Verification of In Vitro Anticancer Activity and Bioactive Compounds in Cordyceps Militaris-Infused Sweet Potato Shochu Spirits

Abstract

Many liqueurs, including spirits infused with botanicals, are crafted not only for their taste and flavor but also for potential medicinal benefits. However, the scientific evidence supporting their medicinal effects remains limited. This study aims to verify in vitro anticancer activity and bioactive compounds in shochu spirits infused with Cordyceps militaris, a Chinese medicine. The results revealed that a bioactive fraction was eluted from the spirit extract with 40% ethanol. The infusion time impacted the inhibitory effect of the spirit extract on the proliferation of colon cancer-derived cell line HCT-116 cells, and a 21-day infusion showed the strongest inhibitory effect. Furthermore, the spirit extract was separated into four fractions, A-D, by high-performance liquid chromatography (HPLC), and Fractions B, C, and D, but not A, exerted the effects of proliferation inhibition and apoptotic induction of HCT-116 cells and HL-60 cells. Furthermore, Fractions B, C, and D were, respectively, identified as adenosine, cordycepin, and N⁶-(2-hydroxyethyl)-adenosine (HEA) by comprehensive chemical analyses, including proton nuclear magnetic resonance (¹H-NMR), Fourier transform infrared spectroscopy (FT-IR), and electrospray ionization mass spectrometry (ESI-MS). To better understand the bioactivity mechanisms of cordycepin and HEA, the agonist and antagonist tests of the A3 adenosine receptor (A3AR) were performed. Cell viability was suppressed by cordycepin, and HEA was restored by the A3AR antagonist MR1523, suggesting that cordycepin and HEA possibly acted as agonists to activate A3ARs to inhibit cell proliferation. Molecular docking simulations revealed that both adenosine and cordycepin bound to the same pocket site of A3ARs, while HEA exhibited a different binding pattern, supporting a possible explanation for the difference in their bioactivity. Taken together, the present study demonstrated that cordycepin and HEA were major bioactive ingredients in Cordyceps militaries-infused sweet potato shochu spirits, which contributed to the in vitro anticancer activity.

Keywords: A3 adenosine receptor; N6-(2-hydroxyethyl)-adenosine; Shochu spirits; adenosine derivatives; anticancer activity; cordycepin; cordyceps militaris.

Figure 1

Inhibitory effect of infusion time (a) and storage period (b) of Cordyceps militaris-infused spirits on the proliferation of HCT-116 cells. The cells (2.2 × 10⁴ cells/cm²) were seeded in 96-well plates for 24 h and then treated for 48 h with indicated dose. Each value represents the mean ± S.D. of triplicate cultures. Columns with asterisk letters denote significant differences (* p < 0.05, ** p < 0.01).

Figure 2

Chemical profile of fractions separated by HPLC-UV/Vis.

Figure 3

Morphological changes (a), proliferation inhibition effects (b), and DNA fragmentation induction (c) of HCT-116 cells by treatment with Fractions B to D. The cells (2.2 × 10⁴ cells/cm²) were seeded in 96-well plates for 24 h and then treated for 48 h with the indicated dose. The microscope magnification was 10× and the scale bar indicates 10.0 μm. The cell viability was measured by MTT assay. For the DNA fragmentation assay, the cells (3.7 × 10³ cells/cm²) were seeded into 12-well plates and then treated for 48 h with 32 μg/mL of the various fractions. The DNA fragmentation was detected using a Cell Death Detection ELISA^PLUS kit. Each value represents the mean ± S.D. of triplicate cultures. Columns with asterisk letters denote significant differences (** p < 0.01).

Figure 4

¹H-NMR spectra of Fraction B (a), Fraction C (b), and Fraction D (c). Assignment based on ¹H-NMR spectra of standards.

Figure 5

Comprehensive structural analyses of Fraction D. (a) ¹³C−NMR spectrum, (b) FT−IR spectra, and (c) ESI−MS spectrum (positive ion mode: upper panel and negative ion mode: bottom panel) of Fraction D.

Figure 6

Effect of adenosine receptor antagonist on cordycepin (a) and HEA (b)−induced cell proliferation inhibition in HCT−116 cells. MR1523 was used as A3AR antagonist. Each value represents mean ± S.D. of triplicate cultures. Columns with asterisk letters denote significant differences (** p < 0.01).

Figure 7

Prediction of the binding region between human A3AR and adenosine (a), cordycepin (b), or HEA (c). (a–c): Docking simulations using the whole A3 molecule as a binding target. The top five poses predicted to bind using MOE−Dock are overlaid on the human A3AR structure model (gray color). Blue indicates adenosine, red indicates cordycepin, and yellow indicates HEA. Their binding sites for the five poses are circled in red.

Figure 8

Prediction of binding between human A3AR and adenosine (a), cordycepin (b), or HEA (c). Top: Three-dimensional protein poses with their binding sites displayed. Bottom: Two-dimensional view displaying the docking poses of the major compound residues interacting with protein interactions in relation to the top figure. The indicated docking simulation diagram is a typical example from among five simulation poses.