Manuel Fernandes-Ferreira

Luteolin, quercetin and ursolic acid are potent inhibitors of proliferation and inducers of apoptosis in both KRAS and BRAF mutated human colorectal cancer cells.

KRAS and BRAF mutations are frequent in colorectal carcinoma (CRC) and have the potential to activate proliferation and survival through MAPK/ERK and/or PI3K signalling pathways. Because diet is one of the most important determinants of CRC incidence and progression, we studied the effects of the dietary phytochemicals quercetin (Q), luteolin (L) and ursolic acid (UA) on cell proliferation and apoptosis in two human CRC derived cell lines, HCT15 and CO115, harboring KRAS and BRAF activating mutations, respectively. In KRAS mutated HCT15 cells, Q and L significantly decreased ERK phosphorylation, whereas in BRAF mutated CO115 cells the three compounds decreased Akt phosphorylation but had no effect on phospho-ERK. Our findings show that these natural compounds have antiproliferative and proapoptotic effects and simultaneously seem to act on KRAS and PI3K but not on BRAF. These results shed light on the molecular mechanisms of action of Q, L and UA and emphasize the potential of dietary choices for the control of CRC progression.

Salvia Fruticosa, Salvia Officinalis, and Rosmarinic Acid Induce Apoptosis and Inhibit Proliferation of Human Colorectal Cell Lines: The Role in MAPK/ERK Pathway

Epidemiological studies have shown that nutrition is a key factor in modulating sporadic colorectal carcinoma (CRC) risk. Aromatic plants of the genus Salvia (sage) have been attributed many medicinal properties, which include anticancer activity. In the present study, the antiproliferative and proapoptotic effects of water extracts of Salvia fruticosa (SF) and Salvia officinalis (SO) and of their main phenolic compound rosmarinic acid (RA) were evaluated in two human colon carcinoma-derived cell lines, HCT15 and CO115, which have different mutations in the MAPK/ERK and PI3K/Akt signalling pathways. These pathways are commonly altered in CRC, leading to increased proliferation and inhibition of apoptosis. Our results show that SF, SO, and RA induce apoptosis in both cell lines, whereas cell proliferation was inhibited by the two sage extracts only in HCT15. SO, SF, and RA inhibited ERK phosphorylation in HCT15 and had no effects on Akt phosphorylation in CO115 cells. The activity of sage extracts seems to be due, at least in part, to the inhibition of MAPK/ERK pathway.

Evaluation of toxic/protective effects of the essential oil of Salvia officinalis on freshly isolated rat hepatocytes

For this study the essential oil (EO) of sage (Salvia officinalis L.) was isolated from air-dried vegetative aerial parts of the plants by hydrodistillation and analysed by GC and GC-MS. A total yield of 12.07 mg of EO per g of plant dry mass was obtained and more than 50 compounds identified. The major compounds were cis-thujone (17.4%), alpha-humulene (13.3%), 1,8-cineole (12.7%), E-caryophyllene (8.5%) and borneol (8.3%). The EO fraction of sage tea was also isolated by partition with pentane and the respective components identified. The toxic and antioxidant protective effects of S. officinalis EO were evaluated on freshly isolated rat hepatocytes. Cell viability (LDH leakage), lipid peroxidation and glutathione status were measured in experiments undertaken with cells (suspensions of 1 x 10(6) cells per millilitre) exposed to EO alone (toxicity of the EO;t-BHP as positive control); and with cells exposed to EO and an oxidative compound (t-BHP) together (in EO protection evaluation; quercetin as positive control) for 30 min. The results show that the EO is not toxic when present at concentrations below 200 nl/ml; it was only at 2000 nl EO/ml that a significant LDH leakage and GSH decrease were observed indicating cell damage. In the range of concentrations tested, the EO did not show protective effects against t-BHP-induced toxicity.

Antigenotoxic effects of quercetin, rutin and ursolic acid on HepG2 cells: Evaluation by the comet assay

In the present study, the chemoprotective effects of quercetin, rutin and ursolic acid on tert-butyl hydroperoxide (t-BHP)-induced DNA damage in a human hepatoma cell line (HepG2) were investigated by the comet assay. To determine whether protection was due to direct chemical interactions alone or to cellular-mediated responses three different types of treatments were used: simultaneous incubation of cells with individual test compounds and the toxicant; pre-treatment with test compound before addition of the toxicant followed or not by a recovery period. The expression of Hsp70 was quantified by Western blotting to test the involvement of heat shock proteins in the cellular responses to the test compounds. In addition, effects on proliferation were evaluated by the MTT assay. The results show that quercetin and ursolic acid prevented DNA damage and had antiproliferative properties in HepG2 cells suggesting an anticarcinogenic potential for these compounds. The protective effects of quercetin against t-BHP-induced DNA damage seem to be due to both direct effects on t-BHP toxicity and to cellularly mediated indirect effects which reflect the potentiation of the cellular antioxidant defenses. Ursolic acid seems to exert effects only through cellularly mediated mechanisms since it was not protective in simultaneous incubation. Quercetin and ursolic acid also showed to increase the rate of DNA repair. Rutin did not have effects at any level. These results, obtained with liver cells, emphasize and confirm the chemopreventive potential of quercetin and ursolic acid, which may help explain the lower cancer incidence in human population with high dietary intakes of fruits and vegetables. These results also demonstrate that Hsp70 is not involved in the observed effects in HepG2.

Phenolic antioxidant compounds produced by in vitro shoots of sage (Salvia officinalis L.)

In vitro shoots of sage (Salvia officinalis L.) were established under four different cytokinin supplementations by culturing nodal segments excised from aseptically germinated seedlings. The highest rates of shoot proliferation and linear shoot growth occurred with the supplementation of 1.5 mg/l benzyladenine and 0.05 mg/l dichlorophenoxyacetic acid. However, under these conditions, the specific production of total antioxidant phenolics was the lowest. Variation in kinetin (KIN) concentration (1.5; 2.0; 4.0 mg/l), in the presence of 0.05 mg/l 2,4-D, did not influence significantly the rates of shoot proliferation and linear shoot growth but influenced the production of antioxidant phenolics and biomass. Seventeen compounds were identified in the antioxidant phenolic extracts from shoots: gallic acid, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, caffeic acid, and rosmarinic acid, as phenolic acids; hesperetin, apigenin, hispidulin, cirsimaritin, and genkwanin, as flavonoids; epirosmanol, epirosmanol methyl ether, carnosol, epiisorosmanol ethyl ether, rosmadial, carnosic acid, and methyl carnosate, as phenolic diterpenes. With exception of carnosic acid and methyl carnosate, all the other phenolic compounds (15) were also identified in a commercial sample of this species. Rosmarinic acid and carnosol were the main compounds in all the antioxidant phenolic extracts. The increase in concentration of KIN decreased the accumulation of the most of phenolic diterpenes, particularly that of carnosol.

Unusual flavonoids produced by callus of Hypericum perforatum

Abstract 6-C-prenyl luteolin, a new naturally occurring compound, together with luteolin 5,3′-dimethyl ether, luteolin 5-glucoside and luteolin 3′-glucoside were isolated from callus of Hypericum perforatum var. angustifolium . These flavonoids were completely different from those previously observed in in vivo plants of the same species. The total flavonoid content of callus , around 0.5–0.7 mg g −1 dry wt, was much lower than that found in wild growing Hypericum perforatum plants (14–70 mg g −1 dry wt).

Metformin-like effect of salvia officinalis (common sage) : is it useful in diabetes prevention?

Common sage (Salvia officinalis L.) is among the plants that are claimed to be beneficial to diabetic patients, and previous studies have suggested that some of its extracts have hypoglycaemic effects in normal and diabetic animals. In the present study, we aimed to verify the antidiabetic effects of an infusion (tea) of common sage, which is the most common form of this plant consumed. Replacing water with sage tea for 14 d lowered the fasting plasma glucose level in normal mice but had no effect on glucose clearance in response to an intraperitoneal glucose tolerance test. This indicated effects on gluconeogenesis at the level of the liver. Primary cultures of hepatocytes from healthy, sage-tea-drinking rats showed, after stimulation, a high glucose uptake capacity and decreased gluconeogenesis in response to glucagon. Essential oil from sage further increased hepatocyte sensitivity to insulin and inhibited gluconeogenesis. Overall, these effects resemble those of the pharmaceutical drug metformin, a known inhibitor of gluconeogenesis used in the treatment and prevention of type 2 diabetes mellitus. In primary cultures of rat hepatocytes isolated from streptozotocin (STZ)-induced diabetic rats, none of these activities was observed. The present results seem to indicate that sage tea does not possess antidiabetic effects at this level. However, its effects on fasting glucose levels in normal animals and its metformin-like effects on rat hepatocytes suggest that sage may be useful as a food supplement in the prevention of type 2 diabetes mellitus by lowering the plasma glucose of individuals at risk.

THE DEVELOPMENT AND EVALUATION OF AN HPLC-DAD METHOD FOR THE ANALYSIS OF THE PHENOLIC FRACTIONS FROM IN VIVO AND IN VITRO BIOMASS OF HYPERICUM SPECIES

A new HPLC-DAD method for separation and identification of the main phenolics present in in vivo and in vitro biomass of Hypericum perforatum and Hypericum androsaemum has been developed. This method accomplished the direct identification of 22 compounds including flavonoids, hypericins, phloroglucinols, and phenolic acids. The HPLC profiles obtained in the course of this work clearly evidenced a distinct phenolic production between in vivo and in vitro biomass. For example calli and suspended cells produced mainly xanthones while in in vivo plants of both species these compounds were not detected. Some luteolin flavone type compounds were identified in calli and suspended cells of H. perforatum while quercetin was found in suspended cells of H. androsaemum. Flavonoids, namely those related to quercetin, were the major metabolites in methanolic extracts from in vivo plants. Hypericins were detected in in vivo plants and in in vitro shoots of H. perforatum but not in calli or suspended cells of the same sp...

Ursolic acid and luteolin-7-glucoside improve lipid profiles and increase liver glycogen content through glycogen synthase kinase-3.

In the present study, two phytochemicals – ursolic acid (UA) and luteolin‐7‐glucoside (L7G) – were assessed in vivo in healthy rats regarding effects on plasma glucose and lipid profile (total cholesterol, HDL and LDL), as well as liver glycogen content, in view of their importance in the aetiology of diabetes and associated complications. Both UA and L7G significantly decreased plasma glucose concentration. UA also significantly increased liver glycogen levels accompanied by phosphorylation of glycogen synthase kinase‐3 (GSK3). The increase in glycogen deposition induced by UA (mediated by GSK3) could have contributed to the lower plasma glucose levels observed. Both compounds significantly lowered total plasma cholesterol and low‐density lipoprotein levels, and, in addition, UA increased plasma high‐density lipoprotein levels. Our results show that UA particularly may be useful in preventable strategies for people at risk of developing diabetes and associated cardiovascular complications by improving plasma glucose levels and lipid profile, as well as by promoting liver glycogen deposition. Copyright

Xanthone biosynthesis and accumulation in calli and suspended cells of Hypericum androsaemum

Calli and suspended cells of Hypericum androsaemum accumulated high levels of 1,3,5,6 and 1,3,6,7 oxygenated xanthones. The major compounds include simple oxygenated xanthones or derivatives with prenyl, pyran or methoxyl groups, four of them being new natural compounds. A hypothetical biosynthetic scheme is proposed based on the isolated compounds and statistical analysis. Xanthone accumulation was influenced greatly by medium factors, namely hormone supplementation. Calli grown with 4.5 mM a-naphtaleneacetic acid (NAA)2.3 mM kinetin (KIN) had the highest specific xanthone production (1.3% biomass dry weight (DW)), whereas suspended cells grown in similar medium accumulated a lower amount (0.87% DW). Calli displayed a negative linear relationship between total xanthone accumulation and NAA concentration, in the range of 4.5‐22.5 mM. However, in this range the xanthone 1,3,6,7:1,3,5,6 ratio and the biomass production showed a positive linear relationship with NAA concentration. Substitution of 4.5 mM of NAA by the same molar amount of 2,4-dichlorophenoxyacetic acid (2,4-D), in the presence of 2.3 mM of KIN, caused a decrease in xanthone accumulation in calli. The use of N 6 -benzyladenine (BA) instead of KIN reduced xanthone production, independently of the auxin used. This effect was attenuated when both hormones were present.