L.R. Comini

Comparisons between conventional, ultrasound-assisted and microwave-assisted methods for extraction of anthraquinones from Heterophyllaea pustulata Hook f. (Rubiaceae).

This work reports a comparative study about extraction methods used to obtain anthraquinones (AQs) from stems and leaves of Heterophyllae pustulata Hook (Rubiáceae). One of the conventional procedures used to extract these metabolites from a vegetable matrix is by successive Soxhlet extractions with solvents of increasing polarity: starting with hexane to eliminate chlorophylls and fatty components, following by benzene and finally ethyl acetate. However, this technique shows a low extraction yield of total AQs, and consumes large quantities of solvent and time. Ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) have been investigated as alternative methods to extract these compounds, using the same sequence of solvents. It was found that UAE increases the extraction yield of total AQs and reduces the time and amount of solvent used. Nevertheless, the combination UAE with benzene, plus MAE with ethyl acetate at a constant power of 900 W showed the best results. A higher yield of total AQs was obtained in less time and using the same amount of solvent that UAE. The optimal conditions for this latter procedure were UAE with benzene at 50 °C during 60 min, followed by MAE at 900 W during 15 min using ethyl acetate as extraction solvent.

Photodynamic activity of anthraquinones isolated from Heterophyllaea pustulata Hook f. (Rubiaceae) on MCF-7c3 breast cancer cells.

Searching for agents that could be effective in the treatment of cancer, special highlight has focused on the study of numerous plant-derived compounds. We previously demonstrated that anthraquinones (AQs) isolated from a vegetal species: Heterophyllaea pustulata Hook f. (Rubiaceae), such as rubiadin, rubiadin-1-methyl ether, soranjidiol, soranjidiol-1-methyl ether exhibit photosensitizing properties without antecedents as photodynamic agents in malignant cells. In the present study, we investigated the potential role of these AQs as a phototoxic agent against human breast carcinoma using MCF-7c3 cells. All AQs exhibited significant photocytotoxicity on cancer cells at the concentration of 100 μM with 1 J/cm(2) light dose, resulting soranjidiol-1-methyl ether in complete cell destruction. The observed cellular killing by photoactivated AQs exhibited close relation with singlet oxygen production, except for soranjidiol-1-methyl ether, where cell viability decrease is in relation to uptake by tumor cells.

Photochemotherapy using natural anthraquinones: Rubiadin and Soranjidiol sensitize human cancer cell to die by apoptosis

Over the past decade the science has studied synthetic photosensitizers used in photodynamic therapy (PDT) or photochemotherapy as anticancer candidates. In this context, compounds extracted from vegetable species present interesting potential in the cancer field. In our laboratory, we studied Heterophyllaea pustulata a phototoxic shrub that habit the northwest of Argentina. From this vegetal, by in vitro germination, we obtained Rubiadin and Soranjidiol, two anthraquinones that exhibited significant photocytotoxicity on human cancer cells. In addition, the fraction obtained from callus cultures allowed us to get a satisfactory content of these compounds compared to those found from the original plant. Under PDT regimen, we found that cell destruction resulted in a dose-dependent manner and occasioned apoptosis on photosensitized cells. Biochemical analysis revealed the involvement of caspase-3, PARP cleavage and DNA fragmentation in Rubiadin induced apoptosis. Moreover, Soranjidiol-PDT led to μ-calpain-induced apoptosis involving caspases-3-independent DNA fragmentation. We also showed that both anthraquinones are cytoplasmatically distributed and out of nucleus. In addition, we demonstrated a synergic cytotoxic effect when we combined them. Our data demonstrated that Rubiadin and Soranjidiol could be further considered as natural photocytotoxic compounds against cancer cells and callus cultures are a plausible source of these anthraquinonic compounds.

Apoptosis and mutagenicity induction by a characterized aqueous extract of Baccharis articulata (Lam.) Pers. (Asteraceae) on normal cells

In a previous study we have demonstrated that cold aqueous extract of Baccharis articulata (Ba-CAE) induced the death of human peripheral blood mononuclear cells (PBMCs) and exerted low mutagenic effects on mice at 6h after administration. The aim of this work was to investigate whether the PBMCs death induced by Ba-CAE is due to apoptosis, and whether this extract exerts mutagenic effects on mice at 24 and 48h after administration. In addition, Ba-CAE was chemically characterized. PBMCs from healthy volunteers were exposed to extract (10, 20, 40, 80, 160, 320, 640 and 1280μg/mL) for 18-24h. Cell viability was determined by staining of trypan blue dye exclusion method. Apoptosis was determined by Hoechst 33258 staining, TUNEL, and DNA fragmentation analysis by agarose gel electrophoresis. BALB/c mice were injected with extract (1800, 900 and 450mg/kg) and sacrificed at 24 and 48h postinjection. Bone marrow samples were used to assess chromosome mutations by the micronucleus test. The extract induced PBMCs death by apoptosis and increased the frequency of micronuclei in bone marrow. The phytochemical study of Ba-CAE showed the presence of flavones as luteolin and acacetin, caffeoylquinic acids as chlorogenic acid, and tannins.

Evaluation of the cytotoxicity, genotoxicity and apoptotic induction of an aqueous extract of Achyrocline satureioides (Lam.) DC

Achyrocline satureioides is widely consumed as infusion or aperitif and shows important therapeutic properties. Previously, we reported absence of genotoxicity of cold aqueous extract (CAE) of A. satureioides by Allium test. However, one test cannot predict the genotoxic effects of a substance. Thus, the aim of this work was to investigate cytotoxicity, genotoxicity and apoptotic ability of CAE of A. satureioides. In addition, CAE was chemically characterized. The cytotoxicity was evaluated by Trypan blue and MTT assays. The apoptotic capacity was evaluated by Hoechst staining and DNA fragmentation-analysis. The genotoxicity was studied by comet assay (CA) and micronucleus test. The identification and quantification of flavonoids were performed by HPLC-ESI-MS/MS. The cytotoxicity studies indicated low toxicity of CAE. In addition, CAE did not induce apoptotic effects on human PBMCs. CAE did not show genotoxicity in vitro against Vero cells, at 10-50 μg/mL. CAE did not induce in vivo genotoxic effects, but it showed at high concentrations cytotoxicity by micronucleus assay. CAE presented flavonoids such as quercetin, 3-O-methylquercetin and luteolin. In conclusion, A. satureioides at popularly concentrations used, in aperitif or infusion, can be consumed safely because did not show any cytotoxic or genotoxic effects.

Polyphenols as possible bioprotectors against cytotoxicity and DNA damage induced by ochratoxin A

The present study aimed to investigate the protective effects of luteolin (L), chlorogenic acid (ChlA) and caffeic acid (CafA) against cyto-genotoxic effects caused by OTA. Vero cells and rat lymphocytes were used and viability was measured by neutral red uptake, MTT and trypan blue dye exclusion method. L (50 and 100μg/mL), ChlA (100 and 200μg/mL) and CafA (10-50μg/mL) reduced the damage induced by OTA (10μg/mL) on both cells type shown a good protective effect. The comet and micronucleus tests in Balb/c mice were performed. ChlA (10mg/kg bw) reduced OTA (0.85mg/kg bw)-induced DNA damage on blood and bone marrow cells, CafA (10mg/kg bw) showed protective effect only in blood cells and luteolin (2.5mg/kg bw) failed to protect DNA integrity on cells. In conclusion, polyphenols tested reduced the toxicity caused by OTA on different target cells with good protective effect, being ChlA the compound that showed the best effects.

In Vitro and In Vivo Cytogenotoxic Effects of Hot Aqueous Extract of Achyrocline satureioides (Lam.) DC.

In this work we extend the toxicological studies of hot aqueous extract of A. satureioides (As-HAE) evaluating cytotoxic and apoptotic effects on human peripheral blood mononuclear cells (PBMCs). We also determine genotoxic action of this extract in vivo. In addition, the extract was chemically characterized. Finally, we established a comparison with previous data of cold aqueous extract. The As-HAE induced cytotoxicity on PBMCs determined by trypan blue dye exclusion (CC50 = 653 μg/mL) and MTT (CC50 = 588 μg/mL) assays being more toxic than cold extract. However, As-HAE as well as cold extract did not induce apoptosis measured by Hoechst 33258 staining, TUNEL assay, and DNA fragmentation analysis. The in vivo micronucleus test showed that As-HAE exerted cytogenotoxic effects on bone marrow of mice, contrary to what was observed with cold extract. The chemical study of As-HAE allowed identifying the flavonoids found in cold extract: luteolin, quercetin, and 3-O-methylquercetin, but at higher concentrations. We suggest that toxic effects induced by As-HAE could be due to high concentrations of these flavonoids. Given that As-HAE is the most used in folkloric medicine, its administration should be controlled in order to prevent potential cell damage.