Phenolic constituents from Wissadula periplocifolia (L.) C. Presl. and anti-inflammatory activity of 7,4′-di-O-methylisoscutellarein

Abstract This study reports the first phenolics from Wissadula genus (Malvaceae) and the anti-inflammatory activity of 7,4′-di-O-methylisoscutellarein. Using chromatographic methods, five phenolic compounds were isolated from aerial parts of Wissadula periplocifolia (L.) C. Presl. The compounds were identified as 4-hydroxybenzoic acid, 3-hydroxybenzoic acid, trans-cinnamic acid, tamgermanetin and 7,4′-di-O-methylisoscutellarein using spectroscopic methods. The flavone 7,4′-di-O-methylisoscutellarein showed anti-inflammatory activity by inhibiting neutrophils recruitment in a mice model of pleurisy and by decreasing significantly the production of cytokines IL-1β and TNF-α.


Introduction
Species in the Malvaceae family are known to produce phenolic compounds. Phytochemical investigations have described the isolation of flavonoids (Gomes et al. 2011), coumarins, ABSTRACT This study reports the first phenolics from Wissadula genus (Malvaceae) and the anti-inflammatory activity of 7,4′-di-Omethylisoscutellarein. Using chromatographic methods, five phenolic compounds were isolated from aerial parts of Wissadula periplocifolia (L.) C. Presl. The compounds were identified as 4-hydroxybenzoic acid, 3-hydroxybenzoic acid, trans-cinnamic acid, tamgermanetin and 7,4′-di-O-methylisoscutellarein using spectroscopic methods. The flavone 7,4′-di-O-methylisoscutellarein showed anti-inflammatory activity by inhibiting neutrophils recruitment in a mice model of pleurisy and by decreasing significantly the production of cytokines IL-1β and TNF-α. phenolic acids (Silva et al. 2006) along with non-phenolics (Teles et al. 2014). The use of these plants in folk medicine may be due to their anti-inflammatory properties, and they have been reportedly used for the treatment of gastrointestinal diseases, bee stings, asthma, bronchitis and skin infections (Chaves et al. 2013). Wissadula periplocifolia (L.) C. Presl. is used in folk medicine to treat snake bites and indigestion (Rahmatullah et al. 2010). The species is reported to possess better antioxidant potential among several studied Malvaceae plants (Oliveira et al. 2012), supporting the search for its phenolic constituents and the antiinflammatory investigation.
This work reports the first phenolic compounds from W. periplocifolia and the antiinflammatory evaluation of the isolated flavone 7,4′-di-O-methyl isocutellarein. Isoscutellarein derivatives, including new glucosides, have been recently identified from Lamiaceae species (Venditti, Serrilli et al. 2013;Leporini et al. 2015), and studies have demonstrated relevant biological activities for these compounds, i.e. leishmanicide and antiofidic (Urs et al. 2014;Teles et al. forthcoming) justifying the interest on the pharmacological potential of 7,4′-di-O-methyl isocutellarein.

Neutrophil recruitment inhibition in LPS-induced pleurisy
An intrapleural injection of LPS in mice induced an intense influx of neutrophils into the pleural cavity that was associated with the development of pleural inflammation. Macrophages and neutrophils play key roles in inflammation conditions by producing mediators that amplify the inflammatory response, including cytokines, lipid mediators and reactive oxygen species (Rainsford 2007). To demonstrate the anti-inflammatory effects, the animals were pre-treated orally with compound 5 (2.5 mg/kg) or dexamethasone (deXA) (2.5 mg/ kg) 1 h before the LPS challenge. This treatment significantly reduced the total number of neutrophils in the pleural lavage ( Figure 2) compared with the untreated LPS-challenged group, demonstrating that compound 5 plays an inhibitory role on neutrophil recruitment.

The effects of 7,4′-di-O-methylisoscutellarein (5) on cytokine production in vitro
Compound 5 was examined for a direct inhibitory effect on macrophage activation by analysing the production of inflammatory cytokines in vitro. As shown in Figure 3(A) and (B), the addition of LPS in peritoneal macrophage culture induced an expressive production of IL-1β and TNF-α that was significantly inhibited by pre-treatment with compound 5, indicating its inhibitory effect on neutrophil recruitment may be associated with inhibition of cytokine production (Libby 2007).

Conclusions
The phytochemical investigation of W. periplocifolia has led to the first phenolic constituents from Wissadula genus: 4-hydroxybenzoic acid, 3-hydroxybenzoic acid, trans-cinnamic acid, tamgermanetin and 7,4′-di-O-methylisoscutellarein. Isoscutellarein derivatives have been previously reported from some Malvaceae species; thus, this study contributes to the chemotaxonomy of the family by supporting the close botanical relationship between Wissadula, Sidastrum, Abutilon, Malope and Althaea genera. Furthermore, the flavone 7,4′-di-O-methylisoscutellarein showed anti-inflammatory activity by inhibiting the recruitment of neutrophils and decreasing the production of IL-1β and TNF-α in vitro, thus it could be a potential candidate for anti-inflammatory drug development.

Disclosure statement
No potential conflict of interest was reported by the authors. Figure 3. the effects of compound 5 on cytokine production in vitro. + significantly different (p < 0.05) from non-stimulated cells; *significantly different from the untreated, lPs-stimulated cells.