Cytogenotoxic evaluations of leaves and stems extracts of Rubus rosifolius in primary metabolically noncompetent cells.

Plants with medicinal potential may also produce adverse effects in humans. This seems to be the case for the species Rubus rosifolius, where preliminary studies demonstrated genotoxic effects attributed to extracts obtained from leaves and stems of this plant using on HepG2/C3A human hepatoma cells as a model. Considering the beneficial properties of this plant as an antidiarrheal, analgesic, antimicrobial, and antihypertensive and its effects in the treatment of gastrointestinal diseases, the present study was developed with the aim of determining the cytotoxic and genotoxic potential of extracts of leaves and stems of R. rosifolius in primary without metabolic competence in human peripheral blood mononuclear cells (PBMC). Cell viability analyses at concentrations of between 0.01 and 100 µg/ml of both extracts did not markedly affect cell viability. In contrast, assessment of the genotoxic potential using the comet assay demonstrated significant damage to DNA within PBMC from a concentration of 10 µg/ml in the stem extract, and a clastogenic/aneugenic response without cytokinesis-block proliferation index (CBPI) alterations at concentrations of 10, 20, or 100 µg/ml for both extracts. Under our experimental conditions, the data obtained demonstrated genotoxic and mutagenic effects attributed to extracts from leaves and stems of R. rosifolius in cells in the absence of hepatic metabolism.

Rubus rosifolius (Rosaceae) stem extract induces cell injury and apoptosis in human hepatoma cell line.

Rubus rosifolius, popularly known as "red mulberry", is a common medicinal plant in southern Brazil and is used as an antidiarrheal, analgesic, antimicrobial and antihypertensive, and to treat stomach diseases. The aim of this study was to analyze the R. rosifolius stem extract (RrSE) for possible in vitro cytotoxic and genotoxic effects, using the comet assay and the micronucleus test to assess genotoxicity, and flow cytometry to assess the impact on the cell cycle and apoptosis in HepG2/C3A cells, in addition to evaluating the expression of genes linked to the induction of DNA damage, cell cycle, apoptosis and metabolism of xenobiotics. The MTT assay observed no cytotoxic effects at concentrations between 0.01 and 100 µg/mL of the extract. However, genotoxic effects occurred in treatments with the extract from a 1 µg/mL concentration. Flow cytometry analysis revealed a significant increase in cells in the G2/M phase after treatment with 10 µg/mL, a decrease in cells in the G0/G1 phase in the treatment with 100 µg/mL, and a significant increase in total apoptotic cells. In the gene expression analysis, an increase in the CYP1A2 xenobiotics metabolizing gene expression was observed. Despite the promising pharmacological effects of R. rosifolius, the results revealed that the RrSE has genotoxic effect and induces apoptosis in HepG2/C3A cells, indicating danger in using this plant extract by humans.

Risk assessment via genotoxicity, metabolism, apoptosis, and cell growth effects in a HepG2/C3A cell line upon treatment with Rubus rosifolius (Rosaceae) leaves extract.

RUBUS ROSIFOLIUS: Sm. (Rosaceae) is a plant traditionally used in Brazil and some other countries to treat diarrhea, stomach diseases, and as an analgesic, antimicrobial, antihypertensive, and as well as other pharmacological properties. The aim of this study was to examine cytotoxic and genotoxic effects of R. rosifolius leaves extract on HepG2/C3A cells and correlate these findings with the expression of mRNA to underlying mechanisms of action. At concentrations between 0.01 and 100 µg/ml, cytotoxic effects were not detected by the MTT assay. This was confirmed by mRNA induction of the CYP3A4 gene (by RT-qPCR assay). However, genotoxic effects occurred at treatments from 1 µg/ml extract (comet and micronucleus test). An increase in the number of cells in S phase was observed at 100 µg/ml, and an elevation in apoptotic cell number was found for all tested concentrations (10, 20, or 100 µg/ml) (cell cycle and apoptosis analysis by flow cytometry). The genotoxicity induced by the extract was the main cause of the rise in the number of cells undergoing apoptosis, as indicated by rise in mRNA of CASP7 gene, and elevation of cells in the S phase of the cell cycle at the higher tested concentrations, as an attempt to repair genetic damage that occurred. These observations suggest that, despite its pharmacological potential, the use of R. rosifolius leaves extract may pose a risk to the integrity of the genetic material of human cells.

Antidepressant-like effect and toxicological parameters of extract and withanolides isolated from aerial parts of Solanum capsicoides All. (Solanaceae).

The present work describes the evaluation of the antidepressant-like activity of the extract, fractions, and compounds obtained from the aerial parts of Solanum capsicoides. The methanolic extract (MESC) obtained by conventional maceration was partitioned with solvents of increasing polarities yielding the respective fractions of hexane (HE), dichloromethane (DCM), and ethyl acetate (EA). The dichloromethane and ethyl acetate fractions were submitted to chromatographic and spectroscopic techniques, leading to the isolation and identification of cilistadiol (1), astragalin (2), and cilistol A (3). In relation to the antidepressant activity, the extract was active against the forced swimming test (FST) at a concentration of 300 mg/kg an ED50 (deffective dose that reduces 50% of immobility time) of 120.3 (117.3-123.4) mg/kg. Similar values were observed when evaluated in the tail suspension test (TST). In addition, the results showed no influence on motor behavior when evaluated in the open field test (OFT). Based on the observed profile of the MESC, dichloromethane fraction presenting the best profile, in both FST and TST test. Likewise, the fraction also did not present motor impairment when evaluated by the OFT test. Considering that the dichloromethane fraction was more effective, the isolated compounds cilistadiol and cilistol A were evaluated in the same experimental models. In FST, both compounds had a significant antidepressant-like effect, with ED50 values of 0.22 (0.16-0.28) and 1.03 (0.89-1.18) µmol/kg, respectively. When evaluated in the TST, showed ED50 values of 0.30 (0.18-0.52) and 1.49 (1.27-1.73) µmol/kg, respectively. The isolated compounds also did not present significant differences in the motor behavior when evaluated on OFT test in comparison with the control group. No toxicological parameters were observed until the highest dose of MESC (2000 mg/kg), demonstrating safety in the use of this plant.

Diuretic effect of extracts, fractions and two compounds 2α,3ß,19α-trihydroxy-urs-12-en-28-oic acid and 5-hydroxy-3,6,7,8,4'-pentamethoxyflavone from Rubus rosaefolius Sm. (Rosaceae) leaves in rats.

Although diuretics have been widely used to treat hypertension along with others cardiovascular and renal disorders, no scientific data have been recorded to support the diuretic properties of Rubus rosaefolius Sm. (Rosaceae), a plant popularly used in Brazil to treat hypertension. Male Wistar rats were orally treated with: vehicle; hydrochlorothiazide; aqueous (AERR) and methanolic (MERR) extracts; dichloromethane (DCM), hexane (HEX) and ethyl acetate (EA) fractions; and the isolated compounds 2α,3ß,19α-trihydroxy-urs-12-en-28-oic acid (TUA) and 5-hydroxy-3,6,7,8,4'-pentamethoxyflavone (PMF). At the end of the experiment (after 8 or 24 h), urine volume and other urine or plasma parameters were measured. AERR and MERR, at 100 and 30 mg/kg, respectively, induced diuretic, natriuretic and kaliuretic effect. Additionally, the DCM and HEX, but not EA, at 10 mg/kg, also increased urine volume and Na+ and K+ excretion. Both active constituents, TUA and PMF, at doses of 1 and 3 mg/kg, showed an augmented diuretic and natriuretic index. While TUA revealed a kaliuretic action, PMF did not interfere with potassium excretion. The compounds increased urinary creatinine, but not urea, levels. TUA was able to decrease calcium excretion, as well as HCTZ, while PMF effect was associated with increased urinary prostaglandin E2 levels. The non-selective muscarinic receptor antagonist (atropine) prevented TUA-induced diuresis. In addition, indomethacin (a cyclooxygenase inhibitor) and atropine, exhibited the ability to block the diuretic effects prompted by PMF. Our study demonstrates the diuretic effect of extracts, fractions and two natural compounds obtained from R. rosaefolius leaves in rats.

Rubus imperialis (Rosaceae) extract and pure compound niga-ichigoside F1: wound healing and anti-inflammatory effects.

Here, we evaluate the anti-inflammatory and wound-healing effects of methanolic crude extract obtained from aerial parts (leaves and branches) of Rubus imperialis Chum. Schl. (Rosaceae) and the pure compound niga-ichigoside F1. Anti-inflammatory activity was determined in vivo and in vitro, and the healing effect was evaluated in surgical lesions in mice skin. The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) assay and H2O2-induced oxidative stress were used to determine antioxidant activity. The efferocytosis activity was also determined. The data obtained show that the extract of R. imperialis promote reduction in the inflammatory process induced by lipopolysaccharide (LPS) or carrageenan in the air pouch model; the effects could be reinforced by nitric oxide reduction in LPS-stimulated neutrophils, and an increase in the efferocytosis. The extract showed wound healing property in vitro and in vivo, scavenging activity for DPPH, and cytoprotection in the H2O2-induced oxidative stress in L929 cells. In addition, the compound niga-ichigoside F1 was able to reduce the NO secretion; however, it did not present wound-healing activity in vitro. Together, the data obtained point out the modulatory actions of R. imperialis extract on leukocyte migration to the inflamed tissue, the antioxidant, and the pro-resolutive activity. However, the R. imperialis anti-inflammatory activity may be mediated in parts by niga-ichigoside F1, and on wound healing do not correlated with niga-ichigoside F1.

Antiproliferative and toxicological properties of methanolic extract obtained from Solanum capsicoides All. seeds and carpesterol.

Natural products are considered important sources of potential chemotherapeutic agents. Here, we evaluated the antiproliferative activity and the toxicological effects of the methanolic extract and a pure compound obtained from Solanum capsicoides seeds. The phytochemical profile was analyzed by chromatographic and spectroscopy methods. The acute toxicity was assessed in mice orally treated with the extract (2000 mg/kg), in vitro hemolytic activity and micronucleus test. The mutagenicity, developmental toxicity, and lethal dose (LD50) of carpesterol were estimated by the Toxicity Estimation Software Tool (TEST) software. A sulforhodamine B assay was employed to evaluate the antiproliferative activity. The toxicological assays did not observe signs of toxicity, either during the behavioral observations or in the autopsies, as well as no mutagenicity and hemolytic activity. The carpesterol did not present mutagenic effect and hemolytic activity but presents developmental toxicology and LD50 of 410 mg/kg in toxicity estimations by the TEST software. The S. capsicoides extract exhibited antiproliferative activity mainly in leukemia (K562) cell lineage. However, carpesterol presented antiproliferative activity in glioma (U251), breast (MCF-7), kidney (786-0), ovary (OVCAR-03), and K562 cell lineages. The data obtained show that S. capsicoides extract presents antiproliferative and does not present toxicological effects. In addition, it was shown for the first time the antiproliferative and toxicological parameters of carpesterol.

Antiulcer mechanisms of Vernonia condensata Baker: A medicinal plant used in the treatment of gastritis and gastric ulcer.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves from Vernonia condensata Baker are broadly used in folk medicine for the treatment of gastric ulcers and dyspepsia. The Brazilian Public Health System (SUS) describes this species as having the potential to serve as a new herbal product with therapeutic benefits. AIM OF THE STUDY: The purpose of the study was to evaluate the gastroprotective activity and gastric healing properties of a crude ethanolic extract from leaves of V. condensata (CEEV) in different animal models. MATERIALS AND METHODS: In order to assess the gastroprotective potential of CEEV, ulcer models were established using ethanol and indomethacin. The gastric healing effect was then evaluated in the acetic acid-induced ulcer model, where the tissue was used to assess oxidative levels (reduced glutathione and lipid hydroperoxide levels, as well as superoxide dismutase and catalase activity), inflammatory [myeloperoxidase (MPO)] parameters, and mucin content. Furthermore, the ligature pylorus model, with and without secretagogue stimuli, was employed to investigate the mechanism of action of CEEV. In addition, H(+)K(+)-ATPase activity, MPO activity, and antioxidant activity through the DPPH assay were examined through in vitro trials. Phytochemical analyses were also performed. The ethanol/HCl-induced gastric ulcer method was employed to verify the gastroprotective effect of the main compound in CEEV. RESULTS: CEEV (30 and 300mg/kg, p.o) exhibited gastroprotective activity and prevented both gastric lesions induced by ethanol or indomethacin in rats. The gastric healing effect of CEEV (300mg/kg, p.o. taken twice a day for a duration of seven days) was confirmed by examining the macroscopic and microscopic appearance of chronic gastric ulcers induced by acetic acid in rats. The restorative effect of CEEV was accompanied by a significant increase in mucin content (PAS staining) and by a reduction in oxidative stress and inflammatory parameters at the site of the ulcer. Moreover, CEEV (300mg/kg), administered via an intraduodenal route, significantly reduced the volume, pH, total acidity and pepsin activity of gastric content in the pylorus ligature model in rats. The gastric acid antisecretory effect of CEEV was maintained even in the presence of cholinergic and gastrinergic, but not histaminergic, stimuli. In vitro, CEEV (1-10µg/ml) was able to scavenge free radical DPPH, but did not promote inhibitory effects on MPO or H(+),K(+)-ATPAse activity. Phytochemical analysis of CEEV indicated that luteolin is the main compound present in the extract. However, luteolin (1, 3 and 10mg/kg, p.o or 1mg/kg, i.p.) did not promote gastroprotection against ethanol/HCl in mice. It is also important to mention that oral administration of CEEV did not produce any sign of acute toxicity in animals. CONCLUSIONS: V. condensata extract demonstrates gastroprotective effects through the inhibition of gastric secretion via cholinergic and gastrinergic pathways. Furthermore, it exhibits cytoprotective effects, involving antioxidant activity, an increase in mucin content and inhibition of neutrophil migration. Thus, this medicinal plant may be a suitable natural source for the prevention and treatment of gastric lesions.

In vivo evaluation of the genetic toxicity of Rubus niveus Thunb. (Rosaceae) extract and initial screening of its potential chemoprevention against doxorubicin-induced DNA damage.

ETHNOPHARMACOLOGICAL RELEVANCE: Rubus niveus Thunb. plant belongs to Rosaceae family and have been used traditionally to treat wounds, burns, inflammation, dysentery, diarrhea and for curing excessive bleeding during menstrual cycle. The present study was undertaken to investigate the in vivo genotoxicity of Rubus niveus aerial parts extract and its possible chemoprotection on doxorubicin (DXR)-induced DNA damage. In parallel, the main phytochemicals constituents in the extract were determined. MATERIALS AND METHODS: The animals were exposed to the extract for 24 and 48 h, and the doses selected were 500, 1000 and 2000 mg/kg b.w. administered by gavage alone or prior to DXR (30 mg/kg b.w.) administered by intraperitoneal injection. The endpoints analyzed were DNA damage in bone marrow and peripheral blood cells assessed by the alkaline alkaline (pH>13) comet assay and bone marrow micronucleus test. RESULTS AND CONCLUSION: The results of chemical analysis of the extract showed the presence of tormentic acid, stigmasterol, quercitinglucoronide (miquelianin) and niga-ichigoside F1 as main compounds. Both cytogenetic endpoints analyzed showed that there were no statistically significant differences (p>0.05) between the negative control and the treated groups with the two higher doses of Rubus niveus extract alone, demonstrating absence of genotoxic and mutagenic effects. Aneugenic/clastogenic effect was observed only at 2000 mg/kg dose. On the other hand, in the both assays and all tested doses were observed a significant reduction of DNA damage and chromosomal aberrations in all groups co-treated with DXR and extract compared to those which received only DXR. These results indicate that Rubus niveus aerial parts extract did not revealed any genotoxic effect, but presented some aneugenic/clastogenic effect at higher dose; and suggest that it could be a potential adjuvant against development of second malignant neoplasms caused by the cancer chemotherapic DXR.

Uncommon Trimethoxylated Flavonol Obtained from Rubus rosaefolius Leaves and Its Antiproliferative Activity.

This study shows the evaluation the antiproliferative effect of the extract, fractions, and uncommon compounds isolated from R. rosaefolius leaves. The compounds were identified by conventional spectroscopic methods such as NMR-H(1) and C(13) and identified as 5,7-dihydroxy-6,8,4'-trimethoxyflavonol (1), 5-hydroxy-3,6,7,8,4'-pentamethoxyflavone (2), and tormentic acid (3). Both hexane and dichloromethane fractions showed selectivity for multidrug-resistant ovary cancer cell line (NCI-ADR/RES) with total growth inhibition values of 11.1 and 12.6 µg/ml, respectively. Compound 1 also showed selective activity against the same cell line (18.8 µg/ml); however, it was especially effective against glioma cells (2.8 µg/ml), suggesting that this compound may be involved with the in vitro antiproliferative action.

Reproductive toxicology and clastogenic evaluation in mice of a phytotherapeutic formulation obtained from Cinchona calisaya Weddel (Rubiaceae) used in Brazilian folk medicine as female fertility stimulant.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil, a phytotherapeutic preparation produced from a standardized tincture of Cinchona calisaya Weddel such that each mL of product contains 400µg of quinine, known in Portuguese as Água Inglesa(®) (English water), is indicated by the manufacturer as a tonic, appetite stimulant, and digestive. However, this preparation has long been used in folk medicine as a female fertility stimulant. Despite its widespread use in folk medicine to stimulate female fertility, no study has been undertaken to assess the potential teratogenic and genotoxic effects of this phytotherapeutic preparation. The aim of the present study was to investigate possible toxic reproductive effects in mice caused by exposure to Água Inglesa(®), either before mating or during the pre- and post-embryo implantation periods. The genotoxic potential was evaluated using the micronucleus assay. MATERIAL, METHODS, AND RESULTS: Virgin female mice, with at least one estrous cycle evidenced by vaginal cytology, were divided into five groups of 15 individuals each (Group I - control, Group II - treated with ethanol solution at 16%, Groups III, IV and V treated with phytotherapeutic preparation at 1.5mL/kg/day, 3.0mL/kg/day and 4.5mL/kg/day, respectively). After the first 28 days of treatment, females were caged individually with adult fertile males. Pregnant females continued to receive treatment for seven days (preimplantation period). Body weight was recorded weekly during treatment. Signs of toxicity (weight loss, food intake, piloerection, apathy, prostration, diarrhea, seizures, behavioral changes, and locomotion) were also observed. The females were sacrificed on the 15th day of pregnancy, uterine horns were evaluated for implantation, and the placental index was recorded. In the micronucleus test, 2000 polychromatic erythrocytes (PCE) per animal, obtained from bone marrow, were scored. Results The results showed that exposure of the females during the pre- and post-implantation periods did not significantly alter the reproductive capacity (p<0.05); however, in higher dose (three times human dose)reduction of fetal weight was observed . There was no difference between the control and phytotherapeutic preparation (p>0.05) in terms of the average number of micronucleated polychromatic erythrocytes. CONCLUSIONS: Although folk medicine suggests that the Água Inglesa(®) preparation is useful as a female fertility stimulant, no such effect was confirmed in mice.