Apinun Limmongkon

Microchirita involucrata is a member of the family Gesneriaceae, a group known for producing diverse secondary metabolites, although phytochemical information for this species remains unexplored. In this study, an in vitro root culture system integrated with cyclodextrin (CD) elicitation was developed to enhance secondary metabolite biosynthesis and secretion. Bioactive constituents were isolated from the elicited culture medium extracts and subjected to comprehensive structural characterization and biological activity evaluation. Repeated CD elicitation induced a pronounced, cycle-dependent accumulation of phytochemicals in the culture medium. This work constitutes the first phytochemical investigation of M. involucrata and reports, for the first time, the isolation and structural characterization of five known compounds—α-dunnione (1), 7-hydroxy-α-dunnione (2), 7-hydroxy-6-methoxy-α-dunnione (3), 2-hydroxy-3-methoxy-7-methylanthraquinone (4), and 2,8-dihydroxy-3-methoxy-7-methylanthraquinone (5)—from this species. All isolated compounds exhibited antioxidant and α-glucosidase inhibitory activities, with compound 3 displaying the highest potency. Notably, compound 3 showed the strongest α-glucosidase inhibition (IC₅₀ = 0.73 ± 0.23 µM), which correlated with its superior antioxidant activity as determined by ABTS (59.90 ± 13.75 µmol Trolox g⁻¹ purified fraction) and FRAP (21.43 ± 1.83 mg ascorbic acid g⁻¹ purified fraction) assays. Integrated molecular docking and FMO analyses revealed that compound 3 displayed enhanced inhibitory potency through strong stabilizing interactions with the key catalytic residues Asp214, Glu276, Asp349, and Arg439 in the α-glucosidase active site. Overall, these findings establish M. involucrata as a previously uncharacterized source of bioactive quinone derivatives and demonstrate the effectiveness of repeated CD elicitation as a strategy for extracellular metabolite production in plant in vitro culture systems.

Microchirita involucrata (family Gesneriaceae) has recently been reassigned to the genus Microchirita ; however, its biological properties and potential applications remain unexplored in Thailand. Although the species is valued for its compact growth habit and attractive floral morphology, its phytochemical potential has received little attention. This study established an in vitro culture platform for M. involucrata , identified explants with high secondary metabolite potential, optimized metabolite accumulation through elicitation, and evaluated the biological activities of elicited root cultures. Thin-layer chromatography (TLC) screening revealed that root tissues contained the highest number of antioxidant-active bands; therefore, in vitro –derived root segments were selected for elicitation using methyl jasmonate (MeJA), chitosan (CHT), and β-cyclodextrin (CD), applied individually and in combination (MeJA + CD and CHT + CD). CD alone and combined treatments significantly enhanced antioxidant activity. Root tissue extracts exhibited higher antioxidant capacity, whereas culture medium extracts showed markedly stronger lipoxygenase (LOX) inhibitory and antibacterial activities. Culture media derived from CD and combined elicitor treatments displayed the highest LOX inhibition, with values ranging from 72.04% to 94.35%. Among all treatments, the MeJA + CD–elicited culture medium extract demonstrated the strongest antibacterial activity, with minimum inhibitory and bactericidal concentrations of 0.78 mg/mL against Staphylococcus aureus . Against Escherichia coli , the corresponding MIC and MBC values were 1.56 and 3.13 mg/mL, respectively. Overall, these findings provide new insights into elicitor-induced secondary metabolism in M. involucrata in vitro root cultures, supporting its relevance as a source of biologically active metabolites.

Prenylated stilbenoids, particularly trans -arachidin-1 (Ara-1) and trans -arachidin-3 (Ara-3), have gained attention for their notable bioactivities and potential health-promoting properties. This study presents the first comprehensive investigation into the stability and biological efficacy of these compounds in both peanut hairy root culture crude extracts (PCE) and partially purified fractions derived from elicited peanut hairy root cultures. PCE stored at –20 °C and 4 °C maintained higher antioxidant capacity, total phenolic content compared to samples stored at room temperature. In cytotoxicity assays using SW480 colon cancer cells, the extract stored at –20 °C retained bioactivity with only minor changes in IC₅₀ values over three months, demonstrating superior stability under frozen conditions. Over a six-month period, partially purified fractions of Ara-1 and Ara-3 showed a time-dependent decline in compound content. However, Ara-3 maintained strong cytotoxicity against KKU-100 cholangiocarcinoma cells, while Ara-1 exhibited a significant loss in activity. These findings demonstrate that low-temperature storage, particularly at –20 °C, is crucial for preserving the chemical integrity and bioactivity of stilbenoid-rich extracts. The study underscores the importance of optimizing storage conditions to ensure consistent bioactivity, supporting the potential application of these compounds in the development of stable and effective pharmaceutical or nutraceutical products.

Numerous stressors are known to trigger the synthesis of phytoalexins in plants. Specifically, stilbenoids, notably trans-arachidin compounds, are induced in elicited peanut hairy root cultures. This study employed two distinct elicitation strategies on peanut hairy root cultures and analyzed stilbenoid profiles alongside their respective biological effects. Results from HPLC-UV revealed that a combination of paraquat, methyl jasmonate, and cyclodextrin (PQ + MeJA + CD) over 72 hrs notably stimulated the production of trans-resveratrol (Res), surpassing levels of trans-arachidin-1 (Ara-1) and trans-arachidin-3 (Ara-3) in the culture medium. Conversely, elicitation with chitosan, methyl jasmonate, and cyclodextrin (CHT + MeJA + CD) led to higher concentrations of Ara-1 and Ara-3 compared to Res in the medium. LC-HRMS/MS and subsequent molecular networking revealed chemical diversity among trans-arachidins and related compounds in peanut hairy root samples. Differential comparison of elicitation mixtures using CHT instead of PQ in the elicitation mixture led to increased metabolite abundances in both roots and the culture medium, with higher levels observed in the medium samples. MTT assays on HCT116 human colon cancer cells demonstrated greater cytotoxicity of extracts from the culture medium compared to root tissue extracts. This study underscores that distinct stilbenes with comparable bioactivities can be induced through various elicitation methods in peanut hairy root cultures. However, the use of CHT as an elicitor holds particular interest due to its non-toxic and environmentally friendly nature compared to PQ.