Antibacterial Effect of Cinnamon and Citronella Oils Combination Against Acne-Related Bacteria

Acne vulgaris is a dermatological disease whose pathogenesis is due to high sebum secretion, hyperkeratinization, hormonal changes, or bacterial infections. Staphylococcus epidermidis and Staphylococcus aureus are bacteria that can induce inflammation in acne. Cinnamomum burmannii and Cymbopogon nardus essential oils have been reported to have antibacterial activity against S. epidermidis and S. aureus . This study aimed to obtain the type of interaction of a combination of C. burmannii and C. nardus oils in inhibiting bacteria associated with acne. Essential oil components were identified using gas chromatography-mass spectrometry (GCMS). Optimize the combination of C. burmannii and C. nardus oils using the checkerboard method. Furthermore, the Fractional Inhibitory Concentration Index (FICI) value is calculated to determine the effect of a combination that is synergistic, additive, not different or antagonistic. The main components of C. burmannii oil identified are cinnamaldehyde, eucalyptol, cinnamyl aceta te, α - limonene, and α -terpineol. While C. nardus oil contains the five largest components: geraniol, citronellal, citronellol, citral, and geranyl acetate. Cinnamomum burmannii oil yielded 0.28%, with a refractive index of 1.5237. Meanwhile, the yield of C. nardus oil was 0.26%, with a refractive index of 1.4667. The combination of both oils yielded a FICI value of 1.5. The conclusion of this study shows that the combination of the two essential oils produces an indifferent effect against both S. epidermidis and S. aureus .


INTRODUCTION
Recently, the development of skin care products using essential oils has gained popularity. Developing these compounds for use in skin care products is desirable to keep the skin young, healthy, and fresh and protect itself from environmental pollution.Some of their pharmacological activities, such as antibacterial, antioxidant, and anti-inflammation, can be utilized this way 1 .The stratum corneum is protected by the lipophilic qualities of essential oils, which have distinct positive values 2 .These features can also help maintain the skin microbiota, which is necessary for skin health.Topical application of essential oils, including possible incorporation into anti-acne products, can influence the normal function of skin cells 3 .They could also add antibacterial, anti-inflammatory, and antioxidant characteristics to formulas that could help skin prone to acne.One of the most often used applications of essential oils in skin care is to limit acne outbreaks by using essential oil activities to inhibit the growth of bacteria-related pimples 4 .One of the species of bacteria implicated in the etiology of acne is Staphylococcus sp. 5 .Citronella (Cymbopogon nardus) and cinnamon (Cinnamomum burmannii) oils are extensively researched for their antibacterial, antioxidant, and anti-inflammatory properties.This essential oil is produced by common and reasonably easy plants to grow in Indonesia.The essential oil products are also readily available on the market, making them accessible.
Cinnamomum burmannii oil is isolated from the bark of the genus Cinnamomum, with cinnamaldehyde as its main component (60-90%) 6 .Generally, C. burmannii oil is isolated from C. burmannii in Southeast Asia, including Indonesia.This oil is reported to have greater antibacterial activity against Gram-positive than Gram-negative bacteria.Cinnamomum burmannii oil has a minimum inhibitory concentration (MIC) value of 5-10 mg/mL against Staphylococcus epidermidis and 1 mg/mL against Staphylococcus aureus 6, 7 .The mode of antibacterial action of C. burmannii oil has been reported through changes in membrane permeability and integrity 7 .In addition, C. burmannii oil can release and eradicate existing biofilms 8 .Cinnamomum burmannii oil's antibacterial effect is thought to work by altering the integrity and permeability of membranes.Several protein markers involved in tissue inflammation and inflammation can be inhibited by C. burmannii oil 9 .The cinnamaldehyde may contribute to the acne reduction that skin problems require 10 .Traditionally, people have used C. burmannii to cure skin conditions 11 .Cymbopogon nardus essential oil's ability to fight S. aureus, its potent antioxidant properties, and its capacity to reduce the 5lipoxygenase enzyme activity increase the inflammatory response that leads to the development of acne 12 .According to Mahant et al. 13 , topical use of C. nardus oil demonstrates excellent potential for minimizing the impacts of acne.Cymbopogon nardus oil is isolated from C. nardus, an annual herb that thrives in Indonesia and other Southeast Asia countries 14 .The major constituent in this oil was reported in various percentages.Cymbopogon nardus oil had antibacterial activity, with MIC of C. nardus oil against S. aureus of 2.0 µL/mL and Propionibacterium acnes of 0.005-0.3µL/mL 15 .In addition, C. nardus essential oil has the activity to inhibit methicillin-resistant Staphylococcus aureus in vitro 16 .Cymbopogon nardus oil has lower IC50 antioxidant activity (2 µL/mL) than vitamin C (7.9 µL/mL).Using the 5-lipoxygenase inhibition test, C. nardus oil was found to have anti-inflammatory properties, with an IC50 value of 0.15 µL/mL 15 .According to several studies, combining essential oils improves their antibacterial, antioxidative, and anti-inflammatory benefits 17 .When examined in combination, certain essential oils have been proven to have high medicinal potential in earlier research 18-20 .However, there is still a dearth of information regarding the antibacterial effectiveness of the combined essential oils.This research was intended to investigate the interaction of C. burmannii and C. nardus oils in combination to inhibit S. epidermidis and S. aureus.

Materials
The samples (Figure 1

Isolation and identification of essential oils
Steam and water distillation techniques were used to separate the essential oils.The sample was distillated for 6 to 8 hours using about 2 kg.The essential oil was removed from the aqueous phase using a separatory flask.After that, the oil phase was separated and gathered.When not used, essential oils were kept in a light-protected bottle at 4°C.The yield of the oils obtained was calculated.Organoleptic examination of essential oils included visual observation of the color, odor, and flavor of the oils.An Abbe refractometer (Atago) was used to measure the refractive index of the oils.

Identification of essential oil components using GC-MS
The essential oil components were identified using GC-MS (QP2010S Shimadzu) with a stationary phase in the form of an Rtx-5 column with a length of 30 m, an ID of 0.25 mm, and a film thickness of 0.25 mm.Helium was employed as the carrier gas; the column's temperature was 50°C; injection temperature: 300°C; pressure-based flow control mode: 24.7 kPa; total flow: 90.6 mL/minute; column flow: 0.66 mL/minute; linear velocity: 29.5 cm/second; and split ratio: 129.9.Mass spectra detected by the ionization method: electron impact (70 Ev), time from 5.20 to 60 minutes, starting from m/z 20 and ending at m/z 600.By matching the mass spectra profile of the sample with those from the Wiley Library, the chemical components were identified.

Inoculum preparation
From a 24-hour culture, a bacterial colony was taken and suspended in a 0.9% saline solution.The turbidity was adjusted to 0.5 McFarland at that point.The bacterial suspension was then diluted until its density was 10 6 CFU/mL.

Minimum inhbitory concentration determination
The MIC determination of essential oils was carried out using microbroth dilution 18 .Serial concentrations of the essential oils at 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, and 4 mg/mL were applied.Then, they were added to the bacterial suspension so that the final density was 1-5 x 10 5 CFU/mL.The MIC was measured after an 18 to 24 hours incubation period at 36°C.The MIC determination was based on the smallest concentration that can inhibit bacteria (no turbidity).

Checkerboard method
Using the checkerboard method, C. burmannii and C. nardus oils were tested for their antibacterial effects 21 .Each oil was made in a series of concentrations, combined, and mixed with a bacterial suspension.The cells were then incubated at 37°C for 24 hours.MIC was obtained from the smallest concentration that can inhibit bacteria, as marked by no turbidity.The MIC determination was also confirmed by adding 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).Furthermore, the sum of FIC substances A and B generated the fractional inhibitory concentration index (FICI), as shown in Equation 1. FICI interpretation of the combination of two compounds was based on the following categories: FICI ≤ 0.5 is synergistic; 0.5 < FICI ≤ 1 is additive; 1 < FICI ≤ 4 is indifferent; FICI > 4.0 is an antagonistic effect 22 .

Data analysis
The yield and refractive index data were expressed using the mean and standard deviation.Three replications of the antibacterial data were created.

RESULTS AND DISCUSSION
The color of the C. burmannii oil produced by steam and water distillation was light yellow or pale yellow (Figure 2).The yield produced in this study was 0.28%.This yield is smaller when compared to previous studies, which produced yields ranging from 0.36 to 0.86% 18, 23 .The refractive index of C. burmannii bark obtained is 1.52375, less than from Indonesia's Lombok Island, which is 1.5627 23 .On the other hand, the C. nardus oil was brownish yellow with a yield of 0.26 and a refractive index of 1.4667 (Table I).This index value aligned with the C. nardus oil obtained from Jember, Java Island, Indonesia 24 .Cinnamomum burmannii oil separated by gas chromatography yielded 23 components that be identified by mass spectrometry (Table II).The five main components identified were cinnamaldehyde (24.14%), eucalyptol (14.09%), cinnamic (13.76%), α-limonene (9.73%), and α-terpineol (8.19%).According to earlier research, trans-cinnamaldehyde is abundant (46.31%) in C. burmannii bark oil 23 .Cinnamaldehyde was reported to have antimicrobial effects 9 .Unsaturated carbonyl groups in the cinnamaldehyde molecule have been demonstrated to be crucial for this advantageous effect.It has been previously documented how C. burmannii oil kills bacteria by modifying lipid levels, rupturing cellular membranes, blocking mobility, cell proliferation, transmembrane porin, ATPase, and inhibiting biofilm formation.It also has antiquorum sensing properties  14 , where the main ingredients are citronellol (5%), citronellal (6%), geranyl acetate (10%), cis-citral (14%), trans-citral (23%), and geraniol (36%).In this study, citronellal was not found to be the primary constituent of C. nardus oil, but geraniol was the main compound.Nakahara et al. 14  Staphylococcus epidermidis and S. aureus are significant in the growth and development of acne 29 .To evaluate the effectiveness of natural anti-acne medications, S. epidermidis and S. aureus were chosen as the targets.Essential oils of C. burmannii and C. nardus individually were discovered to be helpful against these troublesome microorganisms.This study investigated the antibacterial effect if these two oils were combined.Cinnamomum burmannii and C. nardus oils have been reported to have high antibacterial activity.In this study, both oils also showed antibacterial activity against S. epidermidis.Table IV shows that the MIC of C. nardus oil alone is 1 mg/mL and 0.06 mg/mL against S. epidermidis and S. aureus, respectively.According to Nuryastuti et al. 8 , C. burmannii oil demonstrated a MIC value of 0.005 mg/mL for S. aureus ATCC 25923 and MIC of 0.5 to 1% for several clinical isolates of S. epidermidis.In our study, C. nardus oil showed a MIC value of 1 mg/mL against S. epidermidis and S. aureus.According to Pontes et al. 12 , C. nardus oil had a MIC value of 0.5 mg/mL against S. aureus 6 .The inhibitory effect of the C. burmannii and C. nardus oils in combination was carried out using the checkerboard method.This method can be carried out using a relatively small amount of oil and a combination of the two oils tested in varied ratios.In addition, this method is quite simple and relatively easy to perform in the laboratory 30 -the activity results of the combination of the two oils data as shown in Table V.The antibacterial activity test with the checkerboard method of each oil was set at eight concentrations: 4, 2, 1, 0.5, 0.25, 0.12, 0.06, and 0.03 mg/mL.As a result of the antibacterial activity of the two oil combinations, it inhibited S. epidermidis with the combined MIC at one concentration ratio: 0.  Combining essential oils with additive or synergistic properties can lower the concentration needed to have the same antibacterial effect as using just one essential oil 31 .According to earlier research, combining C. burmannii and thyme oils had a synergistic action against Bacillus cereus 20 .It may be feasible to prevent the occurrence of antibiotic resistance by combining certain essential oils 32 .The combination of C. burmannii oil and each of the other three antimicrobial agents (gentamicin, chlorhexidine, and triclosan) showed synergistic effects against some clinical isolates of S. epidermidis 8 .The combination of Rosmarinus officinalis, Thymus satureioides, and piperacillin/tazobactam oils provides a strong and reproducible synergistic effect against Salmonella typhi.This combination seems to strengthen the antibacterial activity of essential oils and antibiotics against S. typhi by increasing their potential to damage the integrity of cell membranes 33 .

CONCLUSION
The combination of C. burmannii and C. nardus oils slightly decreased the MIC of the combination against acne-related bacteria compared to the MIC of the individual oils, so the effect was categorized as indifference.

a b Figure 2 .
(a) Cinnamomum burmannii and (b) C. nardus oils obtained with steam and water distillation.

Table I .
Characteristics of C. burmannii and C. nardus oils.
Table II.Identification of C. burmannii oil components using GC-MS.

Table III .
28so reported that the major constituent of the C. nardus oil was geraniol.Citronellal (24.27%) was discovered as the second main compound of C. nardus oil in this study.Wei and Wee 27 discovered a more significant percentage of citronellal (35%), while Nakahara et al.14found a lower amount of 5.8%.The ingredients that constitute C. nardus essential oil, however, were discovered in various compositions in earlier publications.Some factors that can affect the differences in the components and composition of essential oils are physiological and environmental.Physiological factors, such as different harvest seasons, produce different chemical compositions.In comparison, environmental factors are caused by soil variations, nutrients, and geographical differences such as heat, sunlight, rainfall, elevation, and climate28.Identification of C. nardus oil components using GC-MS.
Meanwhile, the combined MIC obtained combined C. nardus and C. burmannii oils, which was lower than each of C. nardus and C. burmannii.Table V displays the FICI values of the combination oil of C. nardus and C. burmannii was 1.5 against S. epidermidis and S. aureus.This value indicated that combining C. nardus and C. burmannii oils produced an indifferent antibacterial effect against both S. epidermidis and S. aureus.The impact of the two oils together results in either no increase or a minor rise in inhibitory activity for the combination.More needs to be understood, however, regarding the processes via which antimicrobial interactions have indifferent effects.Chemicals targeting the same targets in microorganisms and chemical interactions (direct or indirect) between the chemicals are potential explanations.
5 mg/mL for both C. burmannii and C. nardus oils.Whereas the test results for the two oil combinations inhibiting S. aureus produced combined MICs at two concentration ratios: 0.5 and 0.06 mg/mL, and 1 and 0.03 mg/mL for C. nardus and C. burmannii, respectively.

Table IV .
The MIC of C. nardus and C. burmannii againt S. epidermidis and S. aureus.

Table V .
Antibacterial effect of a combination of C. nardus and C. burmannii oils against S. epidermidis and S. aureus using the checkerboard method.