In Vitro Antifungal Activity of Boesenbergia rotundo Linn. and Syzygium aromaticum L. Merr. and Perry Extracts against Aspergillus flavus ^†

Abstract

Aspergillus flavus is a common human pathogen that releases mycotoxin into the host and is frequently treated with synthetic fungicides, but these fungicides have serious human health consequences. Natural products derived from higher plant species have long been investigated as a potential means of controlling pathogenic microorganisms. The indigenous vegetables Boesenbergia rotunda and Syzygium aromaticum are widely distributed in the tropical area. These plants have also been reported in traditional uses for their antimicrobial activity. The purpose of the study was to explore the antifungal susceptibility of dichloromethane and ethanol extracts of B. rotunda rhizomes and S. aromaticum flower buds by Soxhlet’s apparatus against A. flavus using the poison food technique. The effective extract was also subjected to preliminary phytochemical screening tests. The experiment used a completely randomized design with triplications. B. rotunda ethanol extract demonstrated significantly higher potential antifungal activity. The values of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of B. rotunda ethanol extract were 6.25 and 50 mg/mL, respectively, when tested using the macro-dilution method. According to phytochemical tests, the ethanol extract also contained alkaloids, flavonoids, cardiac glycosides, and saponins. The study suggests that a basic guideline for using this as an effective antifungal compound should be separated from the B. rotunda ethanol extract in the future for topical anti-pathogenic fungus.

1. Introduction

Aspergillus flavus can grow rapidly in environments where mycelia can use substrates provided by multiple carbon sources [1]. Moreover, the fungi produce aflatoxin, which has been related to class I liver cancer [2]. Synthesized chemical agents have long been used for the prevention of the fungus. However, these chemicals can accumulate and be harmful to the environment, humans, and animals.

Phytochemicals are also being developed to be a possible way to achieve outcome trends for antimicrobial agents [3,4,5]. Chemical groups from plants including alkaloids, flavonoids, tannins, and phenolics controlling microbial growth have been reported [6]. The indigenous vegetables Boesenbergia rotunda and Syzygium aromaticum are widely distributed in the tropical area. The plants belong to the Zingiberaceae and Myrtaceae families, respectively. Various extracts and essential oils of these plants have also been reported in traditional uses for their antimicrobial activity against Gram-positive and -negative bacteria, filamentous fungi, and Candida species [7,8]. The chemical groups of the plants, including flavonoids, terpenes, terpenoids, aromatic compounds, and alkaloids, have been reported [9].

The purposes of the study were to investigate the antifungal activity of B. rotunda rhizomes and S. aromaticum flower buds obtained by dichloromethane and ethanol against A. flavus and to examine the phytochemical screening of the effective extract. The effective extract can then be isolated further to be used in the discovery of novel friendly topical agents.

2. Methods

2.1. Plant Collection and Authentication

Rhizomes of B. rotunda and flower buds of S. aromaticum were collected from a vegetable farm in Ongkharak province, Nakhon Nayok, Thailand. The plants were identified botanically in the Department of Botany, Faculty of Science, Chulalongkorn University.

2.2. Soxhlet’s Extraction

Ten kilograms of fresh rhizomes of B. rotunda and five hundred grams of flower buds of S. aromaticum were washed and dried at 60 °C until they reached a constant weight. After that, 200 g of each dried sample was powdered, packed in an extract bag, and subjected to Soxhlet’s apparatus. Dichloromethane was firstly used to extract the samples, followed by ethanol. The crude extracts were filtered and then concentrated using a rotating vacuum evaporator until the crude extracts were a constant weight. The crude extracts were stored in bottles covered with aluminum foil at 4 °C until they were studied.

2.3. Fungal Strain

A. flavus was provided by the Center of Excellence in Chemistry of Natural Products, Faculty of Science, Chulalongkorn University. The fungus was maintained on potato dextrose agar (PDA) at 28 °C in darkness.

2.4. Antifungal Susceptibility

The antifungal activity was applied as a method of poisoning food techniques. Each extract was dissolved in 1% v/v DMSO and then 100 µL was added into PDA to give a final concentration of 1000 mg/L. One hundred microliters of each extract was combined with melted potato dextrose agar (PDA) and poured into Petri plates. Combinations of PDA mixed with only 1% v/v DMSO or nystatin (0.05 mg/mL) were used as a negative and positive controls, respectively. Mycelia discs were plugged from the edges of the 5-day-old culture with a cork borer (0.5 cm diameter). The plates were incubated at 28 °C in the dark, and susceptibility was determined by comparing the relative growth of fungus in each treatment [10]. The formula I = (C − T)/C × 100 was used to calculate the percentage of growth inhibition, where I denotes percent inhibition, C denotes control colony diameter (cm), and T denotes treatment colony diameter (cm) [11]. The minimal inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) in various concentrations of the effective extract were also determined using a macro dilution technique [12,13].

2.5. Phytochemical Testing Screening

The samples were examined against the powdered material using regular methodological approaches [14]. Alkaloids, anthraquinones, flavonoids, terpenoids, steroids, cardiac glycosides, saponins, tannins, and phlobatannins were all tested in the phytochemical screening experiment.

2.6. Statistical Analysis

The SPSS program for Windows version 22.0 was used to analyze the data. Duncan’s multiple range test (DMRT) was used to compare the results, and significance was found at the p < 0.05 level. Within a completely randomized design, the experiment was conducted as a generalized linear model with triplications.

3. Results and Discussion

3.1. Percentage Yields

The percentage yields of the extracts are represented in

Table 1. Percentage yields.

Plant	Solvent	% Yield
B. rotundo	Dichloromethane	1.59
	Ethanol	4.49
S. aromaticum	Dichloromethane	2.08
	Ethanol	5.15

. The ethanol extracts of each plant gave a much larger amount than the dichloromethane extracts. The extraction yields using different solvents were increased by the polarity of the solvent used in extraction (increasing polarity followed the order: hexane < ethyl acetate < dichloromethane < acetone < chloroform < ethanol < methanol) [15,16].

3.2. Antifungal Activity

Table 2. Antifungal activity of B. rotundo and S. aromaticum extracts against A. flavus at 1000 mg/L.

Treatment	Extract	% Mycelia Growth Inhibition (%Mean * ± S.D.), n = 3
B. rotundo	Dichloromethane	45.93 ± 0.57 a
	Ethanol	50.93 ± 0.10 a
S. aromaticum	Dichloromethane	25.19 ± 0.14 b
	Ethanol	27.41 ± 0.12 b
1% DMSO	Negative control	0.00 c
Nystatin	Positive control (0.05 mg/mL)	49.25 ± 0.23 a

* Mean values with different superscript letters in each column are significantly different (p < 0.05).

shows the antifungal activity of the B. rotundo and S. aromaticum extracts with the dichloromethane and ethanol displays. The dried rhizome extracts of B. rotundo from both organic solvents revealed higher antifungal activity than the extracts of the S. aromaticum flower buds. The ethanol rhizome extract of B. rotundo demonstrated the highest percentage of mycelia growth-inhibitory efficacy against A. flavus (50.93%) and was comparable to the positive control. Thus, the ethanol extract was chosen for MIC/MFC susceptibility and phytochemical screening.

The MIC and MFC values of the ethanol extract of B. rotunda rhizomes on the fungus compared with the positive control of amphotericin B are shown in

Table 3. The MIC/MFC values of ethanol extracts of B. rotunda against A. flavus.

Treatment	MIC (mg/mL)	MFC (mg/mL)	MFC Indice	Mode of Action
Ethanol extract	6.25	50.00	8.00	Fungistatic
Nystatin	2.15	3.50	1.63	Fungicidal

. The MIC/MFC values revealed 6.25/50 mg/mL, which could be calculated to an MFC index of 8.00. The result was estimated at more than 4, suggesting that the extract was a fungistatic agent. A fungistatic agent is a chemical that inhibits the growth of fungi [17].

3.3. Phytochemical Screening Test

The result of the phytochemical screening test of the ethanol rhizome extract of B. rotunda is shown in

Table 4. Phytochemical test results of ethanol extract.

Phytochemicals	Result *
Alkaloids	+
Anthaquinones	−
Flavonoids	+++
Terpenoids	−
Steroids	−
Cardiac glycoside	++
Saponins	+
Tannins	-
Phlobatannins	-

Note: * (−) = negative test; (+) = weak positive test; (++) = positive test; (+++) = test strongly positive.

. In the ethanol extract, alkaloids, flavonoids, cardiac glycosides and saponins were present.

4. Conclusions

The ethanol rhizome extract of B. rotunda showed significantly potent antifungal activity against A. flavus. Alkaloids, flavonoids, cardiac glycosides, and saponins were discovered as phytochemicals. Furthermore, the ethanol rhizome extract of B. rotunda could isolate the anti-A. flavus compounds for a new generation of topical agents.