Phytochemical profile of Hypericum calabricum Sprengel: its role on prevention of the oxidative damage and on the inhibition of no in lps-stimulated raw 264.7 macrophages

Plant species of the genus Hypericum are well known for their use in traditional medicine due to their therapeutic efficacy. One of the most important and commercially recognized species of the genus is H. perforatum L. (St. John’s wort), which has been used in herbal medicine, externally for the treatment of skin wounds, eczema and burns, and internally for disorders of the central nervous system, the alimentary tract and other ailments (Bombardelli et al., 1995, Fitoterapia 66: 43–58; Barnes et al., 2001, J. Pharm. Pharmcol. 53: 583–600). The main constituents of the Hypericum species are: naphthodianthrones, primarily represented by hypericin and pseudohypericin, flavonoids, e.g. hyperoside, rutin or quercitrin, and phloroglucinol derivatives, e.g. hyperforin and adhyperforin (Nahrstedt et al., 1997, Pharmacopsychiatry 30: 129–134; Smelcerovic et al., 2006, Phytochemistry 67: 171–177). The present study shows for the first time the phenolic composition and the in vitro properties (antioxidant and inhibition of nitric oxide production) of Hypericum calabricum Sprengel collected in Italy. The dried aerial parts of H. calabricum (720 g) were extracted with methanol, and successively fractioned with n-hexane, dichloromethane and ethyl acetate. The ethyl acetate fraction was subjected to HPLC analysis (JASCO – PU-980 pumps; JASCO-MD-910 Multiwavelength Detector with UV-Diode Array). The content of hypericins (hypericin and pseudohypericin), hyperforin, flavonoids (rutin, hyperoside, isoquercetrin, quercitrin, quercetin and biapigenin) and chlorogenic acid of H. calabricum have been determined. The free radical-scavenger activity was determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, as described previously (Silva et al., 2002, Revista de Fitoterapia, 2: 133). Ethyl acetate fraction from the aerial parts of H. calabricum exhibited activity against the radical DPPH with IC50 value of 1.6 microg/ml. The test for inhibition of NO production was performed using the murine monocytic macrophage cell line RAW 264.7. The presence of nitrite, a stable oxidized product of NO, was determined in cell culture media by Griess reagent [1% sulfanamide and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in 2.5% H3PO4] (Green et al., 1982, Anal. Biochem. 126: 131–138). The Ethyl acetate fraction showed significant inhibition of LPS-induced NO production in RAW 264.7 cells in a dose-dependent manner with an IC50 value of 102 microg/ml. Cytotoxic effect of the sample in the presence of LPS (1 microg/ml) was also evaluated using the MTT assay (Tubaro et al., 1996, Toxicon 34: 965-74. H. calabricum extract did not show any cytotoxicity up to 500 microg/ml concentration. In conclusion in this study we have demonstrated that the extract of H. calabricum exhibited significant antioxidant activity and an inhibitory effect on production of NO (an inflammatory mediator) in macrophages.