Topical anti-inflammatory phytomedicine based on Sphagneticola trilobata dried extracts.

CONTEXT: The aerial parts of Sphagneticola trilobata (L.) Pruski (Asteraceae) are popularly used to treat topical inflammation, but have not been fully investigated. OBJECTIVE: To identify polar compounds in S. trilobata extracts and develop a new topical phytomedicine based on the kaurenoic acid (KA) content while monitoring and demonstrating its topical anti-inflammatory activity. MATERIALS AND METHODS: Ethanol spray-dried extract of S. trilobata was analysed by LC-MS while the KA content from semisolid was analysed by LC-UV. The extent of ear edema induced by applying 20 µL of croton oil (2.5%), arachidonic acid (AA; 2 mg/ear) and decanoylphorbol-13-acetate (TPA; 2.5 mg/ear) in mice was used to evaluate the biological activity of the semisolids, which were applied 30 min before the phlogistic agents. RESULTS: Eight phenylpropanoids and four oleanane-type triterpenoid saponins were identified, majority of them reported for the first time in this species, in addition to KA. The semisolid containing 1.0% of dried extract reduced the ear edema induced by croton oil [77.2 ± 4.5%; ID50 = 0.49 (0.28-0.87%)], TPA (81.5 ± 2.4%) and AA (39.1 ± 6.9%), with decreasing effect at higher KA concentrations. This was accompanied by neutrophil migration inhibition as investigated by biochemical and histological assays. DISCUSSION AND CONCLUSION: The anti-inflammatory effects were (at least in part) due to the interference in protein kinase C (PKC) activation, AA-cascade products and neutrophil migration inhibition, demonstrating the efficacy of the folk topical usage of this plant. The results support the development of a novel topical anti-inflammatory phytomedicine properly standardized to treat inflammatory dermatological diseases.

Development and validation of a stability indicative HPLC-PDA method for kaurenoic acid in spray dried extracts of Sphagneticola trilobata (L.) Pruski, Asteraceae.

A gradient stability indicative HPLC-UV method was developed and validated for assay of the marker kaurenoic acid (KA) in spray dried extract of Sphagneticola trilobata (L.) Pruski. The marker, and another unidentified polar component, were separated on a Luna Phenomenex C(18) column (250×4.6 mm, 5 µm) with mobile phase composed of acetonitrile:acidified water pH 3.0 with phosphoric acid, in a gradient run of 40 min; at a flow rate of 1.0 mL min(-1), 35 °C, using wavelengths of 210 and 338 nm. The method was linear over a KA concentration range of 4.5-30.0 µg mL(-1), without interference of the herbal matrix on the linearity of the method. The RSD% values for the intra- and inter-day precision studies were <2.0 and <8.0% for inter-laboratorial study. The method showed excellent KA recovery (99.0%). The LOQ value was found to be 1.13 µg mL(-1) and the method proved to be robust for small, deliberate changes in temperature and pH of the mobile phase with RSD%<2.5% for the KA assay. A forced degradation study of S. trilobata dried extract was conducted under conditions of visible light (1.200.000 l×h(-1)) and UV (200 Whm(-2)) irradiation, acid (0.5 mol L(-1) HCl, 30 min), basic (1 mol L(-1) NaOH, 2 h) and oxidative (30% H(2)O(2), 4h) hydrolysis, in order to develop a gradient stability-indicating LC-UV method for KA quantification, the selected marker, and also to detect the major polar components of the extract, under investigation. The KA contents remaining after these stress conditions were 72.3, 70.0, 97.6, 65.8 and 87.0%, respectively. The alkaline conditions resulted in higher degradation for the unknown polar components of the extract, without interference of supplementary peaks at the retention time of the KA. This method can be used for the KA assay and qualitative analysis of polar components in stability study of spray dried extracts of S. trilobata, for subsequent use in the quality control of dosage forms.