Indirubin

Indigotin and indirubin are bisindole alkaloids derived from plants such as Strobilanthes cusia (Nees) Ktze, with significant industrial application value. Currently, their primary preparation methods rely on plant extraction, which not only involves complex processes but also results in low yields. Although chemical synthesis is efficient, it necessitates the use of large quantities of organic reagents, contributing to environmental pollution. Additionally, both catalysts and by-products can have toxic effects on the environment and human health. With the rise of green production concepts, biocatalysis technologies that align with sustainable development principles offer new pathways for the biosynthesis of indigotin and indirubin. This study improved the flavin-dependent monooxygenase MeFMO, derived from Methylophaga sp. SK1, resulting in a mutant strain MeFMO^Q318L/W319F with a 2.1-fold increase in catalytic activity. By integrating the engineered MeFMO^Q318L/W319F with the β-glucosidase Asbg1 into a one-pot cascade system, a clean production platform was established, enabling efficient conversion of indigotin or indirubin from indican fermentation broth while eliminating intermediate purification steps. Additionally, a tryptophan-based bioconversion process was developed by integrating a heterologous indican biosynthetic module, an NADPH regeneration system, and a subsequent enzymatic oxidation-hydrolysis cascade. To address interference from fermentation broth impurities, a three-step pretreatment strategy was established to obtain a purified and concentrated indican solution. Following process optimization, the indigotin yield reached 413 mg/L with a molar conversion rate of 99.1 %, while indirubin production reached 795 mg/L with a molar conversion rate of 80.0 %.

Indigo and indirubin are the main active components of the traditional Chinese medicine Qingdai, known for their heat-clearing, detoxifying, antibacterial, and anti-inflammatory effects. Indirubin has already been applied clinically with proven safety. Both compounds show potential against drug-resistant Helicobacter pylori infection; however, their strong hydrophobicity limits further application. This study aimed to construct food-grade self-assembled nanoparticles using electrostatic and hydrophobic interactions between ovalbumin and fucoidan to efficiently encapsulate indigo and indirubin, thereby improving their solubility and evaluating the in vitro anti- Helicobacter pylori activity and underlying mechanisms of the three formulations.

At a carrier concentration of 0.75 mg/mL and a drug loading concentration of 40 μg/mL, the nanoparticles exhibited optimal stability, with an encapsulation efficiency of 68.64% and a loading capacity of 3.66%. Indigo, indirubin, and their nanoparticles showed significant inhibitory and bactericidal effects against both sensitive and drug-resistant Helicobacter pylori strains, with minimum inhibitory concentration ranges of 2.5-5, 5-32, and 2.5-5 μg/mL, respectively. The nanoparticles demonstrated superior efficacy and exhibited synergistic or additive effects when combined with antibiotics. The underlying mechanisms involved disruption of bacterial structures; downregulation of virulence genes related to flagella, adhesion, urease, and VacA; inhibition of urease activity and CagA expression; and interference with key metabolic pathways.

Indigo and indirubin exhibit significant anti-Helicobacter pylori activity, and their encapsulation within ovalbumin/fucoidan self-assembled nanoparticles markedly enhances both solubility and anti- Helicobacter pylori efficacy. This approach provides an experimental basis for the development of novel natural therapies targeting Helicobacter pylori infection.