INTRODUCTION:

Indonesia is a tropical country that has a diversity of flora. Therefore, Indonesia has thousands of types of plants that must be conserved and appropriately utilized. Most of these plants can be used as medicine. Medicinal plants are planted in leaves, stems, fruit, flowers, and roots with medicinal properties and are used as raw materials to manufacture modern medicine and traditional medicine. One of the plants that can be used as medicine is the pagoda plant (Clerodendrum paniculatum L.)^1,2. Pagoda plant is commonly used as ornamental plants in the home garden and used as traditional medicine by people in India, China, Korea, Japan, and Indonesia. In addition, this plant is used in therapy, especially for asthma, cataracts, malaria, skin diseases, cancer, typhus, and hypertension^3–6.

Chemical compounds in pagoda leaf plants are flavonoids, terpenoids, tannins, alkaloids, sterols, and glycosides. Flavonoids are polyphenolic chemicals found in plants with antibacterial, antiviral, antihypertensive, antioxidant, antiplatelet, cytotoxic, and anti-inflammatory properties.^5,7–9.

Based on the results of previous research, pagoda leaf extract with different extractions showed antibacterial activity against various gram-negative bacteria, namely Salmonella newport, Escherichia coli, and Vibrio parahaemolyticus. In addition, alcohol extract has good antibacterial activity against Salmonella newport, and water extract has good antibacterial activity against Escherichia coli, and petroleum ether, chloroform, and ethyl acetate extracts show good antibacterial activity against Vibrio parahaemolyticus^9–12.

This article will discuss the antibacterial activity of the water, ethyl acetate, and n-hexane fractions of pagoda leaves in inhibiting the growth of Propionibacterium acnes bacteria. The purpose of this study was to determine the inhibitory activity of pagoda plants against Propionibacterium acnes bacteria and to compare the inhibition diameter between groups of each pagoda leaf fraction.

MATERIAL AND METHODS:

Chemicals and reagents:

The water fraction, ethyl acetate, and n-hexane of pagoda leaves were utilized in this study. As well as MHA (Muller Hinton Agar) (Oxioid), 0.9 percent NaCl (Otsu-NS), H₂SO₄ 0.36 N, BaCl22H2O 1.175 percent, rubbing alcohol, mg powder, antibiotic clindamycin 300 mg (OGB dexa), and Di (Emsure). The bacteria used was Propionibacterium acnes (ATCC 6919) from the Laboratory of Pharmaceutical Microbiology, Faculty of Pharmacy, Univerita Sumatera Utara.

Instrumentation:

The tools used in this research are: analytical scales, stirring rods, porcelain plates, measuring cups (Pyrex), blenders (Miyako), drop pipettes, test tubes (Pyrex), rotary evaporators, 500ml separating funnels (Iwaki), statives, and clamps, heater, condenser, 2000ml round bottom flask (pyrex), 2000ml beaker glass (Pyrex), 250 ml beaker glass (Approx), micro pipette, bunsen, wire loop, matches, spatula, petri dish (Iwaki, Normax, anumbra), calipers, autoclave, oven (Memmert UN110), labels, tissue, sterile gauze (deluxe), flannelette, tweezers, cotton, parchment, water bath, aluminum foil, incubator, disc paper, laminar airflow (Astec HLF 1200 L), volumetric flask, Erlenmeyer (Pyrex), and filter paper (Whatman).

Extraction and Fractionation of Pagoda Leaf:

The harvested pagoda leaves are dried in a drying cabinet (temperature 40^oC) for three days. The dried leaves are then pollinated (called simplicia) and then extracted.

The extraction process used the maceration method with 96% ethanol as a solvent ratio (1: 10). Simplicia is macerated for five days. As much as 800g of simplicia is put into a glass jar then soaked using 6,000ml of 96% ethanol solvent covered with aluminum foil for three days (stirring occasionally), filtered using filter paper, and obtained filtrate one and pulp. The dregs were re-soaked using 2,000ml of 96% ethanol solvent for two days (stirring occasionally), filtered using filter paper, and obtained filtrate two and pulp. Next, combine the filtrate 1 and 2 and concentrate it in a rotary evaporator until a thick extract is obtained^13,14.

The fractionation process carried out refers to the partition method using ethanol, hex, and ethyl acetate as solvents. First, a total of 5g of crude extract was dissolved in 50ml of ethanol solvent. The solution was then partitioned by adding 150ml of n-hexane solvent, stirred/shaken in a separating flask, letting stand for 30-60minutes, and separating the formed layer (bottom ethanol layer, top layer n-hexane layer). The process of adding n-hexane is carried out several times until the layers of n-hexane become clear (indicating that no compounds can be attracted) and the obtained hexane layers are combined as a hexane fraction. Next, the residual ethanol layer from the n-hexane partitioning process was further partitioned with ethyl acetate. A total of 150ml of ethyl acetate solvent was added to the water-methanol layer, shaken in a separating flask, left to stand for 30-60 minutes, and the formed layer was separated (bottom ethanol layer, top layer ethyl acetate layer). Finally, adding ethyl acetate to the ethanol layer is the same as the n-hexane fraction until the ethyl acetate layer becomes apparent. Then, the ethyl acetate layer obtained is combined into one as ethyl acetate fraction. The remaining fraction is called the water fraction. Each fraction obtained was then concentrated using a rotary evaporator at 50°C until the viscous fraction was obtained^15,16.

Antibacterial Activity Study:

Instruments Sterilization:

Non-glass tools are sterilized by using an autoclave for 15 minutes at a temperature of 121°C. Meanwhile, the glassware was sterilized in an oven at 160-170°C for 2 hours^17,18.

Bacterial Media Preparation:

Media MHA (Muller Hinton Agar) weighed as much as 9.5g (38g/1000ml) and put it in the erlenmeyer flask. Next, dissolve 250ml of distilled water and heat it in a water bath covered with cotton. Then sterilized into the autoclave for 15 minutes at a temperature of 121°C. Then cool to a temperature of ±40-45°C, the media is ready to be used for testing the growth and culture of bacteria^17,19.

McFarland Solution Preparation:

99.5ml of H₂SO₄ 0.36 N solution was mixed with 0.5 ml of BaCl₂.2H₂O solution of 0.5ml in erlenmeyer and then shaken until a cloudy solution is formed. This turbidity is used as the standard for the turbidity of the tested bacterial suspension^17,20.

Bacterial Suspension Preparation:

Propionibacterium acnes bacterial suspension was prepared by culturing Propionibacterium acnes taken with a sterile needle, then suspended into a test tube containing 10ml of 0.9% NaCl until turbidity was obtained standard turbidity of the McFarland solution ^17,21.

Inhibition Test:

Each fraction was dissolved in a solution of dimethyl sulfoxide (DMSO) and then diluted into several concentrations, namely 1, 3, 5, 10, 15, and 20%. The inhibition test used the agar diffusion method, namely paper discs. Prepared petri dishes that have been sterilized in the oven. Enter 0.1ml of the tested bacterial suspension into the petri dish. Pour 20ml of homogenized Mueller Hinton Agar (MHA) medium and let stand until it hardens. Then five paper discs were taken using tweezers which were previously heated over a bunsen burner. Pipettes as much as 300µL of each fraction with a predetermined concentration and placed on disc paper that has been determined the concentration is 1%, 3%, 5%, 10%, 15%, and 20%. DMSO Solution as negative control and clindamycin 1% as a positive control for 15 minutes. After that, it is carefully placed on the surface of the medium with tweezers and marks each site of the relevant concentration. Then the media was incubated in an incubator at 37°C for 24 hours. After that, the inhibition zone formed around the disc was measured using a digital caliper and marked by a clear zone around the disc^22–24.

Table 1: Inhibition Diameter of Pagoda Leaf Fractions Antibacterial Activity

Groups	Concentrations	Inhibition Diameter (mm) + Standard Error	Categorize
Negative Control (DMSO)	1%	0.00+0.00	-
Positive Control (Clindamycin)	1%	28.70+0.15*	Very strong
Water Fractions	1%	9.80+0.56*	Moderate
	3%	10.13+0.29*	Strong
	5%	10.40+0.15*	Strong
	10%	12.57+0.29*	Strong
	15%	14.03+0.14*	Strong
	20%	14.20+0.20*	Strong
Ethyl Acetate Fractions	1%	6.67+0.27	Moderate
	3%	7.00+0.80	Moderate
	5%	7.23+1.13	Moderate
	10%	7.86+0.56*	Moderate
	15%	8.73+0.34*	Moderate
	20%	10.13+0.12*	Strong
n-Hexane Fractions	1%	7.10+0.17	Moderate
	3%	7.36+0.06*	Moderate
	5%	7.53+0.14*	Moderate
	10%	10.16+0.38*	Strong
	15%	10.53+0.40*	Strong
	20%	10.56+0.42*	Strong

Explanation: * Significantly different to negative control (p > 0.05)

Data Analysis:

The data obtained is presented in tabular form, which is the result of the inhibition zone measurement. Furthermore, the data obtained from the research results were statistically tested using the ANOVA (Analysis of variance) method.

RESULTS AND DISCUSSIONS:

The study results in the form of inhibitory diameter against the growth of Propionibacterium acnes bacteria showed the activity of each fraction. The water fraction had higher activity than the ethyl acetate and n-hexane fractions based on the inhibition diameter. Inhibitory diameter data for each fraction in various concentrations can be seen in Table 1.

To assess the strength of the inhibition zone, the bacteria were categorized according to Davis and Stout (1971) as very strong (clear zone> 20mm), strong (10-20mm clear zone), moderate (5-10mm clear zone), weak (<5mm)^25–27. Based on these categories, all pagoda leaf fractions had moderate-strong inhibitory values, and clindamycin had solid activity inhibiting the growth of Propionibacterium acnes bacteria.

In this study, the antibacterial activity was tested in inhibiting the growth of the Propionibacterium acne (ATCC 6919) bacteria. The negative control used was Dimethyl sulfoxide (DMSO) solution because DMSO does not provide antibacterial activity against bacteria. Dimethyl sulfoxide (DMSO) is an organosulfur compound that can dissolve both polar and nonpolar compounds and dissolve in various organic and water solvents; besides that, DMSO is not toxic; it will not interfere with observation^28–30.

The positive control used was clindamycin. Clindamycin is bacteriostatic with a mechanism of inhibiting protein synthesis of microorganisms by affecting the 50S ribosome sub-unit in bacteria, thereby interfering with the process of forming bacterial peptidoglycan chains^31,32.

Plant-derived compounds have different polarities due to their different structures and chemical bonds. Polar compounds can be bound by polar and semipolar solvents such as methanol and ethyl acetate, while nonpolar compounds can be bound by solvents such as n-hexane. According to Harborne, phenolic compounds such as flavonoids, tannins, lignin, and melanin have aromatic rings containing one or two hydroxyl groups so that they are polar. In comparison, terpenoid compounds generally have a cyclic structure. They have one or more functional groups (hydroxyl, carbonyl, etc.) that are non-polar and can be dissolved in fat or non-polar compounds n-hexane and chloroform. The non-polar nature of n-hexane so that it contains only a small amount of antibacterial compounds^7,33.

Some of the active antibacterial compounds in the pagoda plant that are thought to have antibacterial inhibition include steroids/triterpenoids and glycosides. The content of glycoside chemical compounds can be antibacterial by penetrating the cell wall, causing damage to the bacterial cell wall. Triterpenoid compounds can cause lysis in bacterial cells by binding to proteins, lipids, and carbohydrates found in the cell membrane. Triterpenoids can inhibit growth and kill microbes by interfering with the formation process of cell membranes or walls^2,22,34.

According to WHO, technical factors such as inoculum concentration, disc insertion time, incubation temperature, plate size, agar media thickness, and spacing of antimicrobial discs potential for antimicrobial discs, and composition all influence the size of the inhibition of the disc diffusion method.^17,35.

ACKNOWLEDGEMENT:

The authors are grateful to the Research and Community Services Center or LPPM, Institut Kesehatan Helvetia.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 17.03.2021 Modified on 10.08.2021

Research J. Pharm. and Tech. 2022; 15(6):2521-2524.

DOI: 10.52711/0974-360X.2022.00421