Bupleurum chinense Roots: a Bioactivity-Guided Approach toward Saponin-Type NF-κB Inhibitors

The roots of Bupleurum chinense have a long history in traditional medicine to treat infectious diseases and inflammatory disorders. Two major compounds, saikosaponins A and D, were reported to exert potent anti-inflammatory activity by inhibiting NF-κB. In the present study, we isolated new saikosaponin analogues from the roots of B. chinese interfering with NF-κB activity in vitro. The methanol-soluble fraction of the dichloromethane extract of Radix Bupleuri was subjected to activity-guided isolation yielding 18 compounds, including triterpenoids and polyacetylenes. Their structures were determined by spectroscopic methods as saikogenin D (1), prosaikogenin D (2), saikosaponins B2 (3), W (4), B1 (5), Y (6), D (7), A (8), E (9), B4 (10), B3 (11), and T (12), saikodiyne A (13), D (14), E (15) and F (16), falcarindiol (17), and 1-linoleoyl-sn-glycero-3-phosphorylcholine (18). Among them, 4, 15, and 16 are new compounds, whereas 6, previously described as a semi-synthetic compound, is isolated from a natural source for the first time, and 13–17 are the first reports of polyacetylenes from this plant. Nine saponins/triterpenoids were tested for inhibition of NF-κB signaling in a cell-based NF-κB-dependent luciferase reporter gene model in vitro. Five of them (1, 2, 4, 6, and 8) showed strong (> 50%, at 30 µM) NF-κB inhibition, but also varying degrees of cytotoxicity, with compounds 1 and 4 (showing no significant cytotoxicity) presenting IC50 values of 14.0 µM and 14.1 µM in the cell-based assay, respectively

Polyacetylenes from Notopterygium incisum–New Selective Partial Agonists of Peroxisome Proliferator-Activated Receptor-Gamma

Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of glucose and lipid metabolism and therefore an important pharmacological target to combat metabolic diseases. Since the currently used full PPARγ agonists display serious side effects, identification of novel ligands, particularly partial agonists, is highly relevant. Searching for new active compounds, we investigated extracts of the underground parts of Notopterygium incisum, a medicinal plant used in traditional Chinese medicine, and observed significant PPARγ activation using a PPARγ-driven luciferase reporter model. Activity-guided fractionation of the dichloromethane extract led to the isolation of six polyacetylenes, which displayed properties of selective partial PPARγ agonists in the luciferase reporter model. Since PPARγ activation by this class of compounds has so far not been reported, we have chosen the prototypical polyacetylene falcarindiol for further investigation. The effect of falcarindiol (10 µM) in the luciferase reporter model was blocked upon co-treatment with the PPARγ antagonist T0070907 (1 µM). Falcarindiol bound to the purified human PPARγ receptor with a Ki of 3.07 µM. In silico docking studies suggested a binding mode within the ligand binding site, where hydrogen bonds to Cys285 and Glu295 are predicted to be formed in addition to extensive hydrophobic interactions. Furthermore, falcarindiol further induced 3T3-L1 preadipocyte differentiation and enhanced the insulin-induced glucose uptake in differentiated 3T3-L1 adipocytes confirming effectiveness in cell models with endogenous PPARγ expression. In conclusion, we identified falcarindiol-type polyacetylenes as a novel class of natural partial PPARγ agonists, having potential to be further explored as pharmaceutical leads or dietary supplements

Plant extracts in cell-based anti-inflammatory assays—Pitfalls and considerations related to removal of activity masking bulk components

Plants used in traditional medicine represent an important source of new lead compounds. However, cell-based in vitro screening assays with plant material are hampered by the complex nature of plant extracts as mixtures of active and inactive components. Bulk constituents, such as chlorophyll and polyphenols were previously shown to interfere with several biological in vitro assays. Their influence on anti-inflammatory cell-based testing systems has not been thoroughly investigated. Hence, the present study was aimed at comparing different procedures for the removal of bulk constituents from plant extracts and examining the influence of their elimination on selected cell-based anti-inflammatory assays. Malva sp. and Glechoma hederacea L., two plants used in traditional European medicine for the treatment of inflammatory disorders, were subjected to three different methods for the removal of chlorophyll and polyphenols, respectively. Removal of bulk constituents was confirmed by HPLC and mass spectrometry. Extracts were tested before and after the purification procedure, to determine their potential to inhibit the activation of the transcription factor NF-κB in reporter gene assay and to interfere with the secretion of the chemokine IL-8 after stimulation of endothelial cells with tumor necrosis factor (TNF-α) or lipopolysaccharide (LPS). Removal of chlorophyll from tested extracts led to a strong decrease in the anti-inflammatory activities, due to loss of bioactive constituents. In contrast, the effect of the polyphenol-free extracts was either not changed or significantly increased, depending on the purification method used. The study concluded that clearance of bulk compounds represents a valuable strategy for cell-based in vitro anti-inflammatory evaluation of plant extracts. Liquid–liquid partitioning was identified as the optimal method for the elimination of both chlorophyll and polyphenols. It is recommended that removal of chlorophyll from extracts always be accompanied by HPLC profiling to detect a possible loss of active constituents

Bioatividade de três espécies vegetais nativas da Floresta Atlântica brasileira frente ao microcrustáceo Artemia salina

Este trabalho teve por objetivo a investigação fitoquímica e propriedades antioxidantes de extratos das folhas de Trigynaea oblongifolia Schltdl (Annonaceae), Ottonia frutescens Trel (Piperaceae), e Bathysa australis (St Hill) Hooz (Rubiaceae), bem como avaliar, in vitro, a letalidade frente ao microcrustáceo Artemia salina Leach. Os extratos foram preparados por maceração em metanol 10% (p/v) por sete dias, à temperatura ambiente. A atividade antioxidante dos extratos foi determinada pela metodologia que utiliza o radical estável DPPH. A toxicidade dos extratos foi avaliada frente ao microcrustáceo A. salina. Os extratos de O. frutescens e B. australis apresentaram as seguintes classes de metabólitos secundários: Alcalóides, Antraquinonas, Cumarinas, Polifenóis (Taninos), Saponinas. Nos extratos de T. oblongifolia, além dos metabólitos citados anteriormente, foi detectada a presença de Flavonóides. A atividade antioxidante, observada em 30 minutos na concentração de 24 µg/mL de extrato, foi de: O. frutescens - 38,3%, T. oblongifolia - 32,3%, e B. australis - 32,1%. A Concentração Letal, CL50, dos extratos em A. salina foi de: O. frutescens - 149,75 ± 1,02 µg/mL, T. oblongifolia - 148,8 ± 1,74 µg/mL, e B. australis - 684 ± 9,04 µg/mL. Neste contexto, destacamos as espécies, nativas da Floresta Atlântica, O. frutescens e T. oblongifolia de grande potencial na bioprospecção de moléculas biologicamente ativas