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Activity-guided fractionation based on in vitro assays was used to identify antibacterial components in the residue wasted in the manufacturing process of aged garlic extract (AGE). Antimicrobial activity of the hexane extract of aged garlic-derived residue (AGR) was evaluated against clinically isolated Gram-positive and -negative bacteria including levofloxacin (LVX)-resistant strains. Among antimicrobial fractions separated from the hexane extract of AGR, several polysulfides, such as diallyl-tetrasulfide (DAS4), -pentasulfide (DAS5) and -hexasulfide (DAS6), were identified as active components, whose chemical structures were determined by means of LC–MS and NMR spectroscopy. We found that those sulfides exhibited antimicrobial activities against Gram-positive bacteria at minimum inhibitory concentrations (MICs) ranging from 32 to 80 μg/mL. Although these sulfides were less active than LVX against most of the bacteria examined, they were more potent against Staphylococcus aureus (S. aureus) including methicillin-resistant S. aureus (MRSA). Surprisingly, DAS5 significantly suppressed the biofilm formation of S. aureus by 60% at a concentration of 1.25 μg/mL, which is extremely lower than its MIC value. These results show that AGR contains several antimicrobial diallyl-polysulfides which have the potential to inhibit the growth and biofilm formation of bacteria and AGR may be a useful bioresource as a natural additive for antibiotic drugs or foods.

Four alkaloids, voacangine (1), isovoacangine (2), coronaridine (3), and coronaridine hydroxyindolenine (4), were isolated from the MeOH extract of Tabernaemontana divaricata aerial parts by activity-guided fractionation for Wnt signal inhibitory activity. Compounds 1-4 exhibited TCF/β-catenin inhibitory activities with IC50 values of 11.5, 6.0, 5.8, and 7.3 μM, respectively. Of these, coronaridine (3) decreased β-catenin levels in SW480 colon cancer cells, while this decrease in β-catenin was not suppressed by a co-treatment with 3 and MG132, a proteasome inhibitor. These results suggested that the decrease observed in β-catenin levels by coronaridine (3) did not depend on a proteasomal degradation process. On the other hand, the treatment of SW480 cells with coronaridine (3) caused a decrease in β-catenin mRNA levels. Thus, coronaridine (3) may inhibit the Wnt signaling pathway by decreasing the mRNA expression of β-catenin.

Wnt signaling plays important roles in proliferation, differentiation, development of cells, and various diseases. Activity-guided fractionation of the MeOH extract of the Ricinus communis stem led to the isolation of four compounds (1-4). The TCF/β-catenin transcription activities of 1 and 3 were 2.2 and 2.5 fold higher at 20 and 30μM, respectively. Cells treated with ricinine (1) had higher β-catenin and lower of p-β-catenin (ser 33, 37, 45, Thr 41) protein levels, whereas glycogen synthase kinase 3β (GSK3β) and casein kinase 1α (CK1α) protein levels remained unchanged. Cells treated with pyrvinium, an activator of CK1α, had lower β-catenin levels. However, the combined treatment of pyrvinium and 1 led to higher β-catenin levels than those in cells treated with pyrvinium alone, which suggested that 1 inhibited CK1α activity. Furthermore, 1 increased β-catenin protein levels in zebrafish embryos. These results indicated that 1 activated the Wnt signaling pathway by inhibiting CK1α.