Researchers from the Czech Republic have identified a group of prenylated phenolic compounds in the underground parts of Glycyrrhiza uralensis that significantly inhibit the NF-κB signaling pathway in human monocyte-like cells. The work demonstrates an effective bioassay-guided approach to separating and identifying these anti-inflammatory molecules, which are distinct from the well-known sweetener glycyrrhizin typically extracted from the plant.
The genus Glycyrrhiza, commonly known as licorice, comprises 39 accepted species within the family Fabaceae. While the primary agricultural use of the plant is the extraction of glycyrrhizin as a sweetener, a significant proportion of bioactive secondary metabolites remain underutilized during industrial processing. Although only three species are currently described in pharmacopoeias for their pharmacological relevance, other species have historically been used in traditional medicines for their anti-inflammatory properties. The study focused on two pharmacopeial species, G. glabra and G. uralensis, alongside four less-studied species: G. foetida, G. echinata, G. pallidiflora, and G. yunnanensis. The team aimed to compare their in vitro anti-inflammatory activities and isolate specific compounds responsible for these effects.
The research team utilized an experimental model based on THP-1 human monocyte-like cells equipped with a secreted embryonic alkaline phosphatase (SEAP) reporter gene. This system allows for the monitoring of NF-κB signaling pathway expression, a key regulator of inflammation. The inflammatory response in these cells is triggered by lipopolysaccharide (LPS) through toll-like receptor 4 (TLR4), leading to the translocation of the NF-κB subunit p65 to the nucleus. By labeling p65 with antibodies, the researchers could quantify the extent of this nuclear translocation, providing a direct measure of the compounds’ ability to interfere with the inflammatory cascade.
For the initial screening, underground parts of the six species were extracted using ethanol and partitioned into n-hexane, chloroform (CHCl3), and ethyl acetate (EtOAc) fractions. HPLC-DAD analysis revealed chemotaxonomic differences among the species, with G. glabra and G. uralensis containing higher amounts of lipophilic phenolics compared to the others. The CHCl3-soluble extracts demonstrated significant inhibition of the NF-κB signaling pathway in the QUANTI-Blue™ assay. At a concentration of 3 μg/mL, the CHCl3-soluble extract from G. uralensis showed the most prominent effect, inhibiting NF-κB expression below 20%. In contrast, the EtOAc-soluble extracts were mostly inactive at the same concentration, suggesting that the anti-inflammatory activity is linked to the lipophilic phenolic content rather than the more polar flavonoid glycosides.
Based on these results, G. uralensis was selected for the bioassay-guided isolation of bioactive constituents. The team processed 2.2 kg of fresh underground parts to yield 186 g of crude extract, from which 18 g of CHCl3-soluble fraction was obtained. This fraction was subjected to flash chromatography on RP-C18, yielding fifteen fractions. Five of these fractions, designated GUC10 through GUC14, exhibited the most significant NF-κB inhibitory activity. These active fractions were further purified using semi-preparative HPLC on an RP-Amide column, resulting in the isolation of nine pure compounds with purity above 95% as confirmed by HPLC-DAD and NMR analysis.
The isolated compounds included three prenylated isoflavanones and six prenylated isoflavones. Specific compounds identified were 3′-(γ,γ-dimethylallyl)-kievitone, gancaonin H, glisoflavanone, isoangustone A, 2′-hydroxyisolupalbigenin, licoisoflavanone C, 6,8-diprenylgenistein, angustone B, and isochandalone. The structures were elucidated using NMR and high-resolution mass spectrometry data, with the configuration of ring B in certain isoflavanones determined by chemical shift analysis. The absolute configurations of the isoflavanones were not determined due to the absence of Cotton effects in ECD analysis, suggesting a racemic mixture. This isolation process highlights the potential of diprenylated isoflavonoids as a class of NF-κB inhibitors.
When tested at a concentration of 1 μM, all isolated compounds except gancaonin H showed significant inhibitory activity on the NF-κB signaling pathway. Compounds 1 and 8 were the most active, downregulating NF-κB expression below 50%. Isochandalone also showed significant inhibition, though less pronounced than the other compounds. The team further evaluated the effects of compounds 1, 4, 5, and 8 on the nuclear translocation of p65 using immunocytochemistry. These compounds were selected based on their stability and prior NF-κB inhibition results. The analysis revealed that all four compounds significantly inhibited LPS-induced p65 nuclear translocation compared to the DMSO control, with compound 5 showing the most pronounced effect in this specific assay.
The study suggests that the lipophilic nature of prenylated phenolics enhances their interaction with biological membranes, thereby increasing their effectiveness in inhibiting inflammatory pathways. The prenyl moieties may facilitate the compounds’ ability to interfere with NF-κB signaling or reduce the expression of downstream inflammatory mediators. Although the exact molecular mechanism was not fully elucidated, the results suggest that the compounds may act on upstream signaling pathways, potentially via the inhibition of the IKK complex. However, the data presented cannot distinguish between direct IKK inhibition and the inhibition of other upstream activators, such as TAK1 or PKC, which remain hypotheses requiring further biochemical investigation.
One notable observation was that some chromatographic fractions exhibited more pronounced inhibitory effects than the isolated pure compounds. In some cases, fractions suppressed NF-κB expression below basal levels. This finding raises the possibility of synergistic interactions among the constituents or the presence of unidentified minority metabolites. However, the study notes that it does not provide sufficient experimental evidence to establish true synergism. The chemical stability of the compounds was also examined, revealing that isoflavanones underwent apparent degradation in DMSO over six months, even at low temperatures, while isoflavones remained stable. This difference in stability suggests that prenylated isoflavones may be more favorable candidates for further development as bioactive food ingredients or lead compounds.
The researchers acknowledge several limitations in the study. All biological experiments were performed in vitro using a single cell line, meaning the results cannot be directly extended to other cell types or in vivo systems. The pure compounds were tested at only one concentration, preventing the determination of dose-dependent effects or IC50 values. Additionally, the chemical profiling of the extracts was qualitative, and analytical standards were not used to confirm the identity of all detected compounds. The chemical stability of the compounds during the assays was not systematically evaluated, leaving open the possibility of changes in compound integrity under experimental conditions. These limitations highlight the need for further studies to identify specific targets and pathways involved in the observed anti-inflammatory effects.
Despite these limitations, the study provides a valuable comparative analysis of Glycyrrhiza species and identifies G. uralensis as a promising candidate for the isolation of anti-inflammatory compounds. The bioassay-guided separation technique demonstrated an effective approach to selectively isolating bioactive components, which can be applied to various medicinal plants. The development of a high-throughput method for quantifying p65 cellular localization by immunocytochemistry also represents a methodological advance in the field. The findings highlight the enormous potential of diprenylated isoflavonoids as NF-κB inhibitors, opening avenues for further research into their detailed anti-inflammatory activity and the possible discovery of new synthetic routes for their preparation.
The study was conducted by a team from the Czech Republic, with plant material identified and voucher specimens maintained at the MENDELU Faculty of Horticulture in Lednice and the Faculty of Pharmacy at Masaryk University in Brno. For the initial screening, fresh material was washed, mechanically disintegrated, and extracted three times with ethanol over 24 hours, assisted by sonication. This process yielded crude extracts that were sequentially partitioned using n-hexane, chloroform, and ethyl acetate. The researchers noted that while the primary agricultural significance of Glycyrrhiza lies in the extraction of glycyrrhizin as a sweetener, a large proportion of bioactive secondary metabolites remain underutilized during industrial processing. The focus on prenylated phenolics stems from their potential to enhance membrane permeability while preserving hydrogen-bonding capacity, which may allow them to interfere with NF-κB signaling more effectively than non-prenylated phenolics.
Statistical analysis of the bioactivity data was performed using PRISM software, with outliers identified by the ROUT method. The QUANTI-Blue™ assay results were expressed as mean ± SD for three independent experiments in six technical replicates. For the p65 nuclear translocation assay, fluorescence images were analyzed using CellProfiler software, which quantified the ratio of mean nuclear to mean cytoplasmic p65 fluorescence intensity for each cell. The groups were compared using a Kruskal–Wallis test followed by Dunn’s post hoc analysis with multiple comparison correction. This rigorous statistical framework supported the finding that the CHCl3-soluble extracts from all six species exhibited highly significant effects on NF-κB inhibition, with p-values less than 0.0001 when compared to the DMSO control. The selection of G. uralensis for further isolation was based on its extract inhibiting NF-κB expression below 20%, a result that was statistically robust and distinct from the other species tested.
The isolation process involved flash chromatography on RP-C18, followed by semi-preparative HPLC on an RP-Amide column. The purity of the isolated compounds was confirmed to be above 95% by HPLC-DAD at detection wavelengths of 254.4 nm for isoflavones and 280 nm for isoflavanones. This purity was further supported by 1H NMR analysis. The configuration of ring B in compounds 6 and 8 was determined by the chemical shifts of the 5-OH proton in acetone-d6, which were measured at 12.41 and 12.77 ppm, respectively. The absolute configurations of the isoflavanones were not determined because the ECD analysis did not show any Cotton effect, suggesting a racemic mixture.
The study also examined the chemical stability of the isolated compounds, noting that isoflavanones underwent apparent degradation in DMSO over six months, even at −80 °C. This degradation may be linked to their spatial conformation and lack of resonance stabilization, as well as a propensity for intramolecular cyclization to pterocarpans or racemization. In contrast, the isoflavones remained stable under these conditions. This difference in stability suggests that prenylated isoflavones may be more favorable candidates for further development as bioactive food ingredients or lead compounds, given their chemical robustness and the well-described routes of synthetic preparation available in the literature. The findings underscore the importance of considering chemical stability when selecting natural products for further pharmacological investigation.
Subject of Research: Bioassay-guided isolation of anti-inflammatory phenolic constituents from Glycyrrhiza species
Article Title: Bioassay-guided isolation of anti-inflammatory phenolic constituents from Glycyrrhiza species
Article References: Markuš, J., Smištík, O., Treml, J., Gorbulina, M., Kulíšková, L., Neugebauerová, J., Skružná, J., Švajdlenka, E., Šmejkal, K., & Malaník, M. (2026). Bioassay-guided isolation of anti-inflammatory phenolic constituents from Glycyrrhiza species. Journal of Agriculture and Food Research, 32, Article 103391. https://doi.org/10.1016/j.jafr.2026.103391
Image Credits: AI Generated
DOI: 10.1016/j.jafr.2026.103391
Keywords: Bioassay-guided, isolation, anti-inflammatory, phenolic, constituents, Glycyrrhiza, species, scientific research
News Source: Alan Morgan. (November 9, 2026). Licorice Compounds Show Potent Anti-Inflammatory Activity in Cell Models. Scienmag.
