Abstract
Given their medical importance, natural products represent a tremendous source of drug discovery. The aim of this study was to investigate Malva sylvestris L. extract and fractions and their pharmacological activities followed by chemical identification. The aqueous fraction (AF) was identified as the bioactive fraction in the in vitro and in vivo assays. The AF controlled the neutrophil migration to the peritoneal cavity by 66%, inhibited the antiedematogenic activity by 58.8%, and controlled IL-1β cytokine expression by 54%. The in vitro viability tests showed a concentration-dependent effect, where the MSE and fractions at concentrations under 10 μg/mL were non-toxic to cells. Transcriptional factors of carbonic anhydrase II (CAII), cathepsin K (Ctsk) and tartrate-resistant acid phosphatase (TRAP) were analyzed by qPCR in RAW 264.7 cell lines. The gene expression analysis showed that the AF was the only treatment that could downregulate all the study genes: CAII, Ctsk and TRAP (p<0.05). TRAP staining was used to evaluate osteoclast formation. AF treatments reduced the number of osteoclastogenesis 2.6-fold compared to the vehicle control group. Matrix metalloproteinase 9 (MMP-9) activity decreased 75% with the AF treatment. Moreover, the bioactive fraction had the ability to regulate the oxidation pathway in the ABTS (2,2-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) assay with an activity equivalent to 1.30 μmol Trolox/g and DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals 1.01 g/L. Positive ion ESI-mass spectrometry for molecular ions at m/z 611 and 633 confirmed rutin as the major compound in the AF. The AF of M. sylvestris presented anti-inflammatory, controlled osteoclastogenic mechanisms and antioxidant abilities in different in vitro and in vivo methods. In addition, we suggest that given its multi-target activity the bioactive fraction may be a good candidate in the therapy of chronic inflammatory diseases.
Highlights
Inflammation, a biological process that involves vascular and cellular events coordinated by mediators like prostaglandin, leukotrienes, cytokines and thromboxanes that include an essential and protective mechanism of the organism in response to injury, infection and trauma [1,2]
Prolonged inflammation may lead to chronic diseases including rheumatoid arthritis and periodontitis, which are associated with tissue injury and bone resorption [2]
A number of the RANK-induced signaling pathways in osteoclasts induce the expression of enzymes involved in regulating the dissolution of mineral and collagen, including carbonic anhydrase (CAII), cathepsin K (Ctsk) and tartrateresistant acid phosphatase (TRAP) [8,9]
Summary
Inflammation, a biological process that involves vascular and cellular events coordinated by mediators like prostaglandin, leukotrienes, cytokines and thromboxanes that include an essential and protective mechanism of the organism in response to injury, infection and trauma [1,2]. Prolonged inflammation may lead to chronic diseases including rheumatoid arthritis and periodontitis, which are associated with tissue injury and bone resorption [2]. The classic pathway for NF-κB stimulation includes an upregulation of RANKL, an osteoclastogenic cytokine that induces bone resorption by promoting osteoclast differentiation and activation [4]. RANKL plays an essential role in the differentiation, recruitment, activation, and survival of osteoclasts by binding to its receptor (RANK) on osteoclasts or progenitor cells [6]. A number of the RANK-induced signaling pathways in osteoclasts induce the expression of enzymes involved in regulating the dissolution of mineral and collagen, including carbonic anhydrase (CAII), cathepsin K (Ctsk) and tartrateresistant acid phosphatase (TRAP) [8,9]. The main role of RANKL is the signaling and the regulation of homeostasis leading to the system to health or disease state. [7]
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