Throughout history, herbal medicines and natural products have played a crucial role as therapeutics for humans, yet their molecular mechanisms of action often remain elusive. Here, we investigate whether primulagenin A (PGA) from the traditionally used herbal substance Primula root acts via the nuclear receptor RORγ, a key regulator of pro-inflammatory Th17 cells, which are linked to autoimmune diseases like psoriasis. Full-length luciferase assays revealed a high potency (IC50 = 119 nmol/L) and efficacy (I max = 87%) of PGA as an inverse agonist of RORγ. To ensure sufficient supply, we established methods to isolate and synthesize PGA. Its binding to the human RORγ ligand binding domain was confirmed by nano differential scanning fluorimetry, and a structure-activity relationship was proposed by docking and site-directed mutagenesis. qPCR revealed PGA-mediated downregulation of RORγ target gene expression. Furthermore, PGA inhibited murine and human Th17 differentiation in a concentration-dependent manner and reduced the proportion of IL-17A-producing Th17 cells, as assessed by flow cytometry. In this work, we identify PGA as a new, potent, and efficacious inverse agonist of RORγ, with potential for modulating immune responses in inflammatory and autoimmune diseases.

Portulaca oleracea L. (purslane) is a spontaneous herb whose shoots are appreciated in the Mediterranean and Asian diets for their fresh flavor and crunchy texture. In addition to PUFA (PolyUnsaturated Fatty Acids), it contains unusual polyphenolic alkaloids called oleraceins. This work aimed at investigating the oleracein profile of different ‘green’ extracts of the leaves of Portulaca oleracea and evaluating their antioxidant capacity. An LC-MS screening of different extracts revealed the infusion as the extract richest in oleraceins. Qualitative and quantitative analysis of oleraceins in this extract resulted in the identification of three polyphenolic alkaloids never reported before and in the definition of oleracein A as the most abundant alkaloid. An oleracein-enriched fraction from the infusion and its hydrolysed derivative exhibited radical scavenging activity in vitro and led to activation of the Nrf2 pathway in cells without apparent cytotoxicity. Thus, its oleracein content may make purslane a potential nutraceutical for alleviating redox distress.

Evodiamine (EVO) is a natural product found in Tetradium ruticarpum. It inhibits vascular smooth muscle cell (VSMC) proliferation, a key mechanism in the pathogenesis of atherosclerosis and restenosis. This study characterizes the mechanism of action behind the antiproliferative activity of evodiamine in platelet derived growth factor (PDGF)-activated VSMC. We confirmed the antiproliferative activity of EVO (0.3 and 1 µmol/L) in cultured primary VSMC by resazurin conversion and bromo-deoxyuridine (BrdU) incorporation assays, respectively, and its ability to arrest VSMC in G2/M by flow cytometric cell cycle analysis. Annexin V- Fluorescein Isothiocyanate (FITC)/propidium iodide (PI) staining and western blot analysis of caspase-3 cleavage detected low levels of apoptosis in response to 3 µmol/L EVO. We demonstrate that EVO (3 µmol/L) induces mitotic catastrophe (MC), as evidenced by characteristic nuclear morphology observed by confocal microscopy and polyploidy detected by flow cytometric DNA content analysis. Mechanistically, we rule out DNA damage as a cause of MC by western blot analysis of phospho-Ser139 histone H2A.X (γH2A.X). Instead, EVO induces centrosome amplification involving polo-like kinase 4 (PLK4) signaling. This is evident in cells co-treated with EVO (3 µmol/L) and the PLK4 inhibitor centrinone B (CENB) at 125 nmol/L by blunted centrosome amplification and cell cycle arrest. The study concludes with a proteomic analysis of purified centrosomes, which identifies candidates involved in this mechanism. In conclusion, evodiamine induces mitotic catastrophe via centrosome amplification in VSMC, positioning it as an antiproliferative agent with a distinct mechanism.

Targeting retinoic acid-related orphan receptor γ (RORγ) with inverse agonists presents a promising therapeutic strategy for treating autoimmune diseases, including psoriasis, rheumatoid arthritis, and multiple sclerosis. Through structure-based virtual screening, we identified a lupane-type pentacyclic triterpenoid, (2Z)-2-(2-furanylmethylene)-3-oxolup-20(29)-en-28-oic acid (15), as a new inverse agonist of RORγ. The compound exhibited IC50 values of 0.4 μM and 0.9 μM in Gal4-RORγ and full-length RORγ luciferase assays, respectively. Compound 15 showed improved potency and efficacy compared to a structurally related known inverse agonist, betulinic acid. Among the four additional analogues tested (15.1-15.4), two (15.2 and 15.3) also demonstrated RORγ inverse agonist activity with low micromolar IC50 values in Gal4-RORγ luciferase assay. Real-time quantitative polymerase chain reaction experiments confirmed that compounds 15, 15.2, and 15.3 downregulated RORγ target genes. Thermal shift assays showed that both betulinic acid and 15 stabilized the RORγ ligand-binding domain. Molecular docking and structure-activity relationship analysis revealed distinct binding modes within the RORγ ligand-binding domains, further supported by site-directed mutagenesis. These findings expand the repertoire of RORγ inverse agonists based on the pentacyclic triterpenoid scaffolds.

The G-protein coupled bile acid receptor 1 (GPBAR1 or TGR5) is the major cell membrane receptor for bile acids regulating metabolic and immunological functions. Its pharmacological modulation has been shown to alleviate inflammatory diseases, such as type 2 diabetes and atherosclerosis. The naturally occurring lignan leoligin and structural analogues have shown anti-inflammatory effects in vitro. However, the underlying molecular targets are still unknown. In this study, we identify the natural product-inspired synthetic structural analogue of leoligin, LT-188A (1), as a novel nonsteroidal TGR5 agonist. LT-188A (1) induced cyclic adenosine monophosphate (cAMP) accumulation and cAMP response element (CRE)-dependent luciferase activity in a concentration- and TGR5-dependent manner. Consistently, LT-188A (1) inhibited activation of the pro-inflammatory transcription factor nuclear factor κB (NFκB) only in TGR5 expressing cells. In macrophages, LT-188A (1) reduced the expression levels of pro-inflammatory cytokines and the production of nitric oxide (NO) as determined by qPCR and the Griess assay, respectively. We showed that LT-188A (1) decreased the levels of production of these inflammatory mediators in macrophages. In conclusion, we demonstrate that LT-188A (1) is a novel natural product-inspired TGR5 agonist with promising anti-inflammatory in vitro bioactivity in relevant cellular assays representing a promising tool compound with potential for further development.

The aim of our study was to determine the PPARγ agonism and hypoglycemic activity of natural phenolics isolated from Paulownia tomentosa and Morus alba. We started with a molecular docking preselection, followed by in vitro cell culture assays, such as PPARγ luciferase reporter gene assay and PPARγ protein expression by Western blot analysis. The ability of the selected compounds to induce GLUT4 translocation in cell culture and lower blood glucose levels in chicken embryos was also determined. Among the thirty-six plant phenolic compounds, moracin M showed the highest hypoglycemic effect in an in ovo experiment (7.33 ± 2.37%), followed by mulberrofuran Y (3.84 ± 1.34%) and diplacone (3.69 ± 1.37%). Neither moracin M nor mulberrofuran Y showed a clear effect on the enhancement of GLUT4 translocation or agonism on PPARγ, while diplacone succeeded in both (3.62 ± 0.16% and 2.4-fold ± 0.2, respectively). Thus, we believe that the compounds moracin M, mulberrofuran Y, and diplacone are suitable for further experiments to elucidate their mechanisms of action.

The phosphodiesterase 4 (PDE4) family comprises isoenzymes that selectively hydrolyse the second messenger cyclic adenosine monophosphate (cAMP). PDE4s are widely expressed and play key roles in various physiologic paradigms, including immune responses, memory, cognition, and metabolism. Marketed PDE4 inhibitors, such as roflumilast and apremilast, treat chronic obstructive pulmonary disease and psoriasis. Identification and characterisation of PDE4 inhibitors offer a promising strategy for targeting diverse pathological processes. In this study, leoligin, a natural lignan found in the roots of Edelweiss (Leontopodium nivale subsp. alpinum (Cass.) Greuter (Asteraceae)) and 165 analogues were screened for their PDE inhibitory activity using a cAMP accumulation assay employing an exchange protein activated by cAMP-based biosensor. Six compounds, including leoligin itself, were identified to cause a significant accumulation of cAMP, and structure-activity relationships were deduced. One analogue, designated LT-104A, showed a concentration-dependent activity with the highest determined potency (EC50 = 1.9 μM) in the cAMP accumulation assay. LT-104A was further characterised using a CRE-Luciferase assay, showing comparable activity to known PDE4 inhibitors in inducing the anti-inflammatory cAMP-PKA-CREB pathway. The inhibitory activity of LT-104A was also confirmed against recombinant PDE4D3 in a cell-free cAMP hydrolysis assay (IC50 = 9.3 μM). The potential binding mode of LT-104A with the catalytic domain of PDE4D was predicted using induced-fit docking. Lastly, a functional study was conducted in LPS-activated macrophages, where LT-104A reduced nitric oxide release and decreased mRNA expression of Il1b and Nos2. In conclusion, extensive in vitro screening of leoligin analogues led to the identification and characterisation of a novel PDE4 inhibitor, LT-104A, with potential in vitro anti-inflammatory properties.

Biochemometric approaches, which integrate bioactivity data with spectroscopic or spectrometric data, offer significant potential to streamline the discovery of bioactive compounds in targeted isolation strategies. However, the complexity of natural extracts and the presence of structurally similar analogs make this process time-consuming and resource intensive. This study introduces a 2D nuclear magnetic resonance (NMR)-based heterocovariance analysis (HetCA) workflow to identify chemical features that correlate positively or negatively with bioactivity in complex mixtures. As a proof-of-concept, the workflow was established using artificially mixed samples of pentacyclic triterpenes which were screened for modulatory activities of the retinoic acid receptor-related orphan receptor gamma (RORγ) and the G protein-coupled bile acid receptor (TGR5). The validated concept was then exemplified using a triterpene-rich Eriobotrya japonica leaf extract. The applied workflow enabled the targeted and accurate identification of bioactive constituents from E. japonica that modulate RORγ and/or TGR5 using this newly developed biochemometric 2D NMR HetCA approach.

BACKGROUND:Fructose (Fru) consumption has been suggested to contribute to metabolic diseases including metabolic dysfunction-associated steatotic liver disease (MASLD), at least in part, by disturbing intestinal barrier function and intestinal nitric oxide (NO) homeostasis. Honokiol (Hon), a neolignan found in Magnolia officinalis, has been suggested to affect intestinal integrity and barrier function. OBJECTIVES:We assessed whether Hon affects Fru-induced small intestinal permeability in settings of early MASLD. METHODS:Female 8-10-wk-old C57BL/6J mice (n = 7/group) received either a 30% Fru solution + vehicle or plain drinking water + vehicle ± Hon (10 mg/kg bw/d) for 4 wk. Liver damage [e.g. nonalcoholic fatty liver disease activity score (NAS), number of neutrophils, interleukin-6 (IL-6) protein concentration], markers of intestinal permeability (bacterial endotoxin, tight junction proteins), and NO homeostasis in the small intestine were determined in vivo as well as ex vivo in an everted sac model and in Caco-2 cells. One-way and 2-way analysis of variance were performed, respectively. RESULTS:Hon diminished the development of MASLD, which was associated with a significant lower NAS (-38%), number of neutrophils (-48%), and IL-6 protein concentrations (-38%) in livers of Fru-fed mice. Hon also attenuated Fru-induced alterations of markers of intestinal barrier function with Fru+Hon-fed mice showing lower bacterial toxin levels in portal plasma (-29%, P = 0.075), higher tight junction protein concentrations (+2.4-fold, P < 0.05), and lower NOx concentration (-44%, P < 0.05) as well as NO synthase activity (-35%) in the small intestine compared with Fru+vehicle-fed mice. Moreover, the decrease in AMP-activated protein kinase phosphorylation found in the small intestine of Fru-fed mice was significantly attenuated (+5.3-fold) by the concomitant treatment with Hon in Fru-fed mice. In support of the in vivo findings, Hon significantly attenuated Fru-induced intestinal permeability ex vivo and in Caco-2 cells. CONCLUSIONS:Our data suggest that Hon diminished the development of Fru-induced early MASLD by alleviating impairments in intestinal barrier function.

The Takeda G protein-coupled receptor 5 (TGR5) is activated endogenously by primary and secondary bile acids. This receptor is considered a candidate target for addressing inflammatory and metabolic disorders. We have targeted TGR5 with structure-based methods for ligand finding using the recently solved experimental structures, as well as structures obtained from molecular dynamics simulations. Through addressing the orthosteric as well as a putative allosteric site, we identified agonists and positive allosteric modulators. While the predicted binding locations were not in line with their efficacy, our work contributes activating small-molecule ligands that we have thoroughly characterized in vitro.

Aberrantly high dietary cholesterol intake and intestinal cholesterol uptake lead to dyslipidemia, one of the risk factors for cardiovascular diseases (CVDs). Based on previous studies, laminarin, a polysaccharide found in brown algae, has hypolipidemic activity, but its underlying mechanism has not been elucidated. In this study, we investigated the effect of laminarin on intestinal cholesterol uptake in vitro, as well as the lipid and morphological parameters in an in vivo model of high-fat diet (HFD)-fed mice, and addressed the question of whether Niemann–Pick C1-like 1 protein (NPC1L1), a key transporter mediating dietary cholesterol uptake, is involved in the mechanistic action of laminarin. In in vitro studies, BODIPY-cholesterol-labeled Caco-2 cells were examined using confocal microscopy and a fluorescence reader. The results demonstrated that laminarin inhibited cholesterol uptake into Caco-2 cells in a concentration-dependent manner (EC50 = 20.69 μM). In HFD-fed C57BL/6J mice, laminarin significantly reduced the serum levels of total cholesterol (TC), total triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C). It also decreased hepatic levels of TC, TG, and total bile acids (TBA) while promoting the excretion of fecal cholesterol. Furthermore, laminarin significantly reduced local villous damage in the jejunum of HFD mice. Mechanistic studies revealed that laminarin significantly downregulated NPC1L1 protein expression in the jejunum of HFD-fed mice. The siRNA-mediated knockdown of NPC1L1 attenuated the laminarin-mediated inhibition of cholesterol uptake in Caco-2 cells. This study suggests that laminarin significantly improves dyslipidemia in HFD-fed mice, likely by reducing cholesterol uptake through a mechanism that involves the downregulation of NPC1L1 expression.

The heartwood extract of the Ayurvedic medicinal plant Pterocarpus santalinus L. f. has previously been shown to significantly suppress the expression of CX3CL1 and other pro-inflammatory molecules in IL-1-stimulated human endothelial cells. Here, we identify the pigment-depleted extract PSD as the most promising yet still complex source of metabolites acting as an inhibitor of CX3CL1 gene expression. For the target-oriented identification of the constituents contributing to the observed in vitro anti-inflammatory effect of PSD, the biochemometric approach ELINA (Eliciting Nature's Activities) was applied. ELINA relies on the deconvolution of complex mixtures by generating microfractions with quantitative variances of constituents over several consecutive fractions. Therefore, PSD was separated into 35 microfractions by means of flash chromatography. Their 1H NMR data and bioactivity data were correlated by heterocovariance analysis. Complemented by LC-MS-ELSD data, ELINA differentiated between constituents with positive and detrimental effects towards activity and allowed for the prioritization of compounds to be isolated in the early steps of phytochemical investigation. A hyphenated high-performance counter-current chromatographic device (HPCCC+) was employed for efficient and targeted isolation of bioactive constituents. A total of 15 metabolites were isolated, including four previously unreported constituents and nine that have never been described before from red sandalwood. Nine isolates were probed for their inhibitory effects on CX3CL1 gene expression, of which four isoflavonoids, namely pterosonin A (1), santal (6), 7,3 '-dimethylorobol (12) and the previously unreported compound pterosantalin A (2), were identified as pronounced inhibitors of CX3CL1 gene expression in vitro.

In this study, the ability of six limonoids from Trichilia prieuriana (Meliaceae) to activate the liver X receptor (LXR) was assessed. One of these limonoids, flindissone, was shown to activate LXR by reporter-gene assays. Flindissone is a ring-intact limonoid, structurally similar to sterol-like LXR ligands. In endogenous cellular settings, flindissone showed an activity profile that is characteristic of LXR agonists. It induced cholesterol efflux in THP-1 macrophages by increasing the cholesterol transporter ABCA1 and ABCG1 gene expression. In HepG2 cells, flindissone induced the expression of IDOL, an LXR-target gene that is associated with the downregulation of the LDL receptor. However, unlike synthetic and similarly to sterol-based LXR agonists, flindissone did not induce the expression of the SREBP1c gene, a major transcription factor regulating de novo lipogenesis. Additionally, flindissone also appeared to be able to inhibit post-translational activation of SREBP1c. The results presented here reveal a natural product as a new LXR agonist and point to an additional property of T. prieuriana and other plant extracts containing flindissone.

Currently, the interest of consumers towards functional foods as source of bioactive compounds is increasing. The sprouts of Raphanus sativus var longipinnatus (Brassicaceae) are "microgreens" popular, especially in gourmet cuisine, for their appealing aspect and piquant flavour. They represent a functional food due to their high nutritional value and health-promoting effects. Herein, the sprouts of daikon were extracted by different solvent mixtures to highlight how this process can affect the chemical profile and the antioxidant activity. An in-depth investigation based on a preliminary LC-ESI/LTQOrbitrap/MS profiling was carried out, leading to the identification of nineteen compounds, including glucosinolates and hydroxycinnamic acid derivatives. An undescribed compound, 1-O-feruloyl-2-O-sinapoyl-β-D-glucopyranoside, was isolated, and its structure was elucidated by NMR spectroscopy. The phenolic content and radical scavenging activity (DPPH and TEAC assays), along with the ability to activate Nrf2 (Nrf2-mediated luciferase reporter gene assay) of polar extracts, were evaluated. The results showed the highest antioxidant activity for the 70% EtOH/H2O extract with a TEAC value of 1.95 mM and IC50 = 93.97 µg/mL in the DPPH assay. Some 50% and 70% EtOH/H2O extracts showed a pronounced concentration-dependent induction of Nrf2 activity. The extracts of daikon sprouts were submitted to 1H NMR experiments and then analyzed by untargeted and targeted approaches of multivariate data analysis to highlight differences related to extraction solvents.

We report a modular synthetic approach towards novel derivatives of the naturally occurring arylated benzophenone selagibenzophenone A. The initial strategy for the construction of the carbon framework of the derivatives relied on the Suzuki reaction of 2,4,6-tribromobenzonitrile, and the addition of the aryl lithium species to nitrile to generate imine. However, the formed imines showed remarkable stability toward hydrolysis. Therefore, Suzuki cross-coupling was carried out with 2,4,6-tribromobenzaldehyde and the subsequent addition of organometallic species to the aldehyde. Oxidation of the resulting alcohol ensured the access to desired ketones. The importance of the developed modular strategy is underlined by the discovery of several derivatives with selective cytotoxic effects and potential anti-inflammatory activity superior to the effect of the natural product.

Cardiovascular diseases are overall the leading cause of mortality and morbidity worldwide. Therefore, treating and preventing coronary heart disease are of high scientific interest. Among several percutaneous coronary intervention procedures, coronary artery stenting displayed potent activity against restenosis, often observed using other invasive therapies. Nowadays, drug eluting stents’ superiority over bare metal stents is increasingly recognizable, since drug eluting stents are able to overcome problems encountered with bare metal stent technology. Within this study, we developed a novel method for performing drug-releasing experiments utilizing an affordable stent model made from a readily available silver-coated copper wire, which was further coated with poly(n-butyl methacrylate). Leoligin, previously reported to inhibit intimal hyperplasia and the regrowth of endothelial cells, was exploited along with several structural analogs in drug-releasing experiments. It was found that compounds exhibiting similar biological activity can have significantly different releasing properties, a crucial parameter to know for the selection of compounds for in vivo studies.

We showed previously that capsaicin, an active compound of chili peppers, can inhibit platelet-derived growth factor-induced proliferation in primary rat vascular smooth muscle cells (VSMCs). The inhibition of BrdU incorporation by capsaicin in these cells was revoked by BCTC, which might be explained by a role of TRPV1 in VSMCs proliferation. To further pursue the hypothesis of a TRPV1-dependent effect of capsaicin, we investigated TRPV1 expression and function. Commercially available antibodies against two different TRPV1 epitopes (N-terminus and C-terminus) were rendered invalid in detecting TRPV1, as shown: i) in western blot experiments using control lysates of TRPV1-expressing (PC-12 and hTRPV1 transfected HEK293T) and TRPV1-downregulated (CRISPR/Cas gene edited A10) cells, and ii) by substantial differences in staining patterns between the applied antibodies using fluorescence confocal microscopy. The TRPV1 agonists capsaicin, resiniferatoxin, piperine and evodiamine did not increase intracellular calcium levels in primary VSMCs and in A10 cells. Using RT qPCR, we could detect a rather low TRPV1 expression in VSMCs at the mRNA level (Cp value around 30), after validating the primer pair in NGF-stimulated PC-12 cells. We conclude that rat vascular smooth muscle cells do not possess canonical TRPV1 channel activity, which could explain the observed antiproliferative effect of capsaicin.