Pakistan Journal of Biological Sciences

Year: 2022 | Volume: 25 | Issue: 10 | Page No.: 922-928
DOI: 10.3923/pjbs.2022.922.928

Antibacterial and Anticancer Properties of Microbispora sp., AL22: An Endophyte of Alpinia galanga (L.) Willd

Thongchai Taechowisan , Thanaporn Chuen-Im and Waya S. Phutdhawong

Abstract: Background and Objective: The AL22 strain was isolated from the rhizosphere soil of Alpinia galanga (L.) Willd (Zingiberaceae) and identified as Microbispora sp., by analysing its morphology, chemotaxonomy and 16S rDNA sequence. Previous studies demonstrated the bactericidal effects of its crude extract against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. The present study aimed to isolate the major compounds and evaluate their biological properties. Materials and Methods: Silica gel column chromatography and thin-layer chromatography were used for the purification and identification of 3,4-dihydro-lactucin (compound 1) and umbelliferone (compound 2) by NMR and mass spectrometry, respectively. Antibacterial and anticancer activities were carried out. Results: The bioassay studies illustrated that compound 1 had antibacterial activity against gram-positive bacteria, with its minimum inhibitory concentration and minimum bactericidal concentration of 16-32 and 64-128 μg mL^–1, respectively. The crude extract and purified compounds showed weak cytotoxic activity on the L929 and Vero cells with IC₅₀ values >512.00 μg mL^–1. The cytotoxicity of compound 1 was observed in the MDA-MB-231 and HeLa cells with IC₅₀ values of 37.62 and 75.34 μg mL^–1, respectively, while its IC₅₀ value against the HepG2 cells was 456.67 μg mL^–1. Conclusion: These findings showed that compound 1 of Microbispora sp., AL22 exhibited antibacterial and anticancer activities. Extensive studies on 3,4-dihydro-lactucin could lead to the development of beneficial approaches for managing bacterial infections and cancer.

Keywords: Microbispora sp., endophytic actinomycetes, bioactive compounds, anticancer activity, antibacterial activity, Alpinia galanga, 4-dihydro-lactucin, 3 and AL22

INTRODUCTION

Alpinia galanga (L.) Willd (Zingiberaceae) is a herbal plant that is widely distributed in Southeast Asia. Globally, it is used for food flavouring and is mostly applied as a folk medicine for treating various diseases¹. Previous studies have reported the different classes of chemical compounds of Alpinia galanga^2-4. The plants exhibited anti-allergic, anti-anxiety, anti-diabetic, anti-emetic, anti-fungal, anti-HIV, anti-hepatotoxic, anti-inflammatory, anti-microbial, anti-oxidant, anti-SAR-CoV-2, anti-tumor, anti-ulcer, cardioprotective, hypoglycaemic, immunomodulatory and neuroprotective activities⁵. Furthermore, this plant is used for the isolation of endophytic actinomycetes⁶. Antimicrobial agents that are involved in a symbiotic association with a host plant are produced by some endophytic actinomycetes⁷. Our previous study, Streptomyces sp., Tc022 was isolated from the roots of Alpinia galanga. The major active ingredient from the crude extract was identified as actinomycin D⁸. Recently, studies on endophytic actinomycetes have led to the isolation of the AL22 strain from the rhizosphere soil of Alpinia galanga (L.) Willd, which demonstrates antibacterial activity against gram-positive bacteria. This study identified the AL22 strain, purified the major compounds from the crude extract and elucidated their structures. Furthermore, the antibacterial activity against the reference strains and anticancer activity against three cancer cell lines [human hepatocellular carcinoma cells (HepG2), human cervical carcinoma cells (HeLa) and human breast carcinoma cells (MDA-MB-231)] was evaluated using an MTT colorimetric assay.

MATERIALS AND METHODS

Study area: The study was carried out at the Departments of Microbiology and Chemistry, Silpakorn University, Nakhon Pathom, Thailand, from October, 2021 to May, 2022.

Isolation, cultivation and antibacterial screening of actinomycetes: Nine samples were isolated from the rhizosphere soil of Alpinia galanga that was obtained from the four Nakhon Pathom, Thailand, from October, 2021 to November, 2021. The soil samples were dried in an oven at 50°C for 24 hrs and a 10-fold dilution was performed using distilled water and spread on the humic-acid vitamin agar containing 100 μg mL^–1 of nystatin and 50 μg mL^–1 of nalidixic acid. The actinomycetes were isolated, purified and identified⁹. Thirty-seven actinomycetes isolates were tested for their antibacterial activity against Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 7064, Staphylococcus aureus ATCC 25923, methicillin-resistant Staphylococcus aureus Sp6, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 28753 using the soft-agar overlay method on the ISP-2 agar plates¹⁰ with slight modifications. Additionally, as described in a previous study, antibacterial screening of actinomycetes was conducted by Taechowisan et al.¹¹. Among the 37 isolates of actinomycetes, isolate AL22 demonstrated the most potent antibacterial activity. This isolate was identified following the techniques previously described by Taechowisan et al.^6,11. The isolated AL22 was grown on the ISP-2 agar at 30°C for 14 days and the culture was extracted using ethyl acetate¹¹.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC): The MIC of the crude extract and purified compounds were conducted by the National Committee for Clinical Laboratory Standards micro broth dilution method¹². The purified compounds and crude extract were initially dissolved in dimethyl sulfoxide. Then, the MIC and MBC were conducted as previously described by Taechowisan et al.¹¹.

MTT assay: The cell lines [African green monkey kidney cells (Vero) and murine epithelial cells (L929)] and three cancer cell lines [human cervical carcinoma (HeLa), human hepatocellular carcinoma (HepG2) and human breast carcinoma cells (MDA-MB-231)] were used to assess the cytotoxicity of the crude extract using MTT assay¹¹.

Structural elucidation and purification of major components: Silica gel column chromatography was used to fractionate ethyl acetate extract (8.54 g) and chloroform-methanol mixture with increasing polarity was used to elute the extract. The crystallisation of fractions was obtained using 10-12% methanol in chloroform, which yielded 21.25 mg of compound 1. The fractions eluted using 16-20% methanol in chloroform were purified using thin-layer chromatography (solvent: Ethyl acetate/chloroform/methanol, 5/3/1), which yielded 25.67 mg of compound 2. The NMR spectroscopy was used to analyse the purified compounds.

Compound 1: White amorphous powder: UV λ_max (MeOH): 248 nm. IR ν_max (KBr): 1784, 1680, 1636, 1620 and 1214 cm^–1. EIMS m/z (rel. abundance %): 279 (2) [M⁺,C₁₅H₁₈O₅], 264 (3.0), 248 (3.2), 234 (9.1), 220 (3.6), 204 (3.4), 178 (9.5), 168 (8), 150 (33), 134 (20), 112 (18) and 58 (100). ¹H-NMR (δ, CD₃OD): 2.47 (3H, s, H-14), 2.50 (1H, m, H-9β), 2.55 (2H, d, J = 7.4 Hz, CH₂-3), 2.53 (1H, m, H-4), 2.75 (H, t, J = 11.0 Hz, H-9α), 3.03 (1H, t, J = 10.1, 3.0 Hz, H-7), 3.12 (1H, t, J = 29 Hz, H-5), 3.64 (1H, dd, J = 10.9, 5.6 Hz, H-15α), 3.76 (1H, dd, J = 10.1, 4Hz, H-6), 3.78 (1H, dd, J = 10.9, 4.0 Hz, H-15β), 3.80 (1H, dt, J = 4.2, 6.6 Hz, H-8), 6.17 (1H, dd, J = 3.1, 1.2 Hz, H-13α) and 6.24 (1H, dd, J = 3.1, 1.2 Hz, H-13β). The ¹³C-NMR (δ, CD₃OD): 153.5 (C-1), 208.7 (C-2), 43.8 (C-3), 38.5 (C-4), 48.2 (C-5), 83.8 (C-6), 59.4 (C-7), 69.1 (C-8), 51.2 (C-9), 135.8 (C-10), 140.4 (C-11), 176.6 (C-12), 123.6 (C-13), 23.8 (C-14) and 66.2 (C-15).

Compound 2: Colourless needles, MP 229°C-230°C. UV λ_max (MeOH): 370 nm. EIMS m/z (rel. abundance %): 164 (8) [M⁺,C₉H₆O₃], 150 (25), 140 (13), 123 (7), 112 (100), 95 (90), 82, (41), 67 (75) and 56 (89). ¹H-NMR (δ, CDCl₃+CD₃OD): 7.61 (1H, d, J = 15.8 Hz, H-4), 7.09 (1H, s, H-8), 6.99 (1H, d, J = 8.1 Hz, H-6), 6.82 (1H, δ, J = 8.1 Hz, H-5) and 6.32 (1H, d, J = 15.8 Hz, H-3). The ¹³C-NMR (δ, CDCl₃ + CD₃OD): 167.5 (C-2), 114.7 (C-3), 145.0 (C-4), 147.9 (C-4α), 114.9 (C-5), 121.4 (C-6), 144.4 (C-7), 113.6 (C-8) and 145.5 (C-8α).

In comparing the spectral data of this study with those of the previous studies. Compounds 1 and 2 were identified as 3, 4-dihydrolactucin¹³ and umbelliferone¹⁴, respectively (Fig. 1).

Statistical analysis: In this study, descriptive statistics are used to describe and summarize the characteristics of the samples.

RESULTS

Isolation, screening and identification of actinomycetes: Thirty-seven isolates were collected from the Alpinia galanga rhizosphere soil and purified on the ISP-2 medium. The isolates were examined for their antibacterial activity using the soft-agar overlay method. The antibacterial activity was calculated by measuring the diameter of the clear zone surrounding the actinomycete colony after 24 hrs incubation at 37°C. The isolate AL22 showed the highest actinomycetes inhibition activity against the gram-positive bacteria, B. cereus, B. subtilis, S. aureus and methicillin-resistant S. aureus. None of the isolates inhibited the growth of gram-negative bacteria, E. coli and P. aeruginosa.

The AL22 isolate was cultured on the ISP-2 medium with a pinkish aerial mass colony (Fig. 2a). The cell wall peptidoglycans of the AL22 isolate contained meso-diaminopimelic acid. As observed under the light microscope, the morphological characteristics of the isolate exhibited typical characteristics, which were consistent with the members of the genus Microbispora and spore chains. A pair of ovular to circular and smooth-surfaced spores were observed on aerial mycelia (Fig. 2b). The BLAST analysis of the 16S rDNA gene (1509 nt) showed that the AL22 isolate was closely related to M. rosea sub sp., rosea ATCC 12950^T (95.0%). The phylogenetic tree revealed that the strain shared a cluster with M. rosea sub sp., rosea ATCC 12950^T and M. rosea sub sp., aerata DSM 43176^T (Fig. 3). The 16S rDNA sequence reported in this article is present in the GenBank under the accession number LC683790.

MIC, MBC and anticancer activity of the crude extract and purified compounds: The antibacterial activities of the purified compounds and crude extract were shown in Table 1. The crude extract inhibited the tested bacteria, with MICs of 32-128 μg mL^–1 and MBCs of 256 to >512 μg mL^–1. Compound 1 showed potent activities against gram-positive bacteria, whereas compound 2 showed moderate activities against B. cereus ATCC 7064, B. subtilis ATCC 6633 and S. aureus ATCC 25923. However, compound 2 showed no MBC activity against any tested bacteria.

The crude extract and purified compounds exhibited weak cytotoxic activities against the Vero and L929 cells, with IC₅₀ values of >512.00 μg mL^–1 (Table 2). Alternatively, the most potent cytotoxicity of compound 1 was observed in the MDA-MB-231 and HeLa cells, with IC₅₀ values of 37.62 and 75.34 μg mL^–1, respectively, whereas the IC₅₀ value against the HepG2 cells was 456.67 μg mL^–1. Interestingly, this suggests that compound 1 is more toxic to some cancer cells than to normal cells, implying its potential as an anticancer agent. Furthermore, a more detailed study is needed to understand its action mechanism in the future.