Acne vulgaris is one of the most common skin disorders and mainly affects adolescents: around 40% of all 14–17-year old girls and 35% of all 16–19-year old boys [1
]. Acne, by definition, is a multifactorial chronic inflammatory disease of pilosebaceous units that affects the skin of the face, neck, and upper trunk. Acne develops when these specialized follicles undergo pathologic alteration that results in the formation of non-inflammatory lesions (comedons) and inflammatory lesions (papules, pustules, and nodules). In general, Staphylococcus epidermidis
and Propionibacterium acnes
are the major skin bacteria that cause this formation of acne. Topical as well as systemic therapies are available for acne treatments. A topical therapy usually includes comedolytic agents, antibiotics, and various anti-inflammatory drugs, whereas systemic therapy covers antibiotics, zinc, and hormones for acne treatments. However, an excessive use of antibiotics over a long period can lead to the increasing resistance of acne bacteria. To overcome antibiotic resistance, essential oils and medicinal plant extracts present alternative solutions that are safer, more efficacious, and multifunctional. In topical acne treatments, medicinal plant extracts have fewer side effects than synthetic agents [2
In some research reports, cinnamon has shown potential activity against acne bacteria. Cinnamaldehyde, a major constituent of cinnamon, shows anti-inflammatory activity. It inhibits the production of nitric oxide, which is responsible for inflammatory conditions in the human body. Moreover, cinnamon has also been shown to prevent the production of COX-2, a pro-inflammatory agent. Therefore, cinnamon has antibacterial and anti-inflammatory properties. The chemistry of cinnamon bark oil primary contains a cinnamaldehyde that is different from that found in cinnamon leaf oil (eugenol) and root-bark oil (camphor) [3
Similar to cinnamon’s biological activity, honey works as a natural antibiotic, killing bacteria that cause acne. The anti-inflammatory properties of honey reduce the redness of acne. Its acidic property does not allow the bacteria to grow [4
]. Honey releases hydrogen peroxide, which is an antibiotic that can also remove bacteria and clear acne [5
]. Moreover, honey contains natural antioxidants, which can scavenge free radicals [6
This paper investigates the antibacterial activity of an ethanolic extract of cinnamon bark, honey, and their combination against acne-causing bacteria. The antibacterial activity of cinnamon bark extract and honey were investigated against P. acnes and S. epidermidis using disc diffusion. Minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were attained using Clinical and Laboratory Standard Institute (CLSI) methods.
3. Results and Discussions
Cinnamon bark was bought from an herbal medicine store in Bandung, Indonesia. Firstly, the bark was selected to ensure that the bark used was not damaged and was free from foreign matter. Then, the selected bark was washed about three times using clean and fast flowing tap water to remove soil and other contaminants. The bark was then cut into small pieces to increase the surface area and to minimize the drying process period. After that, the drying process was conducted in a drying cabinet at 40–42 °C for 3–4 days. The drying process was essential to reduce the water content present in the crude drug, in which water can promote the growth of microorganisms such as yeast and molds. After the drying process was completed, the crude drug was ground into powder. The purpose was to minimize the crude drug particle size, which can increase the surface of the contact area, which makes the extraction process easier. Then, the crude drug was stored in a closed container and protected from light. The microscopic characterization of Cinnamomum
sp. bark powder is shown in Figure 1
The powdered crude drug was extracted with the reflux method. About 300 g of crude drug was dissolved in 1 L of 96% ethanol as the solvent system. The extraction process was repeated three times to obtain an optimum yield. The solvent was then removed using a rotary evaporator to obtain the concentrated extract. Then, characterization of the extract and crude drug was conducted by determining water content, density, total ash, water- and ethanol-soluble extractable matter, and phytochemical screening. Characterization extract and crude drug are important for ensuring the consistency of the plant extract in terms of quality, safety, and efficacy. The result of the characterization of crude drug and extract are shown in Table 1
and Table 2
Based on phytochemical screening (Table 2
), the crude drug and ethanolic extract of cinnamon bark contained alkaloid, flavonoid, steroid/triterpenoid, tannin, and quinone, and there was an absence of saponin. Results were similar to a previous study showing that Cinnamomum cassia
bark extract contained phenols, alkaloids, steroids, and tannins, while saponins and glycosides were not detected in the test extract [12
Furthermore, there are several methods for determining the characteristics of plant extracts. Firstly, the water content was determined by azeotropic distillation. This method was selected to determine the water content of the cinnamon bark crude drug because it contains a volatile compound that, can show inaccurate results when determining water by a gravimetric method. Based on the evaluation, the water content of cinnamon bark crude drug powder was 9.0% (v/w), and the water content of ethanolic cinnamon bark extract was 7.0% (v/w).
An amount of 1 kg of cinnamon bark after grinding was 950 g, so the percentage of the yield was 95% (w/w). Three hundred grams of cinnamon bark powder was extracted by the reflux method and concentrated using a rotary evaporator. The final weight of the extract was 47.67 g, so the yield percentage of the extract was 15.93% (w/w).
The quality test of honey for parameters such as HMF, diastase enzyme activity, moisture content, level of sucrose, glucose level, ash content, water-insoluble solids, and metal contamination including lead and copper fulfilled the requirements based on Standard National Agency of Indonesia.
In this experiment, two tested bacteria strains were chosen: Propionibacterium acnes and Staphylococcus epidermidis, obtained from the Microbiology Laboratory, School of Pharmacy-Bandung Institute of Technology. The bacterium cultures were streaked on a solidified slant nutrient agar and incubated for 24 h at 37 °C. After 24 h of growth, the bacteria were inoculated in nutrient broth (NB) and incubated again for 24 h. The suspension of bacteria was prepared at absorbance ranges between 0.08 and 0.13 using a UV-Vis spectrophotometer at a wavelength of 625 nm. Based on the determination of total CFU, a serial dilution procedure for each bacterium to 107 and 108 was performed afterward. The result for P. acnes at an absorbance of 0.093 was 154 × 107 and 146 × 108, whereas that for S. epidermidis at an absorbance of 0.083 was 271 × 107 and 177 × 108, and both bacteria strains fulfilled the CFU normal range of 30–300.
The antibacterial screening was performed by disc diffusion to determine the inhibition zone of the cinnamon bark extract, honey, tetracycline HCl, and the solvent of 96% ethanol against P. acnes
and S. epidermidis
. The result of the antibacterial screening of cinnamon bark extract, honey, and tetracycline HCl 250 µg/mL is presented in Table 3
. The highest activity of cinnamon bark extract was 17.2 mm of the inhibition zone diameter found against P. acnes
, followed by 16.8 mm against S. epidermidis
, whereas the control disc used for the solvent (96% ethanol) had no zone of inhibition. Furthermore, the activity of honey was 16.2 mm of the inhibition zone diameter observed against P. acnes
, while S. epidermidis
was 16.7 mm. The reference standard tetracycline at 250 µg/mL had an inhibition zone diameter against P. acnes
of 18.9 mm; against S. epidermidis
, 24.8 mm. Based on the inhibition zone diameter of cinnamon bark extract and honey against bacteria more than 16 mm, it can be concluded that cinnamon bark extract and honey can be categorized as potent against both bacteria.
Cinnamon bark extract was shown to have a better inhibitory effect against P. acnes with a MIC of 256 µg/mL, followed by S. epidermidis with a MIC of 1024 µg/mL. Meanwhile, the honey was shown to have the same MIC against both P. acnes and S. epidermidis, which was 50% (v/v).
The MBC was obtained after determining the MICs. The MBC is the least amount of drug required to kill 99.9% of bacteria. The MBC of the sample test was determined by inoculating the sample from the well that showed no apparent bacterial growth. The samples were streaked with an ose needle to the MHA plate. The wells that contained cinnamon bark extract showed growth at the agar plate until the concentration of extract was 2048 µg/mL. Therefore, the MBC could not be obtained. It is assumed that the MBC might be greater than 2048 µg/mL (see Table 4
Antimicrobial combinations were then tested, and a better interaction between the extract of cinnamon and honey against those bacteria was expected. When two drugs are given simultaneously, interactions in the body that cause pharmacological effect, be they synergistic, addictive, or antagonist effect, can occur. The combination of extract and honey was evaluated from the FICI values for each combination using the microdilution checkerboard method.
The concentration of the individual compounds in combination between the extract and honey, which inhibit bacterial growth, was recorded as the MIC of the individual compounds in the respective combination. The combination of cinnamon bark extract and honey against P. acnes
and S. epidermidis
showed an additive effect with an FICI value of 0.625 (see Table 5
). This means that the combination effect of cinnamon bark extract and honey was equal to the sum of the effect of each agent alone. The combination of the ethanolic extract of cinnamon bark and honey had potential activity against acne-causing bacteria. In addition, since both agents have a good flavor and texture, there is a promising prospect to develop them as topical treatments against acne-causing bacteria.