Toxicological potential of Aloysia gratissima: Insights from chemical analysis and in vitro studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Aloysia gratissima leaves are popularly used to treat respiratory, digestive, and nervous system disorders. Several studies have been carried out to determine the biological activity of A. gratissima, such as its antibacterial and anti-edematogenic activities, but despite the beneficial uses of A. gratissima, few studies have examined the toxicological profile of this plant. AIM OF THE STUDY: This study aimed to determine the chemical composition, cytotoxic, genotoxic, mutagenic potential, and antioxidant activity of an aqueous extract of A. gratissima leaves (AG-AEL). MATERIAL AND METHODS: The phytochemical constitution of AG-AEL was assessed by colorimetric analyses and High-performance liquid chromatography (HPLC). The inorganic elements were detected by Particle-Induced X-ray Emission (PIXE). The antioxidant, cytotoxicity, genotoxic, and mutagenic activities were evaluated in vitro by Di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH), Sulforhodamine B (SRB) assay, comet assay, and Salmonella/microsome assays. RESULTS: AG-AEL indicated the presence of terpenoids, flavonoids, and phenolic acids. HPLC detected rutin at 2.41 ± 0.33 mg/100 mg. PIXE analysis indicated the presence of Mg, Si, P, S, K, Ca, Mn, and Zn. The 50% inhibitory concentration was 84.17 ± 3.17 µg/mL in the DPPH assay. Genotoxic effects were observed using the Comet assay in neuroblastoma (SH-SY5Y) cells and mutations were observed in TA102 and TA97a strains. The extract showed cytotoxic activities against ovarian (OVCAR-3), glioblastoma (U87MG), and colon (HT-29) cancer cell lines. CONCLUSIONS: In conclusion, AG-AEL increased DNA damage, induced frameshift, and oxidative mutations, and showed cytotoxic activities against different cancer cells. The in vitro toxicological effects observed suggest that this plant preparation should be used with caution, despite its pharmacological potential.

Protective effect of Hovenia dulcis Thunb. leaf extracts against ethanol-induced DNA damage in SH-SY5Y cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Hovenia dulcis Thunb. has been used as a medicinal herb for the treatment of hepatic diseases and alcohol intoxication. AIM OF THE STUDY: The genotoxic effect and the antigenotoxic potential of ethanolic extract of H. dulcis leaves and its methanolic fraction were evaluated against ethanol-induced damages in SH-SY5Y cells. MATERIALS AND METHODS: The phytochemical analysis and antioxidant activity of H. dulcis extracts were also assessed. In addition, a systems biology analysis was performed to investigate the molecular pathway of action of the H. dulcis leaves compounds. RESULTS: The ethanolic extract and its methanolic fraction presented genotoxicity through comet assay at 0.5 and 0.25 mg/mL. On the other hand, both extracts showed protective action against ethanol at all concentrations. Additionally, an NBT assay was performed and demonstrated an ability of the extracts to reduce superoxide anion formation when SH-SY5Y cells were challenged with ethanol. HPLC analysis indicated the presence of quercitrin, isoquercitrin, and rutin. Further, system biology assays indicated a molecular action pathway, where the compounds from the leaves of H. dulcis, in addition to performing free radical scavenging activity, activate PP2A, and may inhibit the apoptosis pathway activated by ethanol-induced oxidative stress. CONCLUSIONS: This work is important to indicate potential antigenotoxic and antioxidant properties of H. dulcis leaves, and its use can be investigated against DNA damage induced by ethanol.

Antioxidant evaluation of the aqueous extract of hulls of Campsiandra laurifolia in colitis induced by acetic acid in Wistar rats / Avaliação antioxidante do extrato aquoso de cascas de Campsiandra laurifolia na colite induzida por ácido acético em ratos Wistar

Abstract Due to the ethnopharmacological use of Campsiandra laurifolia (Fabaceae), popularly known as Acapurana, to treat wounds and ulcers, associated with the lack of alternative treatments for intestinal inflammations such as ulcerative colitis (UC), the present work sought to characterize its phytochemical and antioxidant activities, and to evaluate remedial action in experimental colitis with acetic acid. Phytochemical analyzes were performed through qualitative and quantitative colorimetric tests of the main secondary metabolites. In the colitismodel, 24male Wistar rats aged±60 days oldwere used, divided into 4 groups: Control (CO) control+aqueous extract of C. laurifolia 50mg/kg (CO+A50); Colitis (CL); and Colitis+aqueous extract of C. laurifolia 50 mg/kg (CL+ A50).Measurement of sphincter anal pressure and histological tests of the large intestine, lipoperoxidation (LPO), enzymeactivity of superoxide dismutase (SOD), and levels of glutathione (GSH)were performed. For statistical analysis, the oxidative stress (OS) results were expressed as means±standard error, adopting a significance level of p < 0.05. The screening indicated the presence of flavonoids, saponins and tannins in the extract, with high levels of phenolic

Resumo Devido ao uso etnofarmacológico de Campsiandra laurifolia (Fabaceae), popularmente conhecida comoAcapurana, para tratar feridas e úlceras, associado à falta dealternativas de tratamentos para as inflamações intestinais como a retocolite ulcerativa (RCU), o presente trabalho buscou caracterizar sua constituição fitoquímica, sua atividade antioxidante, e avaliar sua ação reparadora na colite experimental com ácido acético. As análises fitoquímicas foram realizadas por meio de ensaios colorimétricos qualitativos e quantitativos dos principaismetabólitos secundários.Nomodelo de colite, foramutilizados 24 ratos machos Wistar de±60 dias de idade, divididos em 4 grupos: Controle (CO), controle+ extrato aquoso de C. laurifolia 50mg/kg (CO+A50); Colite (CL); e Colite+extrato aquoso de C. laurifolia (CL+ A50). Foram realizadas aferições da pressão anal esfincteriana e avaliações histológicas do intestino grosso, lipoperoxidação (LPO), atividade da enzima superóxido dismutase (SOD) e níveis da glutationa (GSH). Para a análise estatística, resultados do estresse oxidativo (EO) foram expressos em médias±erro padrão, adotando um nível de significância de p < 0,05. O screening indicou no extrato a presença de flavonoides, saponinas e taninos com altos teores de compostos fenólicos e taninos, relacionando-os a uma elevada capacidade antioxidante. Na análise histológica, o grupo CL apresentou perda das criptas, do edema e do infiltrado inflamatório. O uso do extrato de C. laurifolia reestruturou as criptas, diminuiu o edema e aumentou a pressão anal esfincteriana, com diminuição da LPO, da SOD, e aumento da GSH. Sugere-se que o uso do extrato de C. laurifolia diminui o EO por seu poder antioxidante, conferido pelos compostos fenólicos presentes no extrato.

In vivo and in vitro toxicological evaluations of aqueous extract from Cecropia pachystachya leaves.

CECROPIA PACHYSTACHYA: leaves are popularly used to treat asthma and diabetes. Despite the widespread consumption of this plant, there are few scientific studies regarding its toxicological potential. In order to conduct a thorough study concerning the potential adverse effects, the aim of this study was to assess acute and subacute toxicity tests of crude aqueous extract from C. pachystachya leaves (CAE-Cp) using in vivomodel, as well as in vitro cytotoxicity, genotoxicity and antioxidant activity. In addition, genotoxicity, and cytotoxicity of chlorogenic acid (CGA) and cytotoxicity of isoorientin (ISOO) were also evaluated. The antioxidant activity was verified by DPPH, cytotoxicity using sulforhodamine B (SRB) assay and genotoxicity by comet assay on V79 cells. The phytochemical analysis of CAE-Cp detected flavonoids and tannins, CGA and ISOO as the major compounds utilizing HPLC. The total flavonoid content (6.52 mg/g EQ) and antioxidant activity (EC50 = 62.15 µg/ml) of CAE-Cp were determined. In vitro evaluations with CAE-Cp showed genotoxic effects at 0.31 to 2.5 mg/ml and an expressive cytotoxicity on HT-29 (IC50 = 4.43 µg/ml) cells. CGA was genotoxic against V79 cells at 0.07 mg/ml and cytotoxic against to HT-29 (IC50 = 71.70 µg/ml), OVCAR-3 (IC50 = 80.07 µg/ml), MCF-7 (IC50 = 45.58 µg/ml) and, NCI-H460 (IC50 = 71.89 µg/ml) cancer cell lines. Wistar rats treated with a single dose (2,000 mg/kg) CAE-Cp decreased hemoglobin levels after 14 days, although no significant toxicity was observed in animals after 28 days. In view of the in vitro cytotoxicity and genotoxicity detected, further studies are necessary to establish the safe use of CAE-Cp.

Genotoxic and chemopreventive assessment of Cynara scolymus L. aqueous extract in a human-derived liver cell line.

Cynara scolymus L., popularly known as artichoke, is consumed as food and used as tea infusions for pharmacological purposes to treat liver dysfunctions and other conditions. Scientific data on the safety and protective effect of artichoke in human-derived liver cells is missing. This study investigated the genotoxic and modulatory effect of a liophilized extract suspended in water of C. scolymus L. leaves. Four extract concentrations (0.62, 1.25, 2.5 and 5.0 mg/mL) were evaluated using the comet assay on human hepatocyte cultures, HepG2 cells. Genotoxicity was assessed after two treatment periods, 1 and 24 h. Antigenotoxicity was evaluated against oxidative lesions induced by hydrogen peroxide in pre-, simultaneous and post-treatment protocols. Artichoke leaves aqueous extract induced genotoxic effects in HepG2 cells after 1- and 24-h treatments. In turn, extract concentrations of 0.62, 1.25 and 2.5 mg/mL, exhibited a protective effect in pretreatment, compared to hydrogen peroxide alone. However, in simultaneous and post-treatment protocols, only the lowest concentration reduced the frequency of DNA damage induced by hydrogen peroxide. In addition, in the simultaneous treatment protocol, the highest artichoke extract concentration increased hydrogen peroxide genotoxicity. It can be concluded that artichoke is genotoxic, in vitro, to HepG2 cells, but can also modulate hydrogen peroxide DNA damage.

Protective effects of acerola juice on genotoxicity induced by iron in vivo.

Metal ions such as iron can induce DNA damage by inducing reactive oxygen species (ROS) and oxidative stress. Vitamin C is one of the most widely consumed antioxidants worldwide, present in many fruits and vegetables, especially inMalpighia glabra L., popularly known as acerola, native to Brazil. Acerola is considered a functional fruit due to its high antioxidant properties and phenolic contents, and therefore is consumed to prevent diseases or as adjuvant in treatment strategies. Here, the influence of ripe and unripe acerola juices on iron genotoxicity was analyzed in vivo using the comet assay and micronucleus test. The comet assay results showed that acerola juice exerted no genotoxic or antigenotoxic activity. Neither ripe nor unripe acerola juices were mutagenic to animals treated with juices, in micronucleus test. However, when compared to iron group, the pre-treatment with acerola juices exerted antimutagenic activity, decreasing significantly micronucleus mean values in bone marrow. Stage of ripeness did not influence the interaction of acerola compounds with DNA, and both ripe and unripe acerola juices exerted protective effect over DNA damage generated by iron.

Evaluation of the mutagenicity and genotoxicity of Arrabidaea chica Verlot (Bignoneaceae), an Amazon plant with medicinal properties.

Arrabidaea chica Verlot (Bignoniaceae) is an important folk medicine plant native to the Amazon region and used to treat anemia, hemorrhage, inflammation, intestinal colic, hepatitis, and skin affections. Although studies showed its therapeutic properties, little knowledge regarding genotoxic properties of this plant is available. The aim of this study was to determine the potential mutagenic and genotoxic/antigenotoxic effects of an A. chica chloroformic fraction (Ac-CF) obtained from leaves containing bioactive metabolites. The mutagenic effects were evaluated using the Salmonella mutagenicity assay, with TA98, TA97a, TA100, TA102, and TA1535 strains, with and without metabolic activation. In vivo mutagenic and genotoxic/antigenotoxic effects were investigated using the micronucleus (MN) test in bone marrow and alkaline comet assay in blood and liver after administration of 100, 500, or 1000 mg/kg Ac-CF in CF-1 mice by gavage (once a day for 3 d). In vitro antioxidant potential was evaluated using DPPH and xanthine/hypoxanthine assays. Ac-CF was not mutagenic in any of the Salmonella typhimurium strains tested and showed negative responses for mutagenicity and genotoxicity in mice. Further, Ac-CF displayed antigenotoxic effects by decreasing the oxidative DNA damage induced by hydrogen peroxide by greater than 50% in blood and liver. The antioxidant action detected in the in vitro assays demonstrated IC50 of 0.838 mg/ml in the xanthine/hypoxanthine assay and IC50 of 28.17 µg/ml in the DPPH assay. In conclusion, Ac-CF did not induce mutagenic and genotoxic effects and was able to protect DNA against oxidative damage in vivo, suggesting that this fraction may not pose genetic risks, although further toxicology assays are necessary.

Treatment with aqueous extract from Croton cajucara Benth reduces hepatic oxidative stress in streptozotocin-diabetic rats.

Croton cajucara Benth is a plant found in Amazonia, Brazil and the bark and leaf infusion of this plant have been popularly used to treat diabetes and hepatic disorders. The present study was designed to evaluate the oxidative stress as well as the therapeutic effect of Croton cajucara Benth (1.5 mL of the C. cajucara extract i.g.) in rats with streptozotocin-induced diabetes. Croton cajucara Benth was tested as an aqueous extract for its phytochemical composition, and its antioxidant activity in vitro was also evaluated. Lipid peroxidation and superoxide dismutase, catalase, and glutathione reductase activities were measured in the hepatic tissue, as well as the presence activation of p65 (NF-κB), through western blot. Phytochemical screening of Croton cajucara Benth detected the presence of flavonoids, coumarins and alkaloids. The extract exhibited a significant antioxidant activity in the DPPH-scavenging and the hypoxanthine/xanthine oxidase assays. Liver lipid peroxidation increased in diabetic animals followed by a reduction in the Croton-cajucara-Benth-treated group. There was activation of p65 nuclear expression in the diabetic animals, which was attenuated in the animals receiving the Croton cajucara Benth aqueous extract. The liver tissue in diabetic rats showed oxidative alterations related to the streptozotocin treatment. In conclusion the Croton cajucara Benth aqueus extract treatment effectively reduced the oxidative stress and contributed to tissue recovery.

Neurobehavioral and toxicological activities of two potentially CNS-acting medicinal plants of Piper genus.

Plants from the genus Piper are economically useful and some species have been indicated because of their medicinal properties in the central nervous system. However, few studies about toxicity and neurobehavioral effects have been conducted. In this study, two Piper species, P. amalago and P. mikanianum were investigated in rats to determine acute toxicity and to evaluate the ansiogenic/ansiolytic properties in the elevated plus-maze and the effects on locomotion and exploration in an open field. Additionally, genotoxic activities were evaluated, using the comet assay in several tissues and the micronucleus assay in bone marrow. The phytochemical analysis of both Piper species leaves suggests the presence of amide, essential oils, flavonoids and phenolic compounds. The LD(50) of P. amalago and P. mikanianum were estimated as 2,545 and 1,661 mg/kg, respectively. The behavioral and genotoxic parameters were determined after an intraperitoneal administration of P. amalago (250 or 420 mg/kg) or P. mikanianum (160 or 270 mg/kg). Both plants decreased the number of entries and time spent in the open arms in the plus-maze test, indicating an anxiogenic effect. Only P. mikanianum affected locomotion and exploration in the open field behavior test. No genotoxic or mutagenic effect was observed. Our results suggest that these Piper species act on the central nervous system, without induce genetic toxicity.

Antigenotoxicity and antioxidant activity of Acerola fruit (Malpighia glabra L.) at two stages of ripeness.

Genotoxic and antigenotoxic effects of acerola fruit at two stages of ripeness were investigated using mice blood cells. The results show that no ripeness stage of acerola extracts presented any genotoxic potential to damage DNA (Comet assay) or cytotoxicity (MTT assay). When antigenotoxic activity was analyzed, unripe fruit presented higher DNA protection than ripe fruit (red color) extract. The antioxidant capacity of substances also showed that unripe samples inhibit the free radical DPPH more significantly than the ripe ones. The results about determination of compounds made using HPLC showed that unripe acerola presents higher levels of vitamin C as compared to ripe acerola. Thus, vitamin C and the complex mixture of nutrients of Malpighia glabra L., and especially its ripeness stages, influenced the interaction of the fruit extract with the DNA. Acerola is usually consumed when ripe (red fruit), although it is the green fruit (unripe) that has higher potential as beneficial to DNA, protecting it against oxidative stress.