INTRODUCTION:

A quarter of the global population is dependent on medicinal plants for treating various diseases¹, including those caused by microorganism infections. Several plants possess antimicrobial potential, which contributes to their popularity as an alternative treatment for infectious diseases².

Among these plants is ginggiang (Leea aequata L.), which belongs to the genus Leea with around 70 other species³. Ginggiang is recognized for its diverse pharmacological activities, including anticancer^4,5, antioxidant⁵, antiplatelet⁶, antibacterial^7,8, and anticonvulsant properties⁹.

In Indonesia, Karo people use ginggiang leaf as a remedy for leprosy¹⁰. The indigenous inhabitants of Kuta, Ciamis, West Java, Indonesia, also apply leaf as a preservative in bamboo sap reservoir, which is crucial for preserving palm sap used in palm sugar production. This process allows the chemical compounds in the leaf to infuse the sap, enhancing the quality of the resulting product. Consequently, the palm sap retains its natural sweetness without sourness, producing palm sugar with a desirable brownish-red hue, a manageable consistency for molding, and a robust texture that remains easily sliceable or cuttable. Furthermore, the discovery shows the potential of ginggiang leaf for application as a preservative in various food products. In Myanmar and Bangladesh, leaf is applied as an antiseptic for treating an array of skin conditions¹¹, showing antimicrobial activity that can be further developed as a preservative and antiseptic.

The antimicrobial activity test was conducted on foodborne pathogens and microorganisms responsible for various skin infections. Foodborne pathogens constitute the primary factors contributing to food spoilage, leading to illnesses¹² caused by bacteria, viruses, fungi, and parasites¹². Furthermore, these pathogens include Escherichia coli, Staphylococcus aureus¹³, Pseudomonas aeruginosa¹⁴, Bacillus subtilis¹⁵, Candida albicans¹⁶, as well as Aspergillus brasiliensis and Aspergillus flavus¹⁷. For example, E. coli is a major contributor to afflictions such as thrombotic thrombocytopenic purpura (TTP), hemorrhagic colitis, and hemolytic uremic syndrome (HUS), which frequently contaminate dairy products, meats, fruits, and vegetables¹². The fungal species C. albicans has the potential to induce foodborne illnesses, specifically causing mastitis in cattle, leading to diminished milk production and quality. This significant reduction increases the risk of congenital infections for consumers who ingest milk¹⁸.

Among the microbes with the potential to cause skin infections is Propionibacterium acnes¹⁹ and Staphylococcus epidermidis²⁰. Therefore, this study aimed to determine the effectiveness of ginggiang leaf extract, establishing its potential as a preservative and potent antiseptic for bacteria. This analysis is essential because effective antimicrobial agents are needed to preserve and safeguard public health by combating skin infections.

MATERIALS AND METHODS:

Materials:

Fresh ginggiang leaf obtained from Ciamis, Indonesia, in January 2022 was sorted to separate from impurities. Subsequently, the leaf was dried in an oven at 50°C for four days and processed using a herb grinder.

The 96% ethanol, dimethyl sulfoxide (DMSO), ethyl acetate, and demineralized water were sourced from Brataco Chemika, Indonesia. Mueller Hinton Broth (MHB), Mueller Hinton Agar (MHA), Potato Dextrose Broth (PDB), and Potato Dextrose Agar (PDA) were procured from Himedia, Indonesia. Furthermore, Roswell Park Memorial Institute (RPMI) 1640 (Corning, USA) was obtained from Intralab Ekatama, Bogor, Indonesia. The well plate used was a single-pack flat-bottomed 96-hole sterile well plate from Nest Biotechnology Co., Ltd. The paper discs used were MN 827 ATD with a diameter of 6mm.

The test microbes consisted of seven species of bacteria and three fungi. The bacteria included S. epidermidis (ATCC 14990), S. aureus (ATCC 6538), Methicillin-Resistant Staphylococcus aureus (MRSA) (ATCC 33591), P. aeruginosa (ATCC 9027), P. acnes (ATCC 11828), B. subtilis (ATCC 6051), and E. coli (ATCC 8739). Meanwhile, fungi species tested were C. albicans (ATCC 10231), A. brasiliensis (ATCC 16404), and A. flavus (ATCC 13697). All the microbial strains were sourced from the Microbiology Laboratory, School of Pharmacy, Institut Teknologi Bandung, Indonesia.

Preparation of Extract:

The test extract preparation was aqueous extract (AE) and 96% ethanol extract (EE) of ginggiang leaf. AE was obtained through the reflux method, followed by freeze-drying for preservation. Meanwhile, EE was derived using the maceration method and concentrated through a rotary evaporator. The resulting viscous substance was dried using an oven. The antimicrobial activity was also assessed against the liquid-liquid extraction fractions derived from EE: n-hexane, ethyl acetate, and residual (20%-methanol fraction).

Phytochemical Screening:

Phytochemical screening of Simplicia, AE, and EE of ginggiang leaf was conducted using the Farnsworth method²¹ with slight modifications.

Alkaloid Test:

Approximately 1mL extract was mixed with Dragendorff reagents, leading to the development of an orange coloration, which showed the presence of alkaloid compounds. Alkaloid tests were also conducted by mixing the extract with three drops of Mayer's reagent. Subsequently, the formation of cream-colored deposits confirmed the presence of alkaloids²².

Flavonoid Test:

The flavonoid test was conducted by mixing 1mL extract with magnesium (Mg) powder and 2mL HCl-ethanol (1:1), followed by shaking and adding 10mL amyl alcohol. The appearance of an orange, yellow, or red color in the amyl alcohol layer showed the presence of flavonoids in the extract.

Saponin Test:

The saponin test was conducted by vigorously shaking 0.5g extract with 10mL water in a test tube. The formation of stable foam when heated in a water bath for 5 minutes showed the presence of saponins in the test sample²³.

Tannin Test:

The test began by preparing 2mL of each extract in separate test tubes. Subsequently, one of the tubes was mixed with a 1% gelatin solution, and white precipitation indicated the presence of tannins in the test sample. In a separate test tube, the extract was mixed with 1% FeCl_3,and the development of a green or dark blue solution or precipitation confirmed the presence of tannins^22,24.

Steroid and Terpenoid Tests:

Steroid and terpenoid tests were conducted by dissolving 1g simplicia or 100mg extract in chloroform and adding a few drops of anhydrous acetic acid. The mixture was heated in a water bath and promptly cooled in an ice bath. Subsequently, one drop of concentrated H₂SO₄ was introduced, and the changes were observed. The appearance of a greenish-blue color signified the presence of steroids, while a purplish-red color showed triterpenoids in the extract²⁴.

Quinone Test:

The test was performed by dissolving 1 g simplicia or 100mg extract in 100mL hot water and boiling for 5 minutes. Subsequently, 5mL filtrate was mixed with one drop of 1N NaOH, and a red coloration indicated quinones in the extract.

Preparation of Inoculum:

The bacterial inoculum was prepared by suspending a bacteria sample in a saline solution and measuring turbidity. Suspensions with turbidity equivalent to 0.5 McFarland units (absorption between 0.08-0.1) were directly applied in antibacterial activity tests using the disc diffusion method. Simultaneously, the microdilution method included diluting to 1:20 with MHB medium²⁵.

The fungal inoculum was prepared by suspending a fungus sample in a saline solution and measuring turbidity. Suspensions with turbidity equivalent to 0.5 McFarland units were directly used in tests through the paper disc method. Simultaneously, the microdilution method included a 1:50 initial dilution followed by a 1:20 dilution in RPMI 1640 medium²⁶.

Antimicrobial Activity Test:

Antimicrobial activity tests were conducted using paper disc diffusion and microdilution methods. For the paper disc test, sterilized Petri dishes containing MHA and PDA media were used to evaluate antibacterial and antifungal activities. In this medium, 20μL bacterial/fungal suspension was evenly spread, equivalent to 0.5 McFarland units. Subsequently, paper discs saturated with a specific quantity of the test extract were placed on the media surface. Bacterial samples were incubated at 37°C for 24 hours, while fungi were conducted at 35°C for 48 hours.

Microdilution used a sterile 96-well plate with 12 columns and eight rows, where each well was filled with 100μL of MHB or RPMI 1640. Column 1 contained only the media as a negative control, while column 2 comprised bacterial/fungal media and suspension as a positive control. Furthermore, column 3 included media, fungi/bacteria, and a 2% DMSO solution to show the absence of microbial growth inhibition caused by DMSO in the test solution preparation. Column 4 contained media, bacteria/fungi, and ciprofloxacin for bacterial tests or ketoconazole for fungal tests. Ciprofloxacin and ketoconazole served as positive controls inhibiting bacterial/fungal growth.

Rows A and H of columns 5-12 contained the media and dilution series of extract, serving as controls for turbidity in the absence of test organisms. Meanwhile, rows B-G in columns 5-12 contained the media, bacteria/fungi, and extract dilution series, with column 12 comprising the highest extract concentration. The well plates with the test solution were incubated at 37°C for approximately 24hours for bacteria testing. This process was continued for 48hours to carry out fungal testing. Observations were made by assessing the turbidity of the solution, followed by the observation of fungi at 24 and 48hours.

This test was carried out to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC). MBC/MFC was assessed by streaking the test solution on the MIC test, showing the inhibition of microbial growth through the absence of turbidity. All solutions showing no turbidity were tested on MHA and PDA media for bacteria and fungi, respectively. The media streaked with the test solution was incubated at 37°C for 24 hours to assess bacteria and 35°C for 48 hours to evaluate fungi. Subsequently, MBC/MFC was determined by observing the presence or absence of mold/bacteria growth after the incubation period. The smallest concentration showing no growth was recorded as MBC/MFC^25,27.

Time-kill curve:

The time-kill curve was used to determine the bactericidal/bacteriostatic and fungicidal/fungistatic effects of the test extract. Subsequently, the test was conducted by monitoring the growth of microbes exposed to the extract solution at MIC. Microbial growth was quantified by measuring optical density values at 530nm for C. albicans and 620nm for S. aureus. Optical density readings were taken at 0, 2, 4, 6, 12, 18, and 24hours. A time-kill curve was generated by plotting the time values against the optical density²⁸.

RESULT AND DISCUSSION:

Phytochemical Screening:

Phytochemical screening was conducted on simplicia, AE, and EE, and the results are summarized in Table 1. The leaf samples showed positive results for various secondary metabolites, including flavonoids, saponins, tannins, quinones, and terpenoids/steroids. However, there were slight disparities from the results obtained by Islam (2017), showing the presence of alkaloids in the methanol extract of ginggiang leaf²⁹. This discrepancy occurred due to variances in the geographical origin of ginggiang and disparities in the solvents used for extraction. These secondary metabolites are known to possess antimicrobial activity potentially. Specifically, flavonoids such as luteolin, myricetin, and quercetin have been identified for antimicrobial effects against both bacteria and fungi³⁰.

Tannin compounds such as tannic acid have also demonstrated antibacterial properties, serving as adjunct therapy for skin infections in combination with beta-lactam antibiotics³¹. Moreover, studies showed that tannins had bactericidal effects³² and the combination of saponins with antibiotics was found to produce a synergistic effect³³.

Table 1: Phytochemical contents

Constituents	Simplicia	AE	EE
Alkaloids	-	-	-
Flavonoids	+	+	+
Saponins	+	+	+
Tannins	+	+	+
Quinone	+	+	+
Terpenoids/Steroids	+	+	+

Description: - = negative, + = positive, AE = aqueous extract, EE = ethanol extract

Antimicrobial Activity:

Antimicrobial activity tests using the paper disc method showed that AE, EE, ethyl acetate fraction (EAF), and 20%-methanol (RF=residual fraction) provided significant effects on several test microbes. Meanwhile, the n-hexane fraction did not have antimicrobial activity on all tested microbes, as shown in Figure 1.

EE showed antimicrobial activity against all tested bacteria and in two species of fungi. Bacteria susceptible to EE included S. epidermidis, S. aureus, MRSA, P. aeruginosa, P. acnes, B. subtilis, and E. coli (Figure 1). Similarly, susceptible fungi were A. flavus, including C. albicans, which proved to be the most vulnerable to all extracts and test fractions. This is shown by the size of the inhibitory zone on the paper disc and the low MIC value, ranging from 53 to 133μg/mL. As shown in Table 2, EE provided low MIC values for most foodborne pathogen microbes, including S. aureus, P. aeruginosa, B. subtilis, E. coli, and C. albicans, ranging from 53-426 μg/mL (Table 2). This suggested that the test extract had potential for development as a preservative. The extract could also be explored for antimicrobial properties in treating skin diseases or as an antiseptic due to the inhibitory effects on the growth of S. epidermidis and P. acnes, common culprits of skin infections.

The results have significant implications for the development of natural preservatives and antimicrobials. Consequently, identifying the potential of ginggiang leaf as a source substantially contributes to exploring alternatives to chemical preservatives in various industries. The ability of the extract to inhibit the growth of harmful microorganisms, including pathogenic bacteria and fungi, suggests the potential application in the food industry for extending the shelf life of food products. This unique property also shows the potential of ginggiang extract as a natural antimicrobial agent for treating skin infections. This preliminary study serves as a critical initial step in identifying active components in the plant and assessing the potential applications. The results provide a solid foundation for further investigation, which includes formulation and advanced-level testing. The importance of scientifically validating the local community's traditional use of ginggiang leaf is also confirmed, particularly in Kampung Kuta, Ciamis, Indonesia. This promotes the conservation and sustainable use of local plants with significant potential in developing natural antimicrobial products.

Figure 1. Antimicrobial activity of EE, EA, HF, EAF, and RF on each test microorganism

In this study, MBC and MFC values of extract and the test fraction determined were more than 2560 μg/mL, representing the highest concentration tested. The test solution was prepared at a concentration of 5120 μg/mL. However, higher concentrations were not feasible due to the limited solubility.

Table 2. MIC of test extract

Name of microbe	Test Extract	MIC (µg/mL)
S. epidermidis	AE	1280.00±0.00
	EE	168.00±102.14
	EAF	640.00±0.00
	RF	640.00±0.00
	Ciprofloxacine	0.05±0.00
S. aureus	AE	266.67±75.42
	EE	66.67±18.85
	EAF	160.00±0.00
	RF	133.33±37.71
	Ciprofloxacine	0.195±0.00
MRSA	AE	186.67±99.78
	EE	106.67±37.71
	EAF	533.33±150.85
	RF	213.33±75.42
	Ciprofloxacine	0.195±0.00
P. aeruginosa	AE	186.67±99.78
	EE	133.33±37.71
	EAF	426.67±150,85
	RF	240.00±113.14
	Ciprofloxacine	0.195±0.00
P. acnes	AE	533.33±150.85
	EE	426.67±150.85
	EAF	240.00±113.14
	RF	320.00±0.00
	Ciprofloxacine	0.195±0.00
B. subtilis	AE	-
	EE	426.67±150.85
	EAF	533.33±150.85
	RF	-
	Ciprofloxacine	0.097±0.00
E. coli	AE	-
	EE	426.67±150.85
	EAF	-
	RF	-
	Ciprofloxacine	0.195±0.00
C. albicans	AE	106.67±37.71
	EE	53.33±18.85
	EAF	40.00±0.00
	RF	133.33±37.71
	Ketoconazole	15.00±5.00
A. flavus	AE	2560.00±0.00
	EE	2560.00±0.00
	EAF	-
	RF	-
	Ketokonazole	60.00±20.00

Description: - = no value, AE= aqueous extract, EE= ethanol extract, EAF= ethyl acetate fraction, RF= residual fraction

The time-kill curve of S. aureus and C. albicans showed that extract and test fractions effectively inhibited the growth of these microbes. The control group showed significant growth after 4 to 24 hours of incubation, while the test microbes treated with ciprofloxacin, AE, and EE had insignificant effects. All three treatments showed a lower increase rate than the control group (Figure 2).

Figure 2. Growth curve of test microbes exposed to the test solution at a concentration of MIC; A. in Staphylococcus aureus, B. in Candida albicans

The growth curve of S. aureus and C. albicans presented in Figure 2 showed that extract and test fractions reported a bacteriostatic effect against S. aureus and a fungistatic effect against C. albicans. Although both microbes continued to grow after exposure to the extract/test fraction, their growth remained lower than that of control microbes that were not treated with the test extract/fraction. Specifically, EE had a slightly different trend, with S. aureus showing a bactericidal effect.

CONCLUSION:

In conclusion, this study indicated that ginggiang leaf aqueous and ethanolic extract, including ethyl acetate and 20%-methanol fraction from ginggiang leaf ethanolic extract, showed antimicrobial activity. The observed efficacy extended to both against foodborne pathogens (E. coli, S. aureus, P. aeruginosa, C. albicans, B. subtillis, and A. flavus) and microbes causing skin infections (P. acnes and S. epidermidis). Ethanolic extract provided more potent activity than the water extract and the fractions. This showed that the extract could be developed as a preservative and antiseptic.

CONFLICT OF INTEREST:

The authors state no conflicts of interest related to this investigation and publication.

ACKNOWLEDGMENTS:

The authors are grateful to the Indonesian Education Scholarship (Beasiswa Pendidikan Indonesia), Managed by Balai Pembiayaan Pendidikan Tinggi, and The Indonesian Endowment Funds for Education (Lembaga Pengelola Dana Pendidikan, LPDP) with grant number ²⁰²¹⁰¹¹²¹²⁰^7.

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Received on 04.12.2023 Revised on 13.04.2024

Accepted on 17.06.2024 Published on 27.03.2025

Available online from March 27, 2025

Research J. Pharmacy and Technology. 2025;18(3):1154-1160.

DOI: 10.52711/0974-360X.2025.00166

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.