الفهرس 
Potential Role of selected Natural Products against Acne-associated Microbes
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Abstract
Acne vulgaris is a multifactorial inflammatory skin disease with Cutibacterium acnes and Staphylococcus epidermidis playing key roles in pathogenesis. The inclusion of antibiotics in acne treatment has become one of the effective anti-acne treatment options. However, the emergence of antibiotic resistance requires the search for alternative antimicrobials. Essential oils (EOs) have been used in Mediterranean traditional medicine, tea tree oil, clove oil, thyme oil, mentha oil and basil oil and have proved their efficacy as potent, natural, safe and cost-effective agents in the treatment of many diseases including acne. However, EOs antimicrobial mechanisms of action in acne have not been fully explored. Therefore, this study aimed to analyze the use and potential mechanisms of action of selected five EOs commonly used in Mediterranean traditional medicine.Screening the antibacterial activity of the selected EOs using Kirby-Bauer method was performed against C. acnes and S. epidermidis. Determination of the antimicrobial activity of the screened EOs was done using the minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill kinetics, minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC). The compositions of the most effective EOs were determined using gas chromatography-mass spectrometry (GC-MS) and the selected EO was formulated into nanoemulsion. Effect of the most potent EO (thyme EO) on biofilm inhibition in C. acnes and S. epidermidis was further visualized using scanning electron microscopy (SEM). Antimicrobial role of the selected EO was explored through observation of loss of cell membrane integrity of C. acnes and S. epidermidis treated with selected EO, through quantification of leakage of 260 nmabsorbing material (nucleic acids). The mechanism of action of the selected EO on bacteria was also assessed by detecting leakage of intracellular ions (potassium K+, sulphurS2-and phosphorus PO4-). Moreover, cellular alteration and impairment of bacterial cells treated with the selected EO was visualized by transmission electron microscopy (TEM). The anti-inflammatory action of the selected EO formulated asnanoemulsion was assessed in C. acnes-induced inflammatory mouse model. Suppression of inflammatory marker NF-κB after treatment with EO was measured using Enzyme Linked Immunosorbent Assay (ELISA). Reduction in inflammation and bacterial load, morphological, and histopathological changes were assessed.Screening of antibacterial activity showed that thyme, tea tree and clove EOs exhibited the largest inhibition zone among the screened EOs using agar disc diffusion method compared to reference antibiotics (clindamycin and erythromycin). Thyme, tea tree and clove EOs exhibited the strongest antimicrobial activity with MIC against C. acnes of 0.026, 0.053 and 0.137 g/mL, respectively and MIC and against S. epidermidis were 0.053, 0.053 and 0.137 g/mL, respectively. MBC results were the same as MIC values for both bacteria. Moreover, thyme EOshowed both biofilm inhibition and eradication against S. epidermidis with MBIC of 0.053 g/mL and MBEC of 0.107 g/mL. The composition of the three EOs (thyme, tea tree, and clove) was determined using GC-MS.Thymol was the major volatile constituent of thyme EO with total percentage of 75.46%, while 4-terpinenyl acetaterepresented 66.82% of tea tree EO and eugenol represented 78.6% of clove EO. The antibacterial and antibiofilm assaysshowed that thyme EO had the highest antimicrobial activity. Moreover, efficacy of thyme EO bactericidal action against C. acnes and S. epidermidis was detected at 4 MIC concentration within 10h and 6h, respectively. SEM showed that thyme EO effectively inhibited C. acnes and S. epidermidis biofilm formation. Thyme EO affected cell membrane permeability of both bacteria as evident by detection of leakage of intracellular ions (K+, S2- and PO4-). Additionally, the effect of EO on cell membrane integrity was evaluated by detecting leakage of nucleic acids which increased to 21.4-fold and 16-fold after C. acnes and S. epidermidis were incubated, respectively with thyme at its MIC concentration for 60 minutes. The effect of thyme on bacterial membrane damage was confirmed through TEM photographs showing morphological alteration of both bacteria. Thyme, the most effective EO, was formulated as a nanoemulsion and tested in an inflammatory acne animal model. Thyme treated mice showed significantly reduced erythema and edema in mice ears and 5 fold reduction in NF-κB levels compared to untreated controls. In addition, mice treated with thyme nanoemulsion showed significant bacterial load reduction. Histolopathological results showed thyme EO nanoemulsionhealing effects.Collectively, thyme EOinhibited the growth and biofilm of C. acnes and S.epidermidis, its possible mechanism of action is through altering membrane integrity. Formulating thyme EO as nanoemulsion showed potent healing and antimicrobial effects compared to the reference antibiotics suggesting that thyme EO nanoemulsion can be used effectively in acne treatment