Phytochemical profile and antimicrobial activity of essential oils from two Syzygium species against selected oral pathogens

Essential oils (EOs) have shown great interest over the past decade as holistic integrative modalities for traditional medicinal treatments [1]. They have been used by many populations as alternatives to ready-prescribed medications because of their low toxicity, pharmacological activities, and economic viability [1]. EOs are hydrophobic, complex extracts comprised of hundreds of organic, low-molecular-weight volatile components. They could be simply categorized according to their chemical scaffold into terpenoids and aromatic, each of which is subclassified as oxygenated and non-oxygenated [2]. The variability in their chemical composition could be attributed to a set of interrelated factors such as the extraction method, geographic sources, environmental conditions, maturity stage, plant organ, and genetics [3, 4]. The unique chemical blend of volatile components enriched the EOs with a surge of therapeutic values, especially in the field of dentistry and oral hygiene. For instance, several studies have documented the beneficial effect of supplementing mouthwashes with EOs in inhibiting plaque formation, diminishing anxiety, and reducing toothache [5]. Also, dressings treated with EOs enhanced the healing of wounds after oral surgery. Meanwhile, treating the surface of dental implants with EOs prevented biofilm formation and showed more potent inhibitory activity against microbiomes than methylparaben [5]. The antimicrobial potential is urgently essential because, recently, many synthetic antimicrobial drugs have been facing treatment limitations due to the development of antimicrobial drug resistance and acute toxicity [6]. In this regard, the exploitation of new EOs to control multidrug-resistant pathogenic microorganisms can help combat various infectious diseases. Several plant species are well-known for their notable EO content, among them are those of the genus Syzygium [7, 8]. Syzygium is a genus in the myrtle family (Myrtaceae) that comprises about 1193 recognized aromatic species worldwide [7]. Syzygium is restricted to tropical and subtropical regions of the Old World [7]. The genus has unprecedented culinary use, as witnessed by the unexpanded flower buds of S. aromaticum (Clove), which are the most significant economic spices [8]. Traditionally, clove essential oil is used as a pain reliever in dental care as well as in treating tooth infections [8]. Other Syzygium species produce edible fruits that are eaten fresh or used commercially such as, S. malaccense and S. samarangense [7]. Syzygium malaccense (L.) Merr. & L. M. Perry, known as the Malaysian apple or Malay apple, is an evergreen, flowering tree with edible fruits native to the forested lowlands of Malaysia, Southeast Asia, and Australia [9]. Traditionally, the leaves are chewed, or the juice is dripped into the mouth of infants to treat mouth infections and oral thrush [10]. In Malaysia, the dried leaves are used on a cracked tongue, while bark extract is used for abdominal ailments, coughs, and sore throats [10]. In the same context, Syzygium samarangense (Blume) Merr. & L. M. Perry, recognized as wax apple or java apple, is a tropical non-climactic tree found in south to southeast Asia, Taiwan, and other tropical countries [11]. The species is a well-known antioxidant, immunomodulatory, antibacterial, anticancer, anti-inflammatory, analgesic, and antidepressant [11]. Both species promise to support nutritional values and human health, yet the antimicrobial rationale of their EOs in oral infections has not been studied.

The oral cavity harbors diverse microbiomes that, under normal conditions, reside in homeostasis [12]. The imbalance of these microbiomes or their colonization with new microorganisms of viral, fungal, or bacterial origin can infect the oral cavity and its mucosa, influencing oral health and causing several diseases [13]. Dental biofilms are formed due to the attachment of oral microbiomes to the hard and soft tissues of the oral cavity. They are highly associated, embedded in an extracellular matrix [14], and responsible for many oral diseases. Meanwhile, the incidence of primary bacterial infections of the oral mucosa is rare because of the protective role of the epithelium layer, the saliva's antibacterial characteristics, and the immune responses of the phagocytes [13]. However, if the oral mucosa is disrupted due to poor oral hygiene, trauma, smoking, alcohol misuse, or any other stimuli, the risk of primary bacterial infections will be hazardous. Among common oral microbiomes are Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, and Candida albicans. E. faecalis is an obligate anaerobe that has been related to caries, endodontic infections, and peri-implantitis [15, 16]. Even though Enterococci are sensitive to some antibiotics the emergence of multidrug-resistant strains is becoming a matter of concern. S. aureus is another favorable inhabitant of the oral cavity and has been added to the drug-resistant microbiomes due to the excessive prophylactic usage of antibiotics [17, 18]. On the other side, E. coli has been frequently reported from acute dental abscesses, ranging from 0.7% to 15% [19]. Lastly, C. albicans is a ubiquitous commensal organism and by far the principal causative agent of oral candidiasis accounting for up to 95% of cases [20].

Given the incidence of oral infections, increased resistance by microbiomes to antibiotics, adverse effects of some antibacterial agents, and financial considerations in developing countries, there is a need for alternative options. Constituting mainly effective, safe, and economical advantages. These unique features were collectively documented for natural phytochemicals, just like essential oils.

In the current study, the GC profile of the EOs extracted by three different methods from S. samarangense and S. malaccense leaves cultivated in Egypt were comparatively investigated for the first time. Moreover, the ability of the extracted EOs to inhibit the growth of oral-related pathogens and reduce their ability to develop oral biofilm was also examined.

The oral cavity is a common inhabitant of diverse microbiomes that play a fundamental role in overall human health. Imbalances in the microbiome environment shift them to a pathogenic state causing various oral diseases such as dental cavities, gingivitis, candidiasis, and even systemic infection [35]. Despite the availability of various synthetic antimicrobial agents, the emergence of antibiotic-resistant bacteria is an emerging challenge [36]. On the other side, essential oils (EOs) have been studied for many years as potential antimicrobial agents, and several populations still applying them as traditional medicine. More than 3000 EOs have been established to be consumed, and their benefits are being progressively studied due to the need for alternative therapies for resistant oral microbiomes [37]. In the current study, two EO-rich Syzygium species, viz, S. samarangense and S. malaccense, have been investigated for their antimicrobial potential. Syzygium species have traditional practices to manage various illnesses, including mouth infections and oral thrush [8, 10]. Herein, the intended EOs have been extracted using three methods: hydrodistillation (HD), supercritical fluid (SF), and headspace (HS). Interestingly, the HD method has been previously implemented for the extraction of the EO from both species but from different geographical origins [38‐41]. Conversely, this is the first report on the extraction of EOs from both species using state-of-the-art approaches such as supercritical fluid (SF), and head-space (HS). In general, the extraction conditions significantly affect the physical properties and the yield of an EO. As shown from our results, the SF method provided a higher oil yield than the conventional HD because the SF method utilizes supercritical CO₂ as an extraction solvent. Supercritical CO₂ is characterized by being a non-viscous solvent with low surface tension, thereby increasing the penetration rate, enhancing the extraction capacity, and amplifying the oil yield [42]. In addition, the solvation power of supercritical CO₂ leads to the co-solubilization of some fatty constituents, which gives the SF extract a darker color and semi-solid consistency. Subsequently, the extracted EOs were analyzed qualitatively and quantitatively using hyphenated GC/MS, and the abundance of each constituent was estimated from the area of the peaks recorded by GC/FID. Our results showed that the identified volatile components were significantly variable among the two species concurrently with the applied extraction methods. This may be correlated to the differences in the condition of the plant material, the operated temperature, and pressure, in addition to the extraction time. In the SF method, dried leaves were extracted using supercritical CO₂ (SCC) at 40 °C and 150 bar; in the HD method, fresh leaves were extracted using boiling water, while in the HS fresh leaves were heated until the volatilization of the aroma [21‐23]. As depicted, the HD and HS utilized fresh leaves, while SF used dried ones which rationale the significant difference in the identified constituents of the SF EOs from the other two methods. Although HD and HS extracted the EO from a fresh plant sample, differences in the temperature and extraction time greatly affected the stability of the extracted constituents, nevertheless their detected percentage. A factor that may translate the significant differences we observed in the concentration of the identified major components from both methods. Also, the power of the extraction solvent, in each method, plays another crucial role. For instance, SCC behaves similarly to lipophilic solvents but with the advantage of adjustable selectivity. Hence, hydrocarbons are typically reported as majors in SF-EOs. On the other side, in the HD method, the diffusion of the boiling water inside the plant material enhanced the extraction of both oxygenated and non-oxygenated low molecular weight components. However, oxygenated components faced uncontrollable hydrolysis and decomposition due to the high temperature and long exposure time. Hence, HD-EOs were dominated by hydrocarbons and to a lesser extent stable, low molecular weight, oxygenated components [43, 44]. Ultimately, the HS technique is a relatively state-of-the-art extraction method that is designed to extract volatile constituents with a wide range of boiling points without developing artifacts [45]. Lastly, by skimming the available literature we deduced that the HD EOs showed variability in composition and yield from those reported in the literature. A remark that may be traced back to the environmental factors that the species were acclimatized to, such as seasonal conditions, and geographic sources, in addition to probable genetic discrepancies [3, 4].

Concerning the significance of the extracted EOs in oral health, the antimicrobial activity of the extracted EOs (HD and SF) was tested against four common oral pathogens, namely, S. aureus, E. faecalis, E. coli, and C. albicans. The microbes have been selected based on their in-house availability in addition to their pathogenic history in oral infection. For instance, S. aureus is considered a commensal as well as a human pathogen [46]. It is involved in several infective oral pathologies, including dental implant failure [47]. On the other side, E. faecalis plays an important role in human oral cavity infections such as endodontic infections, periodontitis, and peri-implantitis [15, 16, 48], while E. coli is considered a transitory microbiota in the oral cavity. It has been reported in acute dental abscesses cases, ranging from 0.7% to 15%. [19] (https://​www.​WHO.​Int/​news-room/​fact-sheets/​detail/​e-coli). Ultimately, C. albicans is a commensal organism that lives in the oral cavity without causing any problems, but sometimes if the micro-environment changes it aggressively multiply and invade causing candidiasis in the mouth and throat (also called oral thrush) [20]. In the current study, three available assays were implemented to investigate the antimicrobial activity: the agar diffusion, the broth microdilution, and the biofilm formation. The agar diffusion assay is considered a rapid, simple, and prescreening qualitative method to assess the susceptibility of selected microbial strains and the degree of growth inhibition towards tested antimicrobial agents [49]. Herein, our results from the agar diffusion assay showed that the Gram-positive bacteria S. aureus and E. faecalis are the most susceptible organisms to the HD-EO of both Syzygium species, while C. albicans was sensitive only to S. samarangense HD oil. On the other side, the Gram-negative bacteria, E. coli showed resistant to most of the tested concentrations of both oils. One of the reasons that may explain this preference is the structural difference between Gram-positive and Gram-negative bacteria. Gram-positive bacteria possess a peptidoglycan layer that lies outside the bacterial outer membrane, whereas the outer membrane in Gram-negative bacteria, is composed of a double layer of phospholipids linked with lipopolysaccharides inner membrane [50]. Consequently, hydrophobic macromolecules in EOs become unable to penetrate the double membrane of the Gram-negative bacteria, so it develops instant resistance. Another factor that should be taken into consideration, is that even though the agar diffusion assay assumes that the antimicrobial agents diffuse freely in the solid nutrient medium [28], this assumption in many cases leads to significant deviations from the predicted results due to the variability among the volatile components during diffusion. To infer the activity level of our tested samples, we compare our results with the reported ZOI of HD-EOs from other Syzygium species. For instance, S. aromaticum essential oil (clove oil), is traditionally renowned for treating toothache and a panel of oral infections [7, 8]. In agar diffusion assay, it displayed satisfactory antibacterial activity on S. aureus with measured ZOI of 12 mm at 100 mg/mL [51], while 12–20 mm with E. faecalis [52]. Also, the antibacterial activity of the HD EO from S. cumini leaves on S. aureus showed moderate ZOI of 12 mm at 10 μL/mL [53]. In conclusion, the HD EOs from our investigated species displayed acceptable preliminary antimicrobial effects compared to those reported in literature, though further investigation is needed.

Accordingly, the microdilution assay was implemented to provide higher accuracy and better coverage of the agar diffusion drawbacks [27, 28]. microdilution assay is the gold standard test for antimicrobial activity owed to its reproducibility, effective exposure to the tested antimicrobial samples, and the economy of reagents and tools [27, 28]. In this assay our investigated EOs displayed a promising ability to suppress the growth of all tested microbial strains in a dose-dependent manner. In addition, some microorganisms showed higher sensitivity to the tested EOs than the reference standard drug (Amoxicillin). For instance, the growth of E. coli and C. albicans was highly inhibited by the HD-EOs with MIC values being more potent than Amoxicillin. Concurrently, E. faecalis and S. aureus displayed almost double the MIC of E. coli and C. albicans, while still showing higher susceptibility to the administered HD EOs than the reference standard. Similarly, by comparing our results with those reported in the literature, the HD EO from S. cumini leaves displayed MIC values corresponding to 512 µg/mL and ≥ 1024 µg/mL on E. coli and S. aureus, respectively [54] compared to 3.5–15 μL/mL in our investigated oils. The HD EO of clove exhibited good antibacterial activity against S. aureus with a MIC of 0.625 mg/mL [55]. Hence, from the microdilution assay results, we could infer the promising broad-spectrum antimicrobial activity of the tested EOs which was undetectable by the agar diffusion assay. Secondly, the observed antimicrobial activity of the tested EOs is highly conserved, at least in part, to the chemical constituents in each oil. For instance, the HD EO of S. malaccense is abundant with oxygenated sesquiterpenes such as caryophyllene oxide. Reportedly, caryophyllene oxide possessed insecticidal and broad-spectrum antifungal activities [56, 57]. Thirdly, although some tested EOs showed similarities in their major composition, they possessed dissimilar MIC. The latter may be at least in part due to the variation in the percentage of every single component, the chance of synergism between the major components, or between the major and minor components [58, 59]. In this regard, S. samarangense HD EO that possessed potent MIC on E. coli (MIC = 3.7 μL/mL) constitutes considerable proportions of the terpenoids p-cymene (19.5%) and γ-terpinene (17.84%). Marchese et al. have mentioned that p-cymene alone with a concentration equal to 12 mg/mL completely inhibited the growth of E. coli [60]. In addition, Miladi and his research team have documented the synergistic action of p-cymene and γ-terpinene on the inhibition of drug-resistant bacteria [61]. So, synergism between EO components may increase the permeability of the plasma membrane or enhance their binding to transmembrane proteins, although the exact mechanism of action is often quite difficult to determine [62].

It is acknowledged that the phenotypic features of bacteria grown in biofilms are substantially distinctive from those grown in suspension. Because biofilms are the ordinary habitat for the great majority of oral bacteria, including those contributing to oral diseases. In this context, EOs were noticed in the literature as potent antibiofilm agents acting by diverse mechanisms. For instance, EOs exploit their hydrophobic nature in modifying the permeability of the cytoplasmic membrane with subsequent leakage of intracellular content or deactivation of the bacterial enzymes [63]. Also, EOs could block the quorum-sense system, inhibiting the transcription of flagellar genes, and modulating bacterial motility [64], while another study documented their ability in reducing bacterial adherence to inert surfaces [65]. Das and the research team have reported that EO enhanced the accumulation of reactive oxygen species, increasing oxidative stress, and causing cellular apoptosis [66]. Lastly, Fde et al. showed that EOs inhibit the ergosterol synthesis, a major constituent of the fungal plasma membrane [67]. Accordingly, it was deemed of interest to test the inhibition potential of the extracted EOs on biofilm formation. Interestingly, our results highlighted that the SF-EOs favorably inhibit the biofilm of Gram-positive bacteria rather than Gram-negative strains, while the HD-EOs are more potent to the C. albicans biofilm. Reportedly that oxygenated terpenes such as caryophyllene oxide and globulol, which are present in considerable amounts in the HD- and SF-EO of S. malaccense and S. samarangense, respectively, exert antimicrobial action by destroying the microbial cytoplasmic wall, improving its permeability, and allowing the passage of large protons and ions [68]. Moreover, previous reports have highlighted that squalene, the major component of S. malaccense SF-EO, possessed promising antimicrobial activity and antibiofilm formation against S. aureus and E. coli [69, 70]. Hence, we hypothesized that the wide variety of constituents in each EO is a positive factor that may limit the development of resistance which is a common issue for synthetic drugs [71]. In all, although many essential oils are generally considered safe, further investigations including toxicity studies and dosing in clinical settings are required .

The EOs of S. samarangense and S. malaccense leaves were extracted using three different extraction methods: hydrodistillation (HD), supercritical fluid (SF), and headspace (HS), and their composition was compared qualitatively and quantitatively. The setup of each extraction method greatly impacts the profile of the extracted volatile components from the same species, while environmental and genetic factors may contribute to the variations of EOs between species. The SF method demonstrated significant advantages over the conventional HD method in terms of oil yield. The HD and HS volatile profiles revealed qualitative similarities while differed quantitatively. Caryophyllene oxide, p-cymene, and γ-terpinene were the abundant constituents in the HD EOs. They effectively inhibited the growth of Gram-negative bacteria and biofilm formation by C. albicans. The SF-EO of S. malaccense could be considered an economic source for squalene, while S. samarangense is for globulol (each constitutes > 50% of its oil), and they efficiently suppressed the biofilm formation of Gram-positive bacteria. S. samarangense and S. malaccense EOs are promising broad-spectrum antimicrobial hits. They are promoted for adjuvant formulation with other antimicrobial agents to improve the effectiveness of treating candidiasis, dental caries, and abscesses. However, further investigations are required especially to assess their toxicity and efficacy in clinical settings.