INTRODUCTION:

Acne is an inflammatory condition in the pilosebaceous glands^1,2. This condition often occurs in areas of the body that contain many glands, such as the face, chest, and back^3,4. Acne occurs in around 80% of the teenage population, associated with increased androgen hormones in men and women during puberty⁵. In severe cases, acne has profound psychological and social adverse effects⁶. Inflammation in acne is known to occur due to changes in follicle keratinization that lead to comedones, changes in sebum production due to hormonal influences, follicle colonization by Propionibacterium acnes, and complex inflammatory mechanisms⁷.

Colonization or bacterial infection of the pilosebaceous gland by P. acnes is known to be one of the leading causes of acne^8,9. P. acnes secretes lipases, chemotactic factors, metalloproteases, and porphyrins, all of which can interact to cause damage to keratinocytes and trigger inflammatory conditions¹⁰. Using compounds that can inhibit the growth of P. acnes is a common strategy for treating acne^11,12.

One of the natural ingredients known to have antibacterial activity is tea^13,14. Tea is one of the most abundant plantation commodities in Indonesia¹⁵. There are many kinds of tea, such as green, white, black, and oolong¹⁵. White tea is made from very young leaf buds covered with fine hairs, then dried immediately with minimal heat using sunlight in mountainous areas to prevent oxidation¹⁶. White tea contains more catechins and polyphenols than other teas¹⁷. One of the compounds in white tea with antibacterial activity is epigallocatechin gallate (EGCG)¹⁸. The EGCG affects cellular structures such as peptidoglycan and cell membrane. EGCG also can inhibit cellular enzymes such as ATP synthetase, dihydrofolate reductase, DNA gyrase, and fatty acid synthetase¹⁹. Like tea, pomegranate seed oil (PSO) is a natural ingredient with antibacterial activity²⁰. PSO is a vegetable oil rich in natural compounds with numerous pharmacological effects²¹. PSO contains high amounts of polyunsaturated fatty acids²². The main bioactive component of the PSO is punicic acid²³. The previous study showed that the oil has antibacterial activities against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Listeria monocytogenes²³.

This study developed white tea extract and PSO into emulgel preparations²⁴. Emulgel is a combination of emulsion and gel systems^25,26. Due to an emulsion system, emulgel can deliver hydrophilic and hydrophobic substances, making it suitable as a carrier for multicomponent natural compounds²⁷. Emulgel can increase the emulsion system's stability due to a gelling agent presence in the external phase^28,29. In addition, emulgel can improve the spreadability and adhesion of preparation on the skin^30,31.

This study aims to develop emulgel preparations containing white tea extract and PSO with good physical characteristics and stability. The study also determines the antibacterial activity of extracts and emulgel preparation against the acne-causing bacteria P. acnes.

MATERIALS AND METHODS:

Materials:

White tea was obtained directly from Research Institute for Tea and Cinchona, West Java, Indonesia, pomegranate seed oil (Darjeeling aroma, Indonesia), ethanol (Bratachem, Indonesia), propylene glycol (bratachem, Indonesia), Viscolam Mac 10(Lamberty Spa, Italy), Trypton Soya Agar/TSA (Merck, USA), Trypton Soya Broth/TSB (Merck, USA).

Preparation and phytochemical characterization of white extract:

White tea was obtained directly from Research Institute for Tea and Cinchona, West Java, Indonesia. White tea was extracted using 70% ethanol. The extraction process was carried out at a temperature of 60±2⁰C, using a sonicator bath for 60 minutes (ratio of simplicia : solvent of 1:10).^32,33 The extract was then concentrated using a vacuum rotary evaporator. The white tea extract was screened for secondary metabolites using standard procedures³⁴.

In vitro antibacterial activity of white tea extract against P. acnes:

The antibacterial activity test was carried out quantitatively using the agar diffusion method³⁵. The extract was diluted in dimethyl sulfoxide (DMSO) into several concentrations (0.025, 0.05, 0.1, 0.2, 0.4, and 0.8%). A 20ml tryptone soy agar (temperature 40–50°C) was poured into a petri dish containing 100µL of the P. acnes suspension. The mixture was homogenized and then solidified at room temperature. 50μL of each extract solution was added to the well. The agar media were incubated at 37°C for 18–24 hours.³⁶

Optimization formula of PSO emulgel:

The PSO emulgel was prepared using stearic acid and triethanolamine (TEA) as emulsifiers and viscolam Mac 10 as gelling agents (table 1)³⁷. Emulgel were prepared by mixing PSO and stearic acid at a temperature 65±5 ⁰C (oil phase). Distilled water and TEA were also heated at the same temperature with oil phase. The oil and water phases were mixed using ultraturrax to form an emulsion at 8000rpm for 15 minutes.Finally, viscolam, propylene glycol, alpha-tocopherol, and phenoxyethanol were added to the emulsion system and mixed using a mechanical stirrer at 500rpm for 15.³⁸

Table 1. Formulation of PSO emulgel

Composition (%)	F1	F2	F3
PSO	10	10	10
Stearic acid	4	6	8
TEA	0.5	0.75	1
Viscolam mac 10	5	5	5
Phenoxy ethanol	0.5	0.5	0.5
Alpha tocopherol	0.03	0.03	0.03
Propylene glycol	15	15	15
Distilled water ad	100	100	100

Formulation of emulgel containing white tea extract and PSO:

The preparation was made using the optimum formula of PSO emulgel. The procedure for emulsion formation was the same as the previous step. The extract at concentrations 1 and 2% was dissolved in propylene glycol and then added to the emulsion system.³⁹

Physical and stability evaluation of emulgel:

Emulgel preparations were evaluated by organoleptic, pH, viscosity, and spreadability testings. The pH test was carried out using a calibrated pH meter (Mettler Toledo, Seven CompactTM S220) on the preparation without dilution⁴⁰. Viscosity and rheological behavior were determined using a Brookfield viscometer (DV-1 Prime)⁴¹. The spreadability was carried out by placing 0.5grams of the preparation on a glass, and another glass was placed on it. The additional load of 150 grams was placed, allowed to stand for one minute, and measured the diameter of the emulgel⁴². The stability test was carried out in two stages, the centrifugation and the freeze-thaw tests. Centrifugation test was conducted at 3750 rpm for 5hours⁴³. The freeze-thaw test was carried out by placing the emulgel at 4^oC and 40^oC with a minimum storage time of 24 hours at each temperature for six cycles.⁴⁴

In vitro antibacterial activity of emulgel:

The antibacterial activity tests for emulgel were carried out using the same procedure as for white tea extract by the agar diffusion method (35). The samples were diluted 1:2 using distilled water. A 20ml tryptone soy agar (temperature 40–50°C) was poured into a petri dish containing 100µL of the P. acnes suspension. The mixture was homogenized and then solidified at room temperature. 50μL of each diluted sample was added to the well. The agar media were incubated at 37°C for 18–24hours. Antibacterial activity can be seen from the inhibitory diameter value obtained by each sample. Statistical tests were carried out using ANOVA and further Tukey HSD tests on the inhibitory diameter values of the three samples.⁴⁵

RESULT:

Preparation and phytochemical characterization of white extract:

The extraction process of white tea produced a concentrated extract with a yield of 23.70%. The results of the phytochemical screening are shown in Table 2.

Table 2. Phytochemical screening result of white tea extract:

Secondary metabolites	Results
Alkaloids	+
Polyphenol	+
Flavonoid	+
Saponins	-
Tannins	+
Anthraquinones	+
Monoterpenes & sesquiterpenes	+
Triterpenoids & steroids	+

(+) detected (-) undetected

In vitro antibacterial activity of white tea extract against P. acnes:

The result of antibacterial activity of white tea extract was presented in table 3. The minimum inhibitory concentration of the white extract is 0.05%.

Tabel 3. The antibacaterial activity of white tea extract (n=3)

Concentration (%)	Inhibition zone (mm)
0.025	-
0.050	12.87 ± 0.02
0.100	15.63 ± 0.06
0.200	16.67 ± 0.08
0.400	20.07 ± 0.04
0.800	20.73 ± 0.11

Optimization formula of PSO emulgel:

The three PSO emulgel preparations were evaluated and the results are shown in table 4.

Table 4. Physical evaluation of PSO emulgel (n=3)

Parameter	F1	F2	F3
Organoleptic	White, semisolid, oderless	White, semisolid, oderless	White, semisolid, oderless
pH	6.72±0.06	7.09 ±0.02	7.29±0.01
Viscosity (cps)	20336±936	55910±7438	79900±141
Cetrifugation test	unstable	stable	stable

Formulation of emulgel containing white tea extract and pomegranate seed oil:

Formula F2 was used as basic formula of PSO emulgel due to its good physical characteristic and stability. White tea extract at concentration 1 and 2% were added to PSO emulgel system (table 5).

Table 5. Formulation of white tea emulgel

Composition (%)	F2A	F2B
White tea extract	1	2
PSO	10	10
Stearic acid	6	6
TEA	0.75	0.75
Viscolam mac 10	5	5
Phenoxy ethanol	0.5	0.5
Alpha tocopherol	0.03	0.03
Propylene glycol	15	15
Distilled water ad	100	100

The physical evaluation results of white tea extract emulgel are shown in Table 6.

Table 6. Physical characteristics of white tea extract emulgel (n=3)

Parameter	F2A	F2B
Organoleptic	Brownies, odorless	Brownies, odorless
pH	6.79±0.02	6.69±0,02
Viscosity (cps)	19786±1970	12575±601
Spreadability (cm)	5.27±0.12	6.02±0.18
Centrifugation test	Stable	Stable
Freeze-thaw test	Stable	Stable

In vitro antibacterial activity of emulgel:

The result of antibacterial activity tests of emulgel to P. acnes, compared with the marketed product (clindamycin gel 1.5%) are showed in table 7.

Table 7. Antibacterial activity of white tea emulgel (n=3)

Sample	Inhibition zones (mm)
F2A	10.40 ± 0.24*
F2B	12.80 ± 0.20
Clindamycin gel 1.5% (marketed product)	13.86 ± 1.55

*statistically different with marketed product (P<0.05)

DISCUSSION:

This study used white tea ethanol extract as the active ingredient. White tea is known to contain high levels of polyphenolic compounds. Previous studies have extracted white tea using the same solvent, 70% ethanol. Ethanol at a concentration of 70% could optimally attract polyphenolic compounds in white tea by 35.73% ³⁰. In this study, we used the ultrasonic extraction method. The ultrasonication can help accelerate the destruction of plant cells and the release of secondary metabolites, thereby increasing the effectiveness of the extraction process.. The ultrasonic extraction process can provide good extraction results in a short time⁴⁶. The results of the phytochemical screening showed that white tea extract contained various secondary metabolites, namely alkaloids, flavonoids, polyphenols, tannins, anthraquinones, triterpenoids and steroids, as well as monoterpenes and sesquiterpenes. The results showed no saponins in the white tea ethanol extract. That result aligns with previous studies that tested tea extracts with ethanol solvents⁴⁷.

The antibacterial activity test showed that white tea extract has good antibacterial activity against P. acnes with a minimum inhibitory concentration (MIC) value of 0.05%. Previous studies have determined the antibacterial activity of white tea ethanol extract against P. acnes. The results show that the white tea extract has antibacterial activity starting at a concentration of 1%⁴⁸. Tea leaves have long been known to be antibacterial against gram-positive and gram-negative bacteria. Tea leaves contain high amounts of catechins (25-35% in dry weight). They have two benzene rings A and B, which greatly damage the bacterial cell membrane⁴⁹. There are four main types of catechin, namely epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin (EGC), and epigallocatechin3-gallic (EGCG), with ECGC as the highest constituent⁵⁰. The EGCG is known to have intense antibacterial activity. The EGCG acts by changing protein surface polarity, modifying protein enzymes followed by agregation, and inhibiting bacterial growth⁵¹.

White tea extract was developed into an emulgel preparation using PSO as the oil phase. PSO is known to have bacterial growth inhibition activity, including against P. acnes, with a MIC value of 12.5mg/mL⁵². Stearic acid and TEA are used as emulsifiers in the emulgel formulation. Stearic acid, a fatty acid, will react with TEA as a weak base in a saponification reaction to produce surfactant (TEA-stearate) that can stabilize the emulsion system³⁷. Viscolam mac 10 is used as a gelling agent, which is able to produce good gel characteristics, and alsoact as an emollient when used on the skin⁵³.

The test results in Table 4 show that the formulas F2 and F3 have good physical characteristics and stability, referring to the pH, viscosity, and spreadability values, and there are no creaming and phase separation in the centrifugation and freeze-thaw tests. However, the F2 formula was finally chosen due to its moderate viscosity affecting optimal spreadability. Furthermore, 1% and 2% white tea extract was added to the PSO emulgel. The concentrations were increased from the MIC value to optimize its activity. Evaluation of the white tea emulgel included organoleptic, pH, viscosity, spreadability, and physical stability tests. The result in Table 6 shows that white tea emulgels have good characteristics and stability. The pH value of the preparation is appropriate to the skin's pH, which is in the range of 4-7⁵⁴. The viscosity value of the emulgel follows the requirements of SNI 16-4399-1996, which is between 2000-50,000 cps. Spreadability values are suitable for semisolid preparations, which range 5-7cm⁵⁵.

The antibacterial activity test on emulgel showed an increase in activity in line with the concentration of the extract in the preparation (table 7). The statistical test results showed that the inhibitory zone of 2% emulgel was not statistically different from the inhibitory zone of clindamycin marketed product (P>0.05). Emulgel preparation containing white tea extract at a concentration of 2% has the potential to be developed into an anti-acne herbal emulgel.

CONCLUSION:

The white tea extract has potent antibacterial activity against P. acnes with a MIC value of 0.05%. The extract at concentrations of 1% and 2% have been successfully developed into an emulgel system with good physical characteristics based on organoleptic, pH, viscosity, and spreadability tests. The emulgels were stable base on centrifugation and freeze-thaw tests. Emulgel containing 2% white tea extract has good antibacterial activity with an inhibition zone of 12.80±0.20mm.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are highly thankful LPPMBandung Islamic University for funding this research, with the agreement number: 009/B.04/LPPM/I/2023.

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Received on 19.08.2023 Modified on 28.12.2023

Research J. Pharm. and Tech 2024; 17(9):4472-4476.

DOI: 10.52711/0974-360X.2024.00691