Antimutagenic and antitumor activities of a water-soluble fraction of soursop (syn Graviola, Annona muricata L.) fruit pulp.

Soursop (Annona muricata) is a tropical tree whose decoction derived from bark, root, seed, or leaf has been used for medicinal uses. In addition, the fruit itself is considered a food, and the juice is utilized to treat heart and liver diseases. The aim of this study was to determine the phenolic content. In addition, a water-soluble fraction of the soursop fruit pulp (WSSP) was examined for the following properties: antioxidant, mutagenic, and antimutagenicity. UV-visible spectrophotometry determined total phenolic content by the Folin-Ciocalteu method to be 11.22 ± 0.6 mg of gallic acid equivalent per gram dried extract, and free-radical scavenging activity by the 2,2'-diphenyl-1-picryl-hydrazyl (DPPH•) showed an EC50 of 1032 µg/ml. In the Salmonella/microsome assay, no marked mutagenicity was induced following WSSP treatment, and a chemopreventive capacity was observed in the antimutagenic assay. The cytotoxicity assays were carried out using the water-soluble tetrazolium salt and lactate dehydrogenase (LDH) assays demonstrated that WSSP induced significant cytotoxicity in MCF-7 and Caco-2 cells, indicating greater effectiveness of cytotoxic action by destroying cell membrane integrity. Data suggest that WSSP may exert beneficial effects as a DNA chemopreventive and antitumor agent.

Antiviral and Virucidal Activities of Uncaria tomentosa (Cat's Claw) against the Chikungunya Virus.

Uncaria tomentosa (UT) is a medicinal plant popularly known as cat's claw belonging to the Rubiaceae family that has been reported to display antiviral and anti-inflammatory activities. The chikungunya virus (CHIKV) outbreaks constitute a Brazilian public health concern. CHIKV infection develops an abrupt onset of fever, usually accompanied by a skin rash, besides incapacitating polyarthralgia. There is no vaccine available or treatment for CHIKV infection. The present study evaluates the hydroalcoholic extract of UT bark as a potential antiviral against CHIKV. The in vitro antiviral activity of the UT extract against the Brazilian CHIKV strain was assessed using quantitative reverse transcription polymerase chain reaction, flow cytometry, and plaque assay. Results obtained demonstrated that UT inhibits CHIKV infection in a dose-dependent manner. At the non-cytotoxic concentration of 100 µg/mL, UT exhibited antiviral activity above 90% as determined by plaque reduction assay, and it reduced the viral cytopathic effect. Similarly, a significant virucidal effect of 100 µg/mL UT was observed after 24 and 48 h post-infection. This is the first report on the antiviral activity of UT against CHIKV infection, and the data presented here suggests UT as a potential antiviral to treat CHIKV infection.

Quantitative 1H NMR method for analyzing primaquine diphosphate in active pharmaceutical ingredients.

Thermal analysis, Fourier Transform IR, the isotropic chemical shift of 1H NMR in different solvents, their temperature dependence and spin-lattice relaxation time constant (T1), solution 1D and 2D NMR, and solid-state 13C and 31P NMR (magic angle spinning NMR) were employed to obtain full information and elucidate the structures of primaquine diphosphate (PQD) samples used for quality controlling malaria medicines. Additionally, a simple, rapid, specific, and reliable quantitative method (qNMR) was developed to determine the PQD level in the raw material of active pharmaceutical ingredients (APIs). The method was developed using ethylene carbonate (EC) as the internal standard and dimethylsulfoxide-d6 (DMSO-d6) as the NMR solvent. For the API qNMR, 1H NMR signals at 3.82 and 1.22 ppm were used. The qNMR methodology, through the linearity, range, LOD and LOQ, stability, precision, robustness, and accuracy, was validated within the requirements of guidelines. The accuracy of the qNMR was evaluated by comparing it to a pharmacopeial HPLC technique and there were no statistical differences (p > 0.05). The proposed qNMR method authentically supports and endorses the current pharmacopoeial methods used for determining the PQD content.

Evaluation of the acute toxicity, phototoxicity and embryotoxicity of a residual aqueous fraction from extract of the Antarctic moss Sanionia uncinata.

BACKGROUND: Ultraviolet (UV) radiation is the main exogenous inductor of skin damage and so photoprotection is important to control skin disorders. The Antarctic moss Sanionia uncinata is an important source of antioxidants and the photoprotective activity of its organic extracts has been investigated. This study aimed to evaluate the potential photoprotection, cytotoxicity and embryotoxicity of residual aqueous fraction (AF) from the moss S. uncinata. METHODS: UV-visible spectrum and SPF (sun protection factor) were determined by spectrophotometry. Embryotoxicity potential was evaluated by Fish embryo-larval toxicity test using zebrafish (Danio rerio) as organism model. Cell death assays by water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) were investigated using HaCaT keratinocyte cell line cultured in monolayers and three dimensions (3D). Phototoxicity and association with UV-filters were performed by 3T3 neutral red uptake test. RESULTS: The AF showed sharp absorption bands in the UV region and less pronounced in the visible region. The SPF was low (2.5 ± 0.3), but the SPF values of benzophenone-3 and octyl-methoxycinnamate increased ~ 3 and 4 times more, respectively, in association with AF. The AF did not induce significant lethal and sublethal effects on zebrafish early-life stages. In monolayers, the HaCaT cell viability, evaluated by WST-1, was above 70% by ≤0.4 mg AF/mL after 48 and 72-h exposure, whereas ≤1 mg AF/mL after 24-h exposure. The LDH assay showed that the cell viability was above 70% by ≤0.4 mg AF/mL even after 72-h exposure, but ≤1 mg/mL after 24 and 48-h exposure. In 3D cell culture, an increased cell resistance to toxicity was observed, because cell viability of HaCaT cell by WST-1 and LDH was above ~ 90% when using ≤1 and 4 mg AF/mL, respectively. The AF demonstrated values of photo irritation factor < 2 and of photo effect < 0.1, even though in association with UV-filters. CONCLUSIONS: The residual AF absorbs UV-vis spectrum, increased SPF values of BP-3 and OMC and does not induce embryotoxicity to zebrafish early life-stage. The cell death assays allowed establishing non-toxic doses of AF and phototoxicity was not detected. AF of S. uncinata presents a good potential for skin photoprotection against UV-radiation.

Evaluation of nitroreductase and acetyltransferase participation in N-nitrosodiethylamine genotoxicity.

N-Nitroso compounds, such as N-nitrosodiethylamine (NDEA), are a versatile group of chemical carcinogens, being suspected to be involved in gastrointestinal tumors in humans. The intestinal microflora can modify a wide range of environmental chemicals either directly or in the course of enterohepatic circulation. Nitroreductases from bacteria seem to have a wide spectrum of substrates, as observed by the reduction of several nitroaromatic compounds, but their capacity to metabolize N-nitroso compounds has not been described. To elucidate the participation of nitroreductase or acetyltransferase enzymes in the mutagenic activity of NDEA, the bacterial (reverse) mutation test was carried out with the strains YG1021 (nitroreductase overexpression), YG1024 (acetyltransferase overexpression), TA98NR (nitroreductase deficient), and TA98DNP6 (acetyltrasferase deficient), and YG1041, which overexpresses both enzymes. The presence of high levels of acetyltransferase may generate toxic compounds that must be eliminated by cellular processes or can lead to cell death, and consequently decrease the mutagenic effect, as can be observed by the comparison of strain TA98DNP6 with the strains TA98 and YG1024. The slope curves for TA98 strain were 0.66 rev/microM (R(2) = 0.51) and 52.8 rev/microM (R(2) = 0.88), in the absence and presence of S9 mix, respectively. For YG1024 strain, the slope curve, in the presence of S9 mix was 6897 rev/microM (R(2) = 0.78). Our data suggest that N-nitroso compounds need to be initially metabolized by enzymes such as cytochromes P450 to induce mutagenicity. Nitroreductase stimulates toxicity, while acetyltransferase stimulates mutagenicity, and nitroreductase can neutralize the mechanism of mutagenicity generating innoccuos compounds, probably by acting on the product generated after NDEA activation.

Photoprotective and toxicological activities of extracts from the Antarctic moss Sanionia uncinata.

BACKGROUND: The Antarctic moss Sanionia uncinata (Hedw.) Loeske has shown high ultraviolet (UV)-absorbers content after exposition to high levels of UV-B radiation and can be an important source of antioxidants. OBJECTIVE: The aim was to investigate photoprotection and mutagenicity by the aqueous extract (AE) and hydroethanolic extract (HE) from the Antarctic moss S. uncinata. MATERIALS AND METHODS: Photoprotective activities were determined through survival curves of Escherichia coli strains, after UV irradiation in an aqueous solution of thymine and in vitro sun protection factor (SPF). The Salmonella/microsome assays were applied to assess the mutagenicity. RESULTS: Both extracts induced photoprotection against UV-C radiation. The AE showed a higher protection than the hydroethanolic one against UV-induced thymine dimerization. The SPFs were low in both extracts. In association to benzophenone-3 a significant increase in the SPF was detected for the AE, and a significant decrease was induced by the HE. No mutagenicity was found in the both extracts. Furthermore, it was observed absence of cytotoxicity. CONCLUSION: Water-extractable compounds seem to contribute on photoprotection of this Antarctic moss.

Anti-inflammatory effect of Schinus terebinthifolius Raddi hydroalcoholic extract on neutrophil migration in zymosan-induced arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Schinus terebinthifolius is a species of plant from the Anacardiaceae family, which can be found in different regions of Brazil. Schinus is popularly known as aroeirinha, aroeira-vermelha, or Brazilian pepper. In folk medicine, S. terebinthifolius is used for several disorders, including inflammatory conditions, skin wounds, mucosal membrane ulcers, respiratory problems, gout, tumors, diarrhea and arthritis. According to chemical analyses, gallic acid, methyl gallate and pentagalloylglucose are the main components of hydroalcoholic extracts from S. terebinthifolius leaves. In the present study, we demonstrated the ability of a hydroalcoholic extract to inhibit cell migration in arthritis and investigated the mechanisms underlying this phenomenon. MATERIALS AND METHODS: The anti-inflammatory effect of S. terebinthifolius hydroalcoholic leaf extract (ST-70) was investigated in a zymosan-induced experimental model of inflammation. Male Swiss and C57Bl/6 mice received zymosan (100 µg/cavity) via intra-thoracic (i.t.) or intra-articular (i.a.) injection after oral pre-treatment with ST-70. The direct action of ST-70 on neutrophils was evaluated via chemotaxis. RESULTS: ST-70 exhibited a dose-dependent effect in the pleurisy model. The median effective dose (ED50) was 100mg/kg, which inhibited 70% of neutrophil accumulation when compared with the control group. ST-70 reduced joint diameter and neutrophil influx for synovial tissues at 6h and 24h in zymosan-induced arthritis. Additionally, ST-70 inhibited synovial interleukin (IL)-6, IL-1ß, keratinocyte-derived chemokine (CXCL1/KC) and Tumor Necrosis Factor (TNF)-α production at 6h and CXCL1/KC and IL-1ß production at 24h. The direct activity of ST-70 on neutrophils was observed via the impairment of CXCL1/KC-induced chemotaxis in neutrophils. Oral administration of ST-70 did not induce gastric damage. Daily administration for twenty days did not kill any animals. In contrast, similar administrations of diclofenac induced gastric damage and killed all animals by the fifth day. CONCLUSIONS: Our results demonstrated significant anti-inflammatory effects of ST-70, suggesting a putative use of this herb for the development of phytomedicines to treat inflammatory diseases, such as joint inflammation.

Participation of BER and NER pathways in the repair of DNA lesions induced at low N-nitrosodiethylamine concentrations.

In the present work, we evaluated (p < 0.05) the participation of base excision repair (BER) and nucleotide excision repair (NER) mechanisms in repairing DNA lesions induced by N-nitrosodiethylamine (NDEA) at 1.5 ng/mL-36.5 microg/mL, through cell survival, in different single and double Escherichia coli DNA repair mutants (uvrA, uvrB, uvrC, fpg, nth, xthA, fpg/nth, uvrA/fpg, fpg/xthA, mutY, and fpg/mutY), using pre-incubation periods of 90 min. Mutant strains BH20 (fpg) and AB1886 (uvrA) showed microsomal enzyme (S9 mix) independent NDEA cytotoxicity. Cytotoxicity was also detected at lowest NDEA concentrations, in the presence of S9 mix, with strains BH980 (mutY) and BH990 (fpg/mutY). NDEA cytotoxicity, without S9 mix, was detected for mutant strains AB1884 (uvrC) and AB1885 (uvrB). Through SOS chromotest with 90 min of pre-incubation for uvrA and nth strains, only NER was shown to be required for repairing NDEA-induced lesions with or without metabolic activation. PQ37 and PQ66 strains, both uvrA mutants, showed different levels of NDEA sensitivity. The findings suggest that, under the used conditions, and at low concentrations, NDEA-induced lesions require both repair pathways.

Effects of Sanionia uncinata extracts in protecting against and inducing DNA cleavage by reactive oxygen species.

When mosses are exposed to increased quantities of ultraviolet (UV) radiation, they produce more secondary metabolites. Antarctica moss Sanionia uncinata (Hedw.) Loeske has presented high carotenoid contents in response to an increase in UVB radiation. This moss has been recommended as a potential source of antioxidants. In the present work, the protective and enhancing effects of aqueous (AE) and hydroalcoholic (HE) extracts of S. uncinata on the cleavage of supercoiled DNA were evaluated through topological modifications, quantified by densitometry after agarose gel electrophoresis. Total phenolic contents reached 5.89 mg/g. Our data demonstrated that the extract does not induce DNA cleavage. Furthermore, both extracts showed antioxidant activity that protected the DNA against cleavage induced by (i) O(2)(•-), 89% (AE) and 94% (HE) (P<0.05), and (ii) (.)OH, 17% (AE) and 18% (HE). However, the extracts intensified cleavage induced by Fenton-like reactions: (i) Cu(2+)/H(2)O(2), 94% (AE) and 100% (HE) (P<0.05), and (ii) SnCl(2), 62% (AE) and 56% (HE). DNA damages seem to follow different ways: (i) in the presence of Fenton-like reactions could be via reactive oxygen species generation and (ii) with HE/Cu(2+) could have also been triggered by other mechanisms.

The influence of pH on the inhibition of DNA cleavages induced by pyrogallol.

Induction of DNA damage by pyrogallol has been shown at physiological pH, but mutagenesis data also suggest there is inhibition in acidic media. In the present work, the plasmid pBSK was incubated with pyrogallol, under aerobic conditions at 37 degrees C, at pH 7.4, 4.5 or 3.5, for 1, 3 or 5 h, in the absence or presence of Cu(2+). Cleavage of the supercoiled DNA form was analyzed through topology modifications by agarose gel electrophoresis and quantified by densitometry. Independently of the presence of Cu(2+), DNA cleavage at pH 7.4 was significantly (P < 0.001) induced and occurred extensively after 1-h incubation. At pH 4.5, the cleavage was significantly (P < 0.05) induced only after 5 h incubation in the absence of Cu(2+), but was extensive (P < 0.001) after 1-h incubation when the metal ion was present. At pH 3.5, DNA cleavage was inhibited (P > 0.05), after 5-h incubation, even in the presence of Cu(2+). Our results provide evidence that DNA cleavage by pyrogallol is pH-dependent, catalyzed by Cu(2+) , and extensively decreased in acidic pH. Due to the abundant presence of the pyrogallate ion in physiological media, we suggest that this conjugate base form is responsible for DNA cleavage.

Alternative extraction of alkaloid anticarcinogens from Brazilian "vinca rosea" using Ion exchange chromatography

Extracts in ethanol and ethanol-ammonia of dried leaves from Catharanthus roseus, gathered at Rio de Janeiro state, were adsorbed in a strongly acidic cation exchange resin with sulfonic acid group, using the finite bath method, resulting in an alkaloid retained fraction and an acidic and neutral unretained fraction. High Performance Liquid Chromatography showed the isolation of the alkaloid fraction to be highly selective and with good performance, with an absence of alkaloids in the unretained fraction, while the retained fraction presented 1,54-6,35 mg/g of vindoline and 0,12-0,91 mg/g of vinblastine, common for an alkaloid-rich concentrate, usually obtained by classic extraction with several steps using solvents.