INTRODUCTION:

Acne is a chronic inflammation of the pilosebaceous glands characterized by blackheads, pustules, and nodules. Acne can be caused by several factors, including genetics, dietary, psychological, the sebaceous glands' activity, and bacterial infections¹^,2. One of the main bacteria causing acne infection is Propionibacterium acnes ³. Propionibacterium acnes is a normal flora of the pilosebaceous glands of human skin, and these bacteria cause acne by producing lipases that break down free fatty acids from skin lipid and stimulate tissue inflammation. Propionibacterium acnes also induces inflammatory mediators such as interleukin 1α (IL-1α) and tumor necrosis factor-α.^4,5

One of the plants that have antibacterial properties is cinnamon (Cinnamomum burmannii Nees ex BI). Cinnamomum burmannii or Indonesian cinnamon is one of the four types of cinnamon categorized as high economic value besides Cinnamomum verum, Cinnamomum cassia and Cinnamomum loureiroi^6,7. Cinnamon contains essential oils, which are generally obtained from the bark. The main active compound of cinnamon bark oil is cinnamaldehyde which is known to have antibacterial activity⁸. The mechanism of cinnamaldehyde's actions as an antibacterial agent is inhibiting glucose uptake and effects on bacterial membrane permeability⁹.

Currently, many drug delivery systems are being developed for the treatment of acne^10,11. The novel carrier systems for acne application and treatment include liposome, niosome, microsponge, microemulsion, nanoemulsion, microsphere, and solid lipid nanoparticle^12,13,14. In this research cinnamon bark oil as an active agent was developed to nanogel dosage form. Nanogel can be formed by adding a gelling agent to a nanoemulsion system^15,16. Nanoemulsions are stable thermodynamically systems, either oil-in-water (O/W) or water-in-oil (W/O) emulsions that are nano-sized, and have droplet diameters around 10-200 nm¹⁷. Nanoemulsion is a dispersion system, which is two liquids that are not miscible and combined with the help of an emulsifying agent such as surfactants and cosurfactants¹⁸. The advantages of using nanoemulsion for acne treatment are that it is an appropriate carrier for transporting lipophilic compounds into the skin and is considered ideal for acne⁷. Nanoemulsion increases the active component's penetration inside the pilosebaceous unit since it has very small-sized droplets. Surfactant and co-surfactant in nanoemulsion formulation can act as penetration and occlusivity enhancer that improves skin penetration.^19,20 Gelling agents can be added to the nanoemulsion system to form a nanoemulsion gel (nanogel). The addition of a gelling agent can increase the viscosity and adhesion of the nanoemulsion system, thus extending the contact time, in topical application.

The use of cinnamon bark oil may present an adverse risk for topical application. The components of cinnamon bark oil have been demonstrated to cause hypersensitivity in some patients, which can show as dermatitis²¹. In this study, cinnamon bark oil was used at low concentrations and was used in combination with olive oil to reduce the risk of skin irritation. Olive oil is known to have antibacterial activity and also contains active compounds that can help reduce the risk of skin irritation²². Studies have shown that topical application of olive oil gives an anti-inflammation effect, reducing oxidative damage, and promoting dermal reconstruction^23,24.

This study aimed to develop nanogel preparations containing cinnamon bark oil and olive oil and characterize the gels' physical properties and antibacterial activity. An antibacterial activity test will be carried out in vitro against oil and nanogel preparation.

MATERIAL AND METHODS:

Chemicals and reagents:

Cinnamon bark oil (Pavettia essential oil, Indonesia), olive oil (Lansida Group, Indonesia), tween 80 (Bratachem, Indonesia), polyethylene glycol 400 (Bratachem, Indonesia), viscolam mac 10 (Lamberti Spa, Italy), Triethanolamine/TEA (Bratachem, Indonesia), Trypton Soya Agar /TSA (Merck, USA), Trypton Soya Broth/TSB (Merck, USA).

Instrumentation:

Magnetic stirrer (Thermolyne S131120-33Q), viscometer (Brookfield RV), incubator (Memmert), centrifuge (Shanghai instrument factory), pH meter (Mettler toledo, pH electrode in lab expert pro), oven (Memmert), autoclave (Tommy), spectrophotometer UV-Vis (Shimadzu-Uv mini-1240), gas chromatography-mass spectrophotometry/GC-MS (Shimadzu), particle size analyzer/PSA (Beckman coulter LS 13320).

Determination of active compounds of the cinnamon bark oil:

Determination of major active compounds of cinnamon bark oil was carried out using Gas Chromatography-Mass Spectrophotometry/GC-MS²⁵.

Formulation of nanogel containing cinnamon bark oil and olive oil:

The nanogel formulation was developed based on the results of our previous study. Tween 80 was used as a surfactant, PEG 400 as a cosurfactant, and viscolam mac 10 as a gelling agent. Various formulas were made with the ratio of oil and surfactant + co-surfactant (Smix) 1:7 and 1:8 and the surfactant and co-surfactant 1:1, 2:1, and 3: 2 (table 1). Physical appearance and percent transmittance tests were used as an initial screening.

Table 1. Optimization formula of nanogels

Ingredients (%)	Formula
Ingredients (%)	F1	F2	F3	F4	F5	F6
Cinnamon bark oil	4	4	4	4	4	4
Olive oil	2	2	2	2	2	2
Tween 80	21	28	25.2	24	32	28.8
PEG 400	21	14	16.8	24	16	19.2
Viscolam gel 8%	20	20	20	20	20	20
TEA	qs	qs	qs	qs	qs	qs
Distilled water ad	100	100	100	100	100	100

Comparation of Oil : Smix à F1, F2, F3 (1:7); F4, F5, F6 (1:8)

Comparation of S : CoS à F1, F4 (1:1); F2, F5 (2:1); F3, F6 (3:2)

The gel was made by dissolving viscolam into a certain amount of water then neutralized by adding TEA until the pH reaches a range of 6-7. Nanogels were prepared by mixing the oil phase, cinnamon bark oil, olive oil, tween 80, and PEG 400, then heated at 30-40°C. The water phase was made by mixing distilled water and viscolam gel, then heating at 30-40°C. Furthermore, the water phase is gradually put into the oil phase and stirred using a magnetic stirrer at 300 rpm for 10 minutes^26,27. The optimum formula will be used as a final formula for nanogel with various concentrations of the cinnamon bark oil.

Physical evaluation of nanogel containing cinnamon bark and olive oil:

Nanogel preparations were characterized for organoleptic, pH, viscosity, spreadability, percent transmittance, and globule size diameter. The stability of the preparations was assessed by centrifugation, heating-cooling, and freeze-thaw tests.

pH measurement was carried out using a calibrated pH meter without dilution. Viscosity measurement was carried out using a Brookfield viscometer at 50 rpm. The spreadability of preparations was measured by applying 500 grams of load to 0.5 grams gel under the glass. Percent transmittance was measured with a spectrophotometer UV/Vis at a wavelength of 650 nm. The particle size distribution was carried out on the preparations without dilution using a particle size analyzer.²⁸

At the first step of the stability study, the preparations were centrifuged at 3,500 rpm for 30 minutes. The preparations that did not show phase separation were subject to the heating-cooling test. At the heating-cooling stage, the preparations were stored at two different temperatures (45°C and 4°C). The tests were carried out in three cycles with storage at each temperature for not less than 48 hours. The last stage is a freeze-thaw test. The preparations were stored at two different temperatures, -21°C and 25°C, conducted for three cycles with storage at each temperature for not less than 48 hours.²⁹

Antibacterial activity test of cinnamon bark oil and nanogels preparation

The antibacterial activity test of cinnamon bark oil was carried out by the diffusion method using the perforation technique. A 50 μL of Propionibacterium acnes suspension (in TSB) was put into sterile Petri dishes, and then 20 mL of sterile TSA was added (temperature 45–50°C). The mixture is homogenized and allowed to solidify at room temperature. The solidified culture media were perforated. 50 μL of diluted cinnamon oil were added to the holes at concentrations of 0.1; 0.2; 0.5, and 1 % (b/v). The test media were then incubated at 37°C for 18–24 hours and then measured the inhibition zone's diameter formed³⁰. The same procedure was carried out for the nanogels. The tests were conducted to the nanogel without dilution. Marketed clindamycin gel was used as a reference.

RESULTS AND DISCUSSIONS:

Determination of active compounds of the cinnamon bark oil:

The GC-MS results showed that cinnamon bark oil contains several active compounds. The primary active compound of cinnamon bark oil (Cinnamomum burmannii) is cinnamaldehyde at a concentration of 62.9%. Cinnamon bark oil is standardized according to its cinnamaldehyde content. The minimal cinnamaldehyde content is 50% for the National Standard of Indonesia (BSN/SNI. 06-3734-2006, 2005) and 55-78% for the USA Essential Oil Association (EOA)³¹. These results showed that the cinnamon bark oil used in the study appropriate with the national and international standards. Cinnamon bark oil also contains other active compounds besides cinnamaldehyde that can support antibacterial activity, such as alpha-pinene (3.43%), caryophyllene (1,48%), cineole (2,26%), camphene (1,93%), and limonene (1,82%)³²

Antibacterial activity of cinnamon bark oil against Propionibacterium acnes:

The antibacterial activity of cinnamon bark oil against Propionibacterium acnes is shown in table 2. Based on that results, the minimum inhibitory concentration (MIC) value of the oil is 0.2%. These results indicated that cinnamon bark oil has strong antibacterial activity against Propionibacterium acnes. Another study showed that cinnamon bark oil has antibacterial activity against Propionibacterium acnes with a MIC value of 0.25%³³. That result relatively similar to this study. Cinnamaldehyde provides the most significant role in the antibacterial activity possessed by cinnamon bark oil. Mechanism of cinnamaldehyde's actions as an antibacterial agent are by inhibition of glucose uptake, interfere with bacterial membrane permeability, the decline in intracellular ATP concentration, and stimulate a leakage of intracellular constituents, possibly protein or nucleic acids.³⁴

Table 2. Antibacterial activity of cinnamon bark oil

Sample	Inhibition Zone (mm)
Cinnamon oil 0.1 %	-
Cinnamon oil 0.2 %	8.43 ±0.02
Cinnamon oil 0.5 %	11.03 ±0.03
Cinnamon oil 1.0 %	13.80 ±0.01

Formulation of nanogel containing cinnamon bark oil and olive oil:

Nanogel preparations were made using Tween 80 as a surfactant and PEG 400 as a cosurfactant. The excipient selection was based on our previous studies. Cinnamon bark oil showed good miscibility properties with Tween 80 and PEG 400²⁷. The surfactant functions in the nanoemulsion system are to form the interfacial film around the oil globule and reduce interfacial tension. The functions of cosurfactant in the nanoemulsion system are to create a tighter and more flexible interfacial layer and reduce the interfacial tension to almost zero to form a stable system³⁵. The results of the optimization formula of nanogel are showed at table 3.

Table 3. The results of optimization formula of nanogel

Formula	Physical appearance	% transmittan
F1	Cloudy	59.9
F2	Cloudy	85.3
F3	Cloudy	77.2
F4	Clear	96.3
F5	Clear	97.8
F6	Clear	98.4

The optimum formula of nanogel is F6. Formula F6 at the ratio oil: Smix (1:8) and S: Co-S (3:2) has best the percent transmittance value that indicates the formation of nano globule. The next step is to develop nanogel preparations with various concentrations of cinnamon bark oil to determine their effect on the antibacterial activity.

Cinnamon bark oil concentration was varied at 2-4%. The determination of oil concentration is based on MIC value and our previous studies' results. The study showed cinnamon bark oil at a concentration of 5% on the formulation causes skin irritation slightly¹⁷. Another study showed that an oil concentration of 1% was not given the optimum antibacterial activity. The final formulas are F6A (cinnamon bark oil 2%), F6B (cinnamon bark oil 3%), and F6C (cinnamon bark oil 4 %).

Physical evaluation of nanogel containing cinnamon bark oil and olive oil:

The objective of this step was to determine the physical characteristics of the nanogel preparations. Several evaluation types were carried out, including organoleptic, pH, viscosity, spreadability, percent transmittance, globule size diameter, and stability tests. The results are shown in Table 4.

Based on the results, it can be concluded that the nanogel preparations containing cinnamon bark oil and olive oil have good physical characteristics and stability (figure 1). The difference in cinnamon bark oil concentration did not affect the physical properties of the nanogels. The preparations have a clear physical appearance, homogeny, with percent transmittance ~100%, a globule size diameter <200 nm with narrow size distribution (figure 2). These characteristics are appropriate for nanoemulsion systems. The pH, spreadability, and viscosity of the cinnamon bark nanogels were proper for topical application. The nanogels were kinetically stable based on centrifugation tests and also thermodynamically stable base on heating-cooling and freeze-thaw tests. Phase separation, creaming, and sedimentation did not occur after three stages of stability tests ³⁶.

Antibacterial activity of nanogel preparations:

The antibacterial activity test was also carried out on the nanogels to confirm their activity. The results showed that nanogels containing cinnamon bark oil and olive oil have antibacterial activity against Propionibacterium acnes (table 5). Increasing cinnamon oil concentration can increase the antibacterial activity of the preparation. The antibacterial activity of the nanogel, better than marketed clindamycin gel. The high viscosity of the clindamycin gel could affect the antibacterial activity using the diffusion method.

Fig 1. Nanogel preparations

Fig 2. Globule size determination of nanogel

Table 5. Antibacterial activity of nanogels

Sample	Inhibition zone (mm)
F6A (Cinnamon oil 2%)	13.97 ± 1.23
F6B (Cinnamon oil 3%)	18.73 ± 1.61
F6C (Cinnamon oil 4%)	20.93 ± 1.10
Clindamycin gel (1.5%)	11.73 ± 1.5

CONCLUSIONS:

Based on this study, it can be concluded that cinnamon bark oil has antibacterial activity against Propionibacterium acnes with a MIC value of 0.2%. Nanogel preparations containing cinnamon bark oil (2; 3; and 4%) and olive oil (2%) have good physical characteristics and stability with transparent appearance and have an average globule size 129±17 nm; 132±18 nm; and 135±19 nm, respectively. Nanogel preparations have strong antibacterial activity against Propionibacterium acnes, and the activity depends on cinnamon bark oil concentration.

ACKNOWLEDGEMENT:

The authors are highly thankful to the Research Center of Bandung Islamic University (UNISBA) for the financial support.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 15.12.2020 Modified on 08.02.2021

Research J. Pharm. and Tech 2022; 15(1):143-147.

DOI: 10.52711/0974-360X.2022.00023

Parameter	F6A	F6B	F6C
Organoleptic	The yellowish, transparent, cinnamon smell	The yellowish, transparent, cinnamon smell	The yellowish, transparent, cinnamon smell
Homogeneity	Homogeny	Homogeny	Homogeny
pH	6.80 ± 0.16	6.81 ± 0.05	6.86 ± 0.01
Spreadability (cm)	7.30 ± 0.57	7.30 ± 1.04	7.00 ± 0.5
Viscosity (cps)	660.67 ± 20.42	701.00 ± 9.54	692.67 ± 10.26
% transmittance	99.70 ± 0.46	99.07 ± 0.76	97.76 ± 0.81
Globule size diameter (nm)	129 ± 17	132 ± 18	135 ± 19
Centrifugation test	stable	stable	stable
Heating cooling test	stable	stable	stable
Freeze-thaw test	stable	stable	stable