Hydroethanolic Extract of Morus nigra L. Leaves: A Dual PPAR-α/γ Agonist with Anti-Inflammatory Properties in Lipopolysaccharide-Stimulated RAW 264.7

Inhibition of systemic inflammation has been a beneficial strategy in treating several non-communicable diseases, which represent one of the major causes of mortality in the world. The Peroxisome Proliferator-Activated Receptors (PPAR) are interesting pharmacological targets, since they can act both through the metabolic and anti-inflammatory pathways. Morus nigra L. has flavonoids in its chemical composition with recognized anti-oxidant activity and often associated with anti-inflammatory activity. Therefore, this study aimed to evaluate the hydroethanolic extract of M. nigra leaves' ability to activate PPAR and promote anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated murine macrophage cells. The leaf extract was prepared by cold maceration, and the chemical profile was obtained by HPLC-DAD. Activation of PPAR α and γ was evaluated by the luciferase reporter assay. The anti-inflammatory activity was assessed by measuring the reactive oxygen species (ROS), nitric oxide (NO), and Tumor Necrosis Factor-α (TNF-α) in RAW 264.7 cells after stimulation with LPS from Escherichia coli. The HPLC-DAD analysis identified two major compounds: rutin and isoquercitrin. The extract showed agonist activity for the two types of PPAR, α and γ, although its major compounds, rutin and isoquercitrin, did not significantly activate the receptors. In addition, the extract significantly reduced the production of ROS, NO, and TNF-α. Treatment with the specific PPAR-α antagonist, GW 6471, was able to partially block the anti-inflammatory effect caused by the extract.

Hippeastrum stapfianum (Kraenzl.) R.S.Oliveira & Dutilh (Amaryllidaceae) Ethanol Extract Activity on Acetylcholinesterase and PPAR-α/γ Receptors.

Hippeastrum stapfianum (Kraenzl.) R.S.Oliveira & Dutilh (Amaryllidaceae) is an endemic plant species from the Brazilian savannah with biological and pharmacological potential. This study evaluated the effects of ethanol extract from H. stapfianum leaves on acetylcholinesterase enzyme activity and the action on nuclear receptors PPAR-α and PPAR-γ. A gene reporter assay was performed to assess the PPAR agonist or antagonist activity with a non-toxic dose of H. stapfianum ethanol extract. The antioxidant capacity was investigated using DPPH• scavenging and fosfomolybdenium reduction assays. The identification of H. stapfianum's chemical composition was performed by gas chromatography-mass spectrometry (GC-MS) and HPLC. The ethanol extract of H. stapfianum activated PPAR-α and PPAR-γ selectively, inhibited the acetylcholinesterase enzyme, and presented antioxidant activity in an in vitro assay. The major compounds identified were lycorine, 7-demethoxy-9-O-methylhostasine, and rutin. Therefore, H. stapfianum is a potential source of drugs for Alzheimer's disease due to its ability to activate PPAR receptors, acetylcholinesterase inhibition activity, and antioxidant attributes.

In vitro effects of European and Latin-American medicinal plants in CYP3A4 gene expression, glutathione levels, and P-glycoprotein activity.

Many medicinal plants species from European -such as Artemisia absinthium, Equisetum arvense, Lamium album, Malva sylvestris, Morus nigra, Passiflora incarnata, Frangula purshiana, and Salix alba- as well as Latin American traditions -such as Libidibia ferrea, Bidens pilosa, Casearia sylvestris, Costus spicatus, Monteverdia ilicifolia, Persea americana, Schinus terebinthifolia, Solidago chilensis, Syzygium cumini, Handroanthus impetiginosus, and Vernonanthura phosphorica- are shortlisted by the Brazilian National Health System for future clinical use. However, they lack many data on their action upon some key ADME targets. In this study, we assess non-toxic concentrations (up to100 µg/ml) of their infusions for in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). We further investigated the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of Gamma-glutamyl transferase (GGT) in HepG2 cells. Our results demonstrate L. ferrea, C. sylvestris , M. ilicifolia, P. americana, S. terebinthifolia, S. cumini, V. phosphorica, E. arvense, P. incarnata, F. purshiana, and S. alba can significantly increase CYP3A4 mRNA gene expression in HepG2 cells. Only F. purshiana shown to do so likely via hPXR activation. P-gp activity was affected by L. ferrea, F. purshiana, S. terebinthifolia, and S. cumini. Total intracellular glutathione levels were significantly depleted by exposure to all extracts except S. alba and S. cumini This was accompanied by a lower GGT activity in the case of C. spicatus, P. americana, S. alba, and S. terebinthifolia, whilst L. ferrea, P. incarnata and F. purshiana increased it. Surprisingly, S. cumini aqueous extract drastically decreased GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines causes in vitro disturbances to key drug metabolism mechanisms. We recommend active pharmacovigilance for Libidibia ferrea (Mart.) L. P. Queiroz, Frangula purshiana Cooper, Schinus terebinthifolia Raddi, and Salix alba L. which were able to alter all targets in our preclinical study.