental caries results from the interaction of specific bacteria with constituents of the diet within a biofilm termed ‘dental plaque’ [5]. The dental plaque, related to dental caries, functionsin several ways. It is a site of bacterial proliferationand growth, a location of acid/baseregulation at the tooth surface, and a reservoirfor calcium ion exchange between the tooth and the saliva [6]. These dental biofilms are composed of host constituents, cell-free enzymes, polysaccharides and bacteria [7]. S. sobrinus is a major pathogen responsible for biofilm formation [8,9]. Otherwise, any inherited or acquired disorder of the tissuessurrounding and supporting the teeth (periodontium) can be defined as a periodontal disease [10]. Theperiodontal inflammation is created by plaque bacteria and results in the progressive destruction of the tooth-supporting tissues, leading to gingival recession, periodontal pocket formation, or both. Periodontal disease is initiated by periodontal biofilm, which is made up almost entirely of oral bacteria, contained in a matrix composed of salivary glycoproteins and extracellular polysaccharides [11,12]. The “red complex”, composed by Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, presents as a portion of the climax community in the biofilms at sites expressing progressing periodontitis [13].
Among the forty-eight tested samples, ten samples showed inhibitory activity against P. gingivalis and S. sobrinus respectively. Therefore, asunaro (MIC=1 mg/ml) and cypress (0.5 mg/ml) were selected as the most two effective samples for the further antibacterial evaluation against the two mentioned bacteria. Furthermore, four active compounds were isolated: (+)-cuparene, (-)-thujopsene, (+)-cuparenol, (-)-guaioland a novel sesquiterpenoid were obtained from asunaro and cypress against P. gingivalis and S. sobrinus respectively.

Experimental

Materials
Selected essential oils were commercially purchased from a number of companies and kept under refrigerated in the past few years at the Laboratory of Natural Products Chemistry, Gifu University, Japan. Asunaro was purchase from Yuica Co., Ltd., Japan; Aus. blue cypress was purchased from Plant Extracts International Co., Ltd., U.S.A.
Antibacterial assay
MIC was determined by broth dilution method [14]. The essential oil samples were tested for antibacterial activity in sterile 96-well microplate. The inoculums, 106 cells/ml for S. sobrinus and 108 cells/ ml for P. gingivalis, were added to each well containing sample and medium to achieve a final volume of 200 μl. The tested samples were prepared at a concentration ranged around 4000-31 μg/ml using a two-fold dilution method. Chlorohexidine was included in the assays as positive control. The culture was incubated 24 h for S. sobrinus or 72 h for P. gingivalis at 37℃ under an anaerobic condition. Microbial growth was indicated by adding 50 μl of 0.2 mg/ml INT solution to culture and incubated for 3 h at 37℃ under an anaerobic condition. The MIC was defined as the lowest concentration that inhibited the color change of INT. For sequential MBC determination, 10 μl from wells that showed no change in color into100 μl fresh medium then incubated for 24 h for S. sobrinus or 72 h for P. gingivalis under anaerobic condition at 37℃. Microbial growth was indicated by adding 50 μl INT solution to culture and incubated 3 h at 37℃ under an anaerobic condition. The MBC was defined as the lowest concentration that inhibited the color change of INT.
Fraction of asunaro and cypress
Asunaro essential oil (5 g) was subjected onto silica gel column chromatography (75 mmφ×520 mmL), eluted with benzene/hexane (3:1), ethyl acetate/hexane (3:5) and methanol to obtain Afr.1-Afr.4. The Afr.1 was separated by preparative HPLC [ODS-3(20 mmφ×250 mm L), MeOH/0.05% TFA aq. soln.=97/3 (0 min), 100/0 (40 min), 100/0 (60 min)] to obtain compound 1 and compound 2. Moreover, the Afr .4 was separated by silica gel column chromatography (52 mmφ×480 mmL), eluted with acetone/ethyl acetate/hexane (3:5:8) and absolute methanol to obtain Afr.4a-Afr.4g. ThenAfr.4b was separated by preparative HPLC [ODS-3(10 mmφ×250 mm L), MeOH/0.05% TFA aq.soln.=70/30 (0min), 75/25 (40 min), 100/0 (60 min)] to obtain compound 3.
Cypress (10 g) was subjected onto silica gel column chromatography (75 mm φ× φ×520 mm L) for 48 h at 2.5 ml/min,
eluted with acetone/hexane (1:9) and absolute methanol to obtain Bfr.1-Bfr.3. The Bfr.3 was separated by silica gel column chromatography (50 mm φ×465 mm L), eluted with acetone/ chloroform/hexane (1:1:6) and absolute methanol to obtain Bfr.3a- Bfr.3e.Then Bfr.3b was separated by preparative HPLC [ODS-3(20 mmφ×250 mm L), MeOH/0.05% TFA aq.soln.=70/30 (0 min), 100/0 (40 min), 100/0 (60 min)] to obtain compound 4 and compound 5.
Identification of compounds
Compound 1-5 were identified by 1H NMR, 13C NMR, 1H-1H COSY, HMQC and HMBC. Chloroform-D was used as NMR solvent. NMR measurements were performed by using JEOL ECA 600 MHz, Bruker Biospin AVANCEⅢ600. MALDI-TOF-MS measurement was performed by using Shimadzu Biotech Axima Resonance.
(-)-Thujopsene (compound 1): MALDI-TOF-MS data: m/z: 205 [M+1].1H NMR spectrum (CDCl3, 600.17MHz):δ (ppm) 0.60 (3H, s, H-14 or H-15), 0.67 (1H, dd, H2, J=8.91, 4.83, H-2), 0.70 (1H, t, J=4.8, H-2), 1.09 (3H, s, H-14 or H-15), 1.11 (3H, s, H-13), 1.21 (1H, ddd, J=13.08, 13.05, 4.34, H-3), 1.36-1.49 (4H, m, H-6, H-8), 1.72-1.79 (4H, m, H-9, H-10), 1.80 (3H, s, H-12), 5.02 (1H, dd, J=6.87, 1.35, H-5); 13C NMR spectrum (CDCl3, 150.91MHz): δ (ppm) 11.36(C2), 19.66(C9), 22.29(C3), 23.59(C12), 26.87(C14 or C15), 28.67(C13), 29.14(14C or 15C), 31.51(C7), 33.94(C11), 35.03(C1), 36.63(C8), 40.43(C10), 41.24(C6), 114.52(C5), 135.64(C4). [α]20 D=-101.1989 (c=6.98, CDCl3). Spectral data were coincided to that of published report [15,16].
(+)-Cuparene (compound 2): MALDI-TOF-MS data: m/z: 203 [M+1]. 1H NMR spectrum (CDCl3, 600.17MHz):δ (ppm) 7.25 (2H, d, J=8.28, H-2, H-4), 7.09 (2H, d, J=7.56, H-1, H-5), 2.50 (1H, m, H-8), 2.31 (3H, s, H-12), 1.80–1.50 (5H, m, H-8, H-9, H-10), 1.25 (3H, s, H-13), 1.06 (3H, s, H-14 or H-15), 0.56 (3H, s, H-14 or H-1); 13C NMR spectrum (CDCl3, 150.91MHz): δ (ppm) 144.64 (C6), 134.79 (C3), 128.27 (C2 and C4), 127.00 (C1 and C5), 50.33 (C7), 44.28 (C11), 39.83 (C10), 36.3 (C8), 26.53 (C14 or C15), 24.48 (C13), 24.35 (C14 or C15), 20.90 (C12), 19.83 (C9).[α]25 D=66.0067 (c=0.3, CDCl3). Spectral data were coincided to that of published report [17,18].
(+)-Cuparenol (compound 3): MALDI-TOF-MS data: m/z: 217 [M-1].1H NMR spectrum (CDCl3, 600.17MHz):δ (ppm) 7.34 (2H, d, J=8.22, H-1, H-5), 7.27 (2H, d, J=8.22, H-2, H-4), 4.66 (2H, s, H-15), 2.48-2.51 (1H, m, H-11), 1.76-1.81(2H, m, H-10), 1.66-1.72 (2H, m, H-9, H-11), 1.52–1.57 (1H, m, H-9), 1.26 (3H, s, H-12), 1.06 (3H, s, H-13), 0.55 (3H, s, H-14); 13C NMR spectrum (CDCl3, 150.91 MHz): δ (ppm) 144.37 (C6), 137.87 (C3), 127.36 (C1 and C5), 126.42 (C2 and C4), 65.32 (C15), 50.58 (C7), 44.37 (C8), 39.79 (C9), 36.92 (C11), 26.48 (C13 or C14), 24.46 (C12), 24.35 (C13 or C14), 19.81 (C10). [α]25 D=89.7692 (c=0.13, CH2Cl2). Spectral data were coincided to that of published report [19].
(-)-Guaiol (compound 4): MALDI-TOF-MS data: m/z: 221 [M-1].1H NMR spectrum (CDCl3, 600.17MHz):δ (ppm) 2.52-2.53 (H, m, H-4), 2.41-2.44 (2H, m, H-2), 2.28 (H, m, H-10), 2.08-2.15 (2H, m, H-2, H-6), 1.85-1.97 (2H, m, H-6, H-3), 1.77-1.82 (H, m, H-8), 1.69- 1.72 (H, m, H-9), 1.52-1.55 (2H,m, H-7, H-9), 1.43-1.47 (H, m, H-8), 1.24-1.30 (H, m, H-3), 1.18 (3H, s, H-13), 1.15 (3H, s, H-12), 0.98 (3H, d, J=7.56, H-15), 0.94 (3H, d, J=6.84, H-14); 13C NMR spectrum (CDCl3, 150.91 MHz): δ (ppm) 140.16 (C1), 137.94 (C5), 73.76 (C11), 49.69 (C7), 46.38 (C4), 35.46 (C2), 33.84 (C9), 33.76 (C10), 31.01 (C3), 27.94 (C6), 27.46 (C13), 27.40 (C8), 26.03 (C12), 20.01 (C14), 19.85 (C15). [α]25 D=-13.4 (c=0.1, CHCl3). Spectral data were coincided to that of published report [20].
Compound 5: MALDI-TOF-MS data: m/z: 219.0846[M+1]. 1 H NMR spectrum (CDCl3, 600.17MHz):δ (ppm) 6.70 (1H, d, J=10.32, H-5), 5.84 (1H, d, J=9.6, H-4), 4.72 (2H, s, H-13), 2.22-2.28 (1H, m, H-2), 1.92-1.97 (1H, m, H-10), 1.74 (3H, s, H-12), 1.71–1.73 (1H, m, H-9), 1.61-1.66 (4H, m, H-1, H-7, H-8), 1.41-1.46 (1H, m, H-7), 1.21- 1.27 (1H, s, H-9), 1.11 (3H, d, H-14), 1.07 (3H, s, H-15); 13C NMR spectrum (CDCl3, 150.91 MHz): δ (ppm) 200.28 (C3), 160.57 (C5), 149.92 (C11), 126.37 (C4), 108.96 (C13), 48.52 (C1), 45.31 (C10), 42.65 (C2), 37.99 (C7), 36.34 (C6), 29.70 (C9), 26.38 (C8), 20.93 (C12), 17.31 (C15), 11.88 (C14).
The structures of compound 1-5 were shown in Figure 1.
Figure 1: The structures of compound 1-5.

Results and Discussion

Forty-eight plant essential oil samples were tested in this study. There were six samples included Asunaro (AS), Aus. white cypress (AWC), clove Indonesia (CI), cinnamon leaf (CL), lemongrass East India (LEI) and Myrrh Somalia (MS)were effective against P. gingivalis under a concentration at 4 mg/ml. Otherwise, there were six samples includedAS,CL, Aus. blue cypress (ABC), vetiver (VT), patchouli (PC) and sandalwood Australian(SA) were effective against S. sobrinus under a concentration at 4mg/ml. The MIC and MBC values of ten effective samples were shown in Tables 2 and 3.
Table 2: Inhibitory activities (mg/ml) of effective samples against P. gingivalis.
Table 3: Inhibitory activities (mg/ml) of effective samples against S. sobrinus.
Afr.1 showed inhibitory activity (1 mg/ml) against P. gingivalis, however, Afr.4 and Bfr.3 showed inhibitory activity (MIC=0.5 mg/ ml) against S. sobrinus (data were not shown). The inhibitory activities (MIC) of compound 1-5 were shown in Table 4. Chlorhexidine was used as positive control, because chlorhexidine is well-known for antibacterial activities [21,22]. Among them, (+)-cuparenol was the most effective compound against S. sobrinus with MIC=0.125mg/ ml. Moreover, the significant activity (0.5 mg/mL) of thujopsene is notable since this compound represents more than 50% of Asunaro essential oil. Besides, thujopsene has an extensively antibacterial activity against a lot of bacteria and fungi, such as Enterococcus faecalis, Bacillus subtilis, Staphylococcus aureus, Proteus sp, Aspergillus sydowi and Penicillium decumbens [23,24]. However, (+)-cuparene (MIC=4 mg/ml) has weak inhibitory activity against P. gingivalis; otherwise, (-)-guaiol (MIC=0.25 mg/ml) shows fairly good inhibitory activity against S. sobrinus.
Table 4: Inhibitory activities of compound 1-5 against P. gingivalis and S. sobrinus.
Compound 5 was found to be a novel compound. According to the 1H NMR data of compound 5, the signals at 5.84 ppm and 6.70 ppm were geminal and alkene protons because of the chemical shift and HMQC data. The two protons of 4.72 ppm singlet were alkene protons also because of the chemical shift and HMQC data. The nine protons of 1.07 ppm, 1.11 ppm and 1.74 ppm belonged to three methyl groups. According to the HMBC spectrum of compound 5 (Figure 2), correlations were observed between: H-1and C-3, C-9, C-10, C-11, C-15; H-2 and C-1, C-14; H-4 and C-2, C-6; H-5 and C-1, C-3, C-7; H-7 and C-5, C-6, C-9, C-15; H-8 and C-9, C-10; H-9 and C-7, C-8, C-10, C-11; H-10 and C-9, C-11, C-12, C-13; H-14 and C-1, C-2, C-3; H-5 and C-1, C-5, C-6, C-7. These NMR and MS data showed us the compound 5 is a sesquiterpenoid, named 2,6-dimethyl-10-(prop-1- en-2-yl)-1,6,7,8,9,10-hexahydro-naphthalen-3(1H)-one.
Figure 2: Key HMBC correlations of compound 5.
In sum, five effective compounds were isolated against P. gingivalis and S. sobrinus respectively: (+)-cuparene, (-)-thujopsene and (+)-cuparenol, were isolated from T. dolabrata, otherwise, (-)-guiaol and the novel sesquiterpenoid were isolated from C. intratropica. Among all the isolated compounds, (-)-thujopsene and (+)-cuparenol has the most potent inhibitory activity against P. gingivalis and S. sobrinus respectively.
Typically, essential oils are composed of terpene compounds, alcohols, acids, esters, epoxides, aldehydes, ketones and amines. And the antibacterial activities of essential oils directly correlated to their bioactive volatile compounds [25]. However, disruption of bacterial membranes contributes to the antibacterial properties of most essential oils and isolated compounds, such as carvacrol, thymol, β-cymene, cinnamaldehyde, cinnamic acid, eugenol and γ-terpinene [26-31]. Besides, cell content leakage and damage of membrane proteins also took the responsibility to antibacterial properties of essential oils [32,33].
Moreover, there are many studies indicated that essential oils and their isolated compounds are resistant against a wide variety of bacteria, such as some food-borne bacteria. Wen-RuiDiao showed that fennel seeds essential oil has good inhibitory activities against five food-borne bacteria: Staphylococcus albus, Bacillus subtilis, Salmonella typhimurium, Shigella dysenteriae, Escherichia coli, the MIC are all below 0.25 mg/ml [34]. Also, in Pirbalouti’s study, four Iranian medicinal herbs included Thymus daenensis, Dracocephalum multicaule, Satureja bachtiarica, and Tanacetum polycephalum are fairly effective against staphylococcus aureus with the MIC below 0.125 mg/ml [35]. Juglal and Govinden indicated that clove, cinnamon and oregano essential oils are effective against Fusarium moniliforme (pathogen for pitch canker of pine tree) and Aspergillus parasiticus (a potent liver carcinogen) [36]. Anyway, essential oils have great values and are quite potential for antibacterial and other bioactivities studies.

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