Pakistan Journal of Biological Sciences

Year: 2021 | Volume: 24 | Issue: 2 | Page No.: 235-240
DOI: 10.3923/pjbs.2021.235.240

A New Potential Source of Anti-pathogenic Bacterial Substances from Zamioculcas zamiifolia (Lodd.) Engl. Extracts

Surachai Rattanasuk and Tannatorn Phiwthong

Abstract: Background and Objective: The increase of antibiotic-resistant bacteria is a problem for global health that needs to find new antibiotic drugs. The plant is the potential source of antibiotic substances that important to solve the antibiotic-resistant bacteria. This study was aimed to evaluate the antibacterial activity of Zamioculcas zamiifolia stem extracts against nine human pathogenic bacteria. Materials and Methods: Z. zamiifolia stems were extracted with five extraction solvents. The screening of antibacterial activity of stem extract was measured using agar disc diffusion assay. The Minimal Inhibition Concentration (MIC) and Minimal Bactericidal Concentration (MBC) values of extracts were determined using the broth microdilution assay and colorimetric assay. Results: The results indicated that the lowest MIC value of 0.09 mg mL^–1 against Staphylococcus aureus TISTR 1466 was obtained from hexane extraction. The lowest MBCs value of 1.56 mg mL^–1 against Bacillus cereus TISTR 2373, Listeria spp. and Escherichia coli TISTR 527 were obtained from ethanol and methanol extractions. Conclusion: The ethanolic and methanolic stem extracts of Z. zamiifolia demonstrated the highest anti-human pathogenic bacterial activity. This is the first report to demonstrate the high potential of antibacterial substance from Z. zamiifolia stem extracts, which can be developed further as a natural drug for treating bacterial infectious diseases.

Keywords: stem extracts, ZZ plant, Zamioculcas zamiifolia (Lodd.) Engl., human pathogenic bacteria, Anti-bacterial activity, natural drug and plant extraction

INTRODUCTION

Misuses of antibiotics in humans and animals lead to antibiotic resistance in bacteria. The novel, effective and affordable antibiotic drugs to treat infectious diseases are the most major challenge in global health care¹. Plants constitute a good source of antibiotic compounds, in regards to the variety and diversity of their chemical structures² and also found in marine organisms³. Many plants were reported that contained antibacterial activity such as Albizia adianthifolia, Alchornea laxiflora, Laportea ovalifolia², Allium sativum, Bunium persicum⁴, Canarium schweinfurthii ⁵, Rosmarinus officinalis L.⁶, Rhizophora mucronata, Rhizophora apiculata, Rhizophora annamalayana⁷, Chimonanthus salicifolius⁸, Carum copticum L.⁹, Thymus capitatus¹⁰, Uapaca heudelotti ¹¹, Murraya koenigii ¹², Teucrium polium L.¹³, Cremaspora trifloral, Hypericum roeperianum¹, Scutellaria baicalnsis¹⁴ and Spathiphyllum wallisii ¹⁵.

An unusual drought-tolerant plant Zamioculcas zamiifolia (Lodd.) is a native drought-tolerant medicinal plant which found in tropical East Africa and subtropical southeast Africa¹⁶. This forest plant develops short sprouts from a thick underground tuber-like rhizome to reserve water for its survival without water for longer periods¹⁷. It is an ornamental potted foliage plant in the family Araceae, which a high potential to reduce the concentration of many contaminants indoor air including Benzene, Toluene, Ethylbenzene and Xylene (BTEX), respectively¹⁸. The novel natural products main compound of the Z. zamiifolia leaves, apigenin 6-C-(6"-(3-hydroxy-3-methyl-glutaroyl)-β-glucopyranoside was reported recently¹⁶. However, the antibacterial activity of Z. zamiifolia has yet been evaluated.

This study aimed to evaluate the antibacterial activity of Z. zamiifolia extracts against nine pathogenic bacteria including Bacillus subtilis TISTR 008, Escherichia coli TISTR 527, Staphylococcus aureus TISTR 1466, Bacillus cereus TISTR 2373, Staphylococcus epidermidis TISTR 518, Pseudomonas aeruginosa TISTR 2370, Klebsiella pneumoniae subsp. pneumoniae TISTR 1383, Staphylococcus aureus PK and Listeria spp. This is the first report that the antibacterial activity of Z. zamiifolia extracts was demonstrated in vitro which benefits for antibiotic drug development.

MATERIALS AND METHODS

Study area: All the experiments were performed during October, 2019-April, 2020 in the Microbiology Laboratory, Major of General Science, Department of Science and Technology, Faculty of Liberal Arts and Science, Roi Et Rajabhat University, Roi Et, Thailand.

Chemicals and reagents: Hexane, Dichloromethane, Ethyl acetate, Ethanol and Methanol were purchased from QRëC^™ (Republic of New Zealand), Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich Co. (St. Louis, Missouri, U.S.A.), Mueller Hinton Broth (MHB), Nutrient Broth (NB) and Bacterial Agar were purchased from HiMedia (HiMedia Laboratories Pvt. Ltd, India).

Human pathogenic bacteria: A total of 9 human pathogenic bacterial strains were used in this research: Bacillus subtilis TISTR 008 (B. subtilis TISTR 008), Escherichia coli TISTR 527 (E. coli TISTR 527), Staphylococcus aureus TISTR 1466 (S. aureus TISTR 1466), Bacillus cereus TISTR 2373 (B. cereus TISTR 2373), Staphylococcus epidermidis TISTR 518 (S. epidermidis TISTR 518), Pseudomonas aeruginosa TISTR 2370 (P. aeruginosa TISTR 2370), Klebsiella pneumoniae subsp. pneumoniae TISTR 1383 (K. pneumoniae TISTR 1383) were obtained from the Thailand Institute of Scientific and Technological Research culture collection (TISTR culture collection), Thailand. Staphylococcus aureus PK (S. aureus PK) and Listeria spp. were obtained from Major of Microbiology, Faculty of Science, Mahasarakham University, Thailand. All human pathogenic bacteria were cultured using Nutrient Broth (NB) before use.

Plant sample collection and preparation of extracts: Z. zamiifolia stem was collected from Ban Tha Muang, Selaphum, Roi Et, Thailand. The collected stems were washed three times with water, cut into small pieces and dried in a hot air oven (POL-EKO-APARATURA company, Wodzisław Śląski, Poland) at 50°C for 48 hrs. The dried stems were ground and biochemical substances were extracted with five extraction solvents including hexane, dichloromethane, ethyl acetate, methanol and ethanol (QRëC^TM, Republic of New Zealand) at a ratio of 1:10 (w/v). The extractions were carried out on a shaking at room temperature for 24 hrs before filtration. The filtrates were concentrated using a Rotavapor (Buchi, Switzerland) and the percent yield was calculated¹⁵. The extracts were then diluted with dimethyl sulfoxide (DMSO) to the final concentration at 500 mg mL^–1.

Screening for antimicrobial activity of Z. zamiifolia stem extracts using agar disc diffusion assay: The antagonistic activity of each stem extract was tested by the disk diffusion method¹⁹. Overnight bacterial cultures were adjusted the cell concentration at OD₆₀₀-0.1. One hundred microliters of each pathogenic bacterium were spread onto Mueller Hinton Agar (MHA) and a sterile paper dish (0.6 mm) was placed on MHA. Ten microliters of each extract were dropped onto the center of the paper dish and DMSO and Kanamycin were used as a control. The extracts were allowed to diffuse for 15 min before MHA plates were incubated at 37°C for 24 hrs. The zone of inhibition formation around the paper disks was measured.

In vitro antibacterial activity determination using a broth microdilution assay: The broth microdilution assay was used to determine Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values. Z. zamiifolia stem extracts were twofold serial diluted in 96-well plate containing MHB to obtain various concentrations. The overnight bacterial inoculum was adjusted the cell concentration at OD₆₀₀-0.1 before adding 100 μL into each well. Kanamycin (50 mg mL^–1) and DMSO were used as a positive and negative control, respectively. The 96-well plates were incubated at 37°C for 24 hrs. As an indicator of bacterial growth, 50 μL iodonitrotetrazolium chloride (INT) was added in each well of the 96-well plate and was incubated at 37°C for 1 h¹⁵. The well containing the bacterial growth turned into pink color whereas the well without bacterial growth remained yellow color. The MIC was considered as the lowest concentration of the extract that completely inhibits the bacterial growth. The MBC was considered as the lowest concentration of extract that kills bacteria that did not produce a color change after the addition of INT⁵.

Data analysis: In this study, it was used experimental design followed by descriptive analysis.

RESULTS AND DISCUSSION

Extraction yields: The extraction of Z. zamiifolia stem was extracted using 5 extraction solvents with shaking for 24 hrs. The highest yield was obtained from ethanolic extract of 10.98% while the least yield was obtained from hexane extraction (Table 1).

Antibacterial activity determination using agar disc diffusion assay: On the initial test, the results indicated that Z. zamiifolia stem extracts showed antibacterial activity by suppressing bacterial growth with various degrees, which methanol extract exhibited the largest inhibition zones at 10.0 mm against Listeria spp. (Table 2) which similar to previously reported by Srikacha and Ratananikom²⁰ that ethanol is the most suitable solvent for extraction. Hexane extract inhibited the growth of all gram-positive pathogenic bacteria, whereas dichloromethane extract only showed an effect on B. subtilis TISTR 008. Ethyl acetate extract did not have antibacterial activity against any bacteria tested. The result of inhibition zone from this research was.

The results of antibacterial activity assay by disc diffusion suggested that S. aureus PK, K. pneumoniae TISTR 1383 and E. coli TISTR 527 were the most resistant strains to Z. zamiifolia stem extracts followed by Listeria spp., P. aeruginosa TISTR 2370, S. aureus TISTR 1466, B. cereus TISTR 2373, S. epidermidis TISTR 518 while B. subtilis TISTR 008 was the most susceptible strain to the extracted plants, respectively.

Methanol and ethanol were the most effective solvent to extract antibacterial substances out of Z. zamiifolia stems. Thus, follow-up experiments were conducted to determine their MIC and MBC against these bacteria.

In vitro antibacterial activity assay: Antibacterial activity of the extracts was further evaluated by MIC and MBC measurement using broth microdilution assay. The MICs of Z. zamiifolia stem extracts were ranged between 0.09-12.5 mg mL^–1. Among five solvent extracts, hexane extract showed the highest antibacterial activity against gram-positive pathogenic bacteria, which MICs of Z. zamiifolia stem extracts against S. aureus TISTR 1466, S. aureus PK, B. subtilis TISTR 008 and B. cereus TISTR 2373 were 0.09, 0.19, 0.39 and 0.39 mg mL^–1, respectively (Table 3). Dichloromethane extract was only effective against B. subtilis TISTR 008 with MIC value of 6.2 mg mL^–1. Ethanol and methanol were the most effective extracts against both gram-positive and gram-negative bacteria. Achika et al.¹¹ recently reported that ethyl acetate fraction of the stem bark of Uapaca heudelotti was active against E. coli, B. subtilis, K. pneumoniae, S. aureus with MICs value of 3.12-12.5 μg mL^–1. Abuga et al.¹² also found the ethyl acetate extract of the leaves gave the lowest MIC value against S. aureus and E. coli 0157:H7 at 15.63 μg mL^–1. Trinh et al.²¹ reported that A. conyzoides flower showed the most potent effect on uropathogenic E. coli and K. pneumoniae with MIC at 1.25-10 and 5-12.5 mg mL^–1, respectively.

The MBCs of Z. zamiifolia stem extracts against the bacteria were ranged between 1.56-25 mg mL^–1. The ethanol and methanol extract demonstrated the highest antibacterial activity against B. cereus TISTR 2373, Listeria spp. and E. coli TISTR 527 with MBCs value of 1.56 mg mL^–1 (Table 4). The MIC and MBC value of ethanol extracts of Z. zamiifolia stem were similar to previously reported by Dzotam et al.⁵ that ethanol extract of Canarium schweinfurthii against E. coli AG100ATet was at 64 and 1024 μg mL^–1. Diarra et al.²² reported that ethanol extract of Vaccinium macrocarpon at 0.4 and 0.8% concentration did not affect survival of L. monocytogenes in a cooked chicken-breast meat model. Ceruso et al.²³ reported that Baphia racemosa and Sansevieria hyacinthoides extracted using methanol showed the lowest value of MIC 2.5 mg mL^–1 against L. monocytogenes. Susceptibility to plant extracts of these pathogenic bacteria has been documented but this is the first time that antibacterial activity of Z. zamiifolia stem extracts against this group of bacteria is reported. Z. zamiifolia may be an alternative plant for developing new drug formulations and the effects of cytotoxicity on human cells should be studied.

CONCLUSION

The Z. zamiifolia stem was extracted using 5 extraction solvents. The results presented that the methanolic and ethanolic extraction were the suitable extraction solvent for Z. zamiifolia stem extraction. The new finding obtained from this study give novel information on antibacterial activity from Z. zamiifolia stem extracts against human pathogenic bacteria which benefit and useful for antibiotic drug development.

SIGNIFICANCE STATEMENT

This study discovers the novel antibacterial containing Z. zamiifolia stem extracts that can be beneficial for the new antibiotic drug development. This study will help the researcher to uncover the critical areas of the evaluation of the antibacterial activity of plant extracts that many researchers were not able to explore. Thus, a new application using the antibacterial activity obtained from Z. zamiifolia stem extracts may be arrived at.

ACKNOWLEDGMENT

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

REFERENCES