Antibiotic Isoflavonoids, Anthraquinones, and Pterocarpanoids from Pigeon Pea (Cajanus cajan L.) Seeds against Multidrug-Resistant Staphylococcus aureus

Abstract

Cajanus cajan L. (pigeon pea, locally known in the Philippines as kadios) seed is a functional food with health benefits that extend beyond their nutritional value. C. cajan seeds contain highly diverse secondary metabolites with enriched beneficial properties, such as antibacterial, anticancer, and antioxidant activities. However, the antibacterial activities of secondary metabolites from Philippine-grown C. cajan, against multidrug-resistant Staphylococcus aureus have not been thoroughly described. Here, we investigated the in vitro antibacterial properties of C. cajan seed against multidrug-resistant S. aureus ATCC BAA-44 (MDRSA) and three other S. aureus strains (S. aureus ATCC 25923, S. aureus ATCC 6538, and coagulase-negative S. aureus) and, subsequently, identified the antibiotic markers against S. aureus strains using mass spectrometry. Secondary metabolites from C. cajan seeds were extracted using acetone, methanol, or 95% ethanol. Antibacterial screening revealed antibiotic activity for the C. cajan acetone extract. Bioassay-guided purification of the C. cajan acetone extract afforded three semi-pure high-performance liquid chromatography (HPLC) fractions exhibiting 32-64 µg/mL minimum inhibitory concentration (MIC) against MDRSA. Chemical profiling of these fractions using liquid chromatography mass spectrometry (LCMS) identified six compounds that are antibacterial against MDRSA. High-resolution mass spectrometry (HRMS), MS/MS, and dereplication using Global Natural Products Social Molecular Networking (GNPS)™, and National Institute of Standards and Technology (NIST) Library identified the metabolites as rhein, formononetin, laccaic acid D, crotafuran E, ayamenin A, and biochanin A. These isoflavonoids, anthraquinones, and pterocarpanoids from C. cajan seeds are potential bioactive compounds against S. aureus, including the multidrug-resistant strains.

Keywords: Cajanus cajan; anthraquinones; antibiotics; flavonoids; mass spectrometry; metabolomics; multidrug resistant Staphylococcus aureus; pigeon pea; pterocarpanoids.

Figure 1

Antibacterial screening of C. cajan methanol fraction against four S. aureus strains. (A) Photo of C. cajan seeds. (B) Zone of inhibition (±SD) of KADAM fraction at 20 mg/well against the multidrug-resistant S. aureus ATCC BAA-44, S. aureus ATCC 25923, S. aureus ATCC 6538, and S. aureus coagulase (concentration of 20 mg/well, exhibiting), evaluated using agar well diffusion assay. Tetracycline was used as the positive control at a concentration of 0.25 mg/well against S. aureus ATCC BAA-44, S. aureus ATCC 25923, and S. aureus ATCC 6538 and 0.05 mg/well against S. aureus coagulase (concentration of 20 mg/well, exhibiting). DMSO was used as the negative control. Error bars represent the standard deviation. The assay was conducted in triplicates.

Figure 2

Antibacterial activity of solvent-partitioned extracts against the multidrug-resistant S. aureus. (A) Plates of agar well diffusion assay showing the clearing zone when treated with C. cajan seed acetone extract KADA (A1–A3), methanol fraction KADAM (M1–M3), and ethyl acetate fraction KADAME (E1–E3) at 20 mg/well against MDRSA. Tetracycline (0.25 mg/well) and DMSO were used as positive and negative controls, respectively. (B) Zones of inhibition of the samples in mm (±SD) against S. aureus ATCC BAA-44. Error bars represent the standard deviation.

Figure 3

Bioassay-guided fractionation using flash column chromatography. (A) Chromatogram from the purification of KADAME fraction using normal phase chromatography. Peak annotated with black arrow as I3 in blue circle corresponds to the early eluting fraction KADAMEI3, which exhibited bioactivity against MDRSA. (B) Chromatogram from the subsequent purification of fraction KADAMEI3 using reversed phase flash column chromatography. Peak annotated with black arrow as I7 in blue circle corresponds to fraction KADAMEI3I7, which exhibited activity against MDRSA. UV λ_max 254 nm (red curve) and λ_max 365 nm (black curve).

Figure 4

Antibiotic kinetics assay against MDRSA. The data illustrated are the % growth inhibition (± SD) of KADAMEI3I7 (64 µg/mL), tetracycline (32 µg/mL, positive control), vancomycin (2 µg/mL, positive control), and DMSO against S. aureus ATCC BAA-44 at different time interval. Bacterial density was measured every 3 h from 0 to 24 h. Assay was conducted in triplicates. Error bars represent the standard deviation.

Figure 5

HPLC Chromatogram of KADAMEI3I7 (UV λ_max 254 nm, black curve line). HPLC Fractions H2, H3, and H4 (highlighted broken line) showed antibacterial activity against MDRSA.

Figure 6

Microbroth susceptibility assay of sixteen KADAMEI3I7 HPLC fractions (64 µg/mL), tetracycline (32 µg/mL, positive control), and vancomycin (2 µg/mL, positive control) against MDRSA. The data illustrated are average % growth inhibition (± SEM) of the samples against S. aureus ATCC BAA-44. Error bars are standard error of the mean. Assay was conducted in three replicates and in three trials. KADAMEI3I7 is the mother fraction of the sixteen HPLC fractions tested.

Figure 7

Putative identities and chemical structures of the six antibiotic metabolites of interest from C. cajan seeds.