Exploring four South African Croton species for potential anti-inflammatory properties: in vitro activity and toxicity risk assessment.

ETHNOPHARMACOLOGICAL RELEVANCE: The African Continent harbours approximately 26 Croton species. Many Croton species are used in traditional medicine in southern Africa to treat a variety of ailments including malaria, tuberculosis, microbial infection and inflammation. Considering the high diversity of the genus Croton, the ethnopharmacological information available on southern African species is rather limited. Furthermore, the potential for novel anti-inflammatory drug scaffolds has not previously been investigated. AIM OF THE STUDY: The aim of the study was to evaluate the potential of four South African Croton species extracts (Croton gratissimus, Croton pseudopulchellus, Croton sylvaticus, and Croton steenkampianus) for anti-inflammatory activity targeting the TLR4 signalling pathway and to assess the potential risk for hepatotoxicity and genotoxicity using an in vitro cellomics approach. MATERIAL AND METHODS: Leaf extracts of C. gratissimus, C. pseudopulchellus, C. sylvaticus and C. steenkampianus were prepared using methanol and chloroform (1:1, v/v). The anti-inflammatory activity was determined using LPS induced nitric oxide production in RAW 264.7 macrophages, while the hepatotoxicity and genotoxicity was evaluated using multi-parameter end point analysis in C3A and Vero cells, respectively. Mitochondrial membrane potential, mitochondrial mass, oxidative stress, lysosomal content and lipid accumulation were used as markers to assess the risk for hepatotoxicity. RESULTS: All four species attenuated nitric oxide production with negligible cytotoxicity. However, C. gratissimus yielded the most favorable profile. Cell density was significantly reduced in both C3A and Vero cells with the C. gratissimus extract providing a suitable toxicity profile amenable to further high content analysis. While there was no meaningful effect on mitochondrial dynamics, a strong dose dependent increase in lipid content, paralleled by an expansion of the lysosomal compartment, identifies a potential risk for steatosis. Risk for genotoxicity was investigated using the micronucleus assay which revealed a dose dependent increase in micronuclei formation. Changes in nuclear morphology and cell ploidy further strengthens the associated risk for genotoxicity and suggests the extract from C. gratissimus may function as an aneugen. Collectively, the data demonstrates that although the selected species possess anti-inflammatory components, the risk for possible hepatotoxic and genotoxic side effects may negate their prospect towards further drug development.

In Vitro Anti-proliferative Activity and Mechanism of Action of Anemone nemorosa.

Anemone nemorosa is part of the Ranunculaceae genus Anemone (order Ranunculales) which comprises more than 150 species. Various parts of the plant have been used for the treatment of numerous medical conditions such as headaches, tertian agues, rheumatic gout, leprosy, lethargy, eye inflammation as well as malignant and corroding ulcers. The Anemone plants have been found to contain various medicinal compounds with anti-cancer, immunomodulatory, anti-inflammatory, anti-oxidant and anti-microbial activities. To date there has been no reported evidence of its use in the treatment of cancer. However, due to the reported abundance of saponins which usually exert anti-cancer activity via cell cycle arrest and the induction of apoptosis, we investigated the mode of cell death induced by an aqueous A. nemorosa extract by using HeLa cervical cancer cells. Cisplatin was used as a positive control. With a 50% inhibitory concentration (IC50) of 20.33 ± 2.480 µg/mL, treatment with A. nemorosa yielded a delay in the early mitosis phase of the cell cycle. Apoptosis was confirmed through fluorescent staining with annexin V-FITC. Apoptosis was more evident with A. nemorosa treatment compared to the positive control after 24 and 48 h. Tetramethylrhodamine ethyl ester staining showed a decrease in mitochondrial membrane potential at 24 and 48 h. The results obtained imply that A. nemorosa may have potential anti-proliferative properties.

Potential Therapeutic Benefits of Green and Fermented Rooibos (Aspalathus linearis) in Dermal Wound Healing.

The process of wound healing constitutes an ordered sequence of events that provides numerous opportunities for therapeutic intervention to improve wound repair. Rooibos, Aspalathus linearis, is a popular ingredient in skin care products, however, little scientific data exists exploring its therapeutic potential. In the present study, we evaluated the effects of fermented and aspalathin-enriched green rooibos in various in vitro models representative of dermal wound healing. Treatment of RAW 264.7 macrophages with fermented rooibos resulted in increased nitric oxide production as well as increased levels of cellular inducible nitric oxide synthase and cyclooxygenase-2, which are typical markers for classically activated macrophages. In contrast, the green extract was devoid of such activity. Using glycated gelatin as a model to mimic diabetic wounds, only the green extract showed potential to reduce cyclooxygenase-2 levels. Considering the role of reactive oxygen species in wound healing, the effects of rooibos on oxidative stress and cell death in human dermal fibroblasts was evaluated. Both fermented and green rooibos decreased cellular reactive oxygen species and attenuated apoptotic/necrotic cell death. Our findings highlight several properties that support the therapeutic potential of rooibos, and demonstrate that green and fermented rooibos present distinctly different properties with regards to their application in wound healing. The proinflammatory nature of fermented rooibos may have therapeutic value for wounds characterised with a delayed initial inflammatory phase, such as early diabetic wounds. The green extract is more suited to wounds burdened with excessive inflammation as it attenuated cyclooxygenase-2 levels and effectively protected fibroblasts against oxidative stress.

Anticancer potential of selected Fallopia Adans species.

The aim of the present study was to determine the anticancer potential of three species belonging to the Fallopia genus (Polygonaceae): Fallopia convolvulus (F. convolvulus, Fallopia dumetorum (F. dumetorum) and Fallopia aubertii (F. aubertii). For this purpose, crude extracts were obtained and characterized for their phenolic and flavonoid total content and examined for their anticancer activity on three tumor cell lines: breast cancer (MCF7), colon carcinoma (Caco-2) and cervical cancer (HeLa) cells. The cytotoxic potential of the three species was assessed by MTT assay, cell cycle analysis and by the evaluation of mitochondrial membrane potential (MMP). The acute toxicity of the extracts was evaluated using one in vitro cell model (Vero cells, an African Green monkey kidney cell line) and two invertebrate in vivo models (Daphnia magna and Artemia salina). The highest total phenolic and flavonoid content was found in the F. aubertii flower extracts. The cytotoxic effects of the extracts from F. aubertii and F. convolvulus on all three cell lines were examined at concentrations ranging from 3 to 300 µg/ml. G2/M cell cycle arrest was induced by all the extracts, and a significant increase in the subG1 cell population was observed. The hydroethanolic extract from the flowers of F. aubertii induced cell apoptosis more rapidly than the other extracts. The MMP indicates the involvement of the mitochondria in the induction of apoptosis. A positive correlation between the total phenolic content of the extracts and the IC50 values against the HeLa cells was also noted. None of the extracts exhibited significantly toxic effects. Considering the antitumor potential of F. aubertii and F. convolvulus, these two species may represent a good source of plant extracts with anticancer properties.

Antiproliferative mechanism of the methanolic extract of Enterolobium cyclocarpum (Jacq.) Griseb. (Fabaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Enterolobium cyclocarpum (Jacq.) Griseb. is a tropical tree that has folkloric implications against many ailments and diseases including cancer. MATERIALS AND METHODS: To explore the ethnopharmacological claims against cancer, the cytotoxicity of the methanolic extract of the leaves, was investigated using the brine shrimp lethality assay, MTT assay using cervical (HeLa) and breast (MCF7) cancer cell lines, cell cycle analysis and Annexin V-FITC/PI assay. RESULTS: In the brine shrimp lethality assay, the extract showed cytotoxic activity with LC50 value of 31.63 µg/mL. Significant growth inhibition was observed in both cell lines with IC50 values of 2.07 ± 1.30 µg/mL and 11.84 ± 1.18 µg/mL for HeLa and MCF7, respectively. Cell cycle analysis indicated that HeLa cells were arrested in the G2/M phase while MCF7 cells arrested in the G1/G0 phase. The Annexin V-FITC/PI assay revealed phosphatidylserine translocation in both cell lines and thus apoptosis induction upon treatment with the extract. CONCLUSION: The study demonstrated the potential antiproliferative activity of Enterolobium cyclocarpum thereby supporting the traditional claim and provides basis for further mechanistic studies and isolation of active constituents.