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.

Evaluation of the wound healing properties of South African medicinal plants using zebrafish and in vitro bioassays.

ETHNOPHARMACOLOGICAL RELEVANCE: In South Africa, medicinal plants have a history of traditional use, with many species used for treating wounds. The scientific basis of such uses remains largely unexplored. AIM OF THE STUDY: To screen South African plants used ethnomedicinally for wound healing based on their pro-angiogenic and wound healing activity, using transgenic zebrafish larvae and cell culture assays. MATERIALS AND METHODS: South African medicinal plants used for wound healing were chosen according to literature. Dried plant material was extracted using six solvents of varying polarities. Pro-angiogenesis was assessed in vivo by observing morphological changes in sub-intestinal vessels after crude extract treatment of transgenic zebrafish larvae with vasculature-specific expression of a green fluorescent protein. Subsequently, the in vitro anti-inflammatory, fibroblast proliferation and collagen production effects of the plant extracts that were active in the zebrafish angiogenesis assay were investigated using murine macrophage (RAW 264.7) and human fibroblast (MRHF) cell lines. RESULTS: Fourteen plants were extracted using six different solvents to yield 84 extracts and the non-toxic (n=72) were initially screened for pro-angiogenic activity in the zebrafish assay. Of these plant species, extracts of Lobostemon fruticosus, Scabiosa columbaria and Cotyledon orbiculata exhibited good activity in a concentration-dependent manner. All active extracts showed negligible in vitro toxicity using the MTT assay. Lobostemon fruticosus and Scabiosa columbaria extracts showed noteworthy anti-inflammatory activity in RAW 264.7 macrophages. The acetone extract of Lobostemon fruticosus stimulated the most collagen production at 122% above control values using the MRHF cell line, while all four of the selected extracts significantly stimulated cellular proliferation in vitro in the MRHF cell line. CONCLUSIONS: The screening of the selected plant species provided valuable preliminary information validating the use of some of the plants in traditional medicine used for wound healing in South Africa. This study is the first to discover through an evidence-based pharmacology approach the wound healing properties of such plant species using the zebrafish as an in vivo model.

Comprehensive in vitro antidiabetic screening of Aspalathus linearis using a target-directed screening platform and cellomics.

The antidiabetic potential of Aspalathus linearis has been investigated for over a decade, however, its characterisation remains incomplete with results scattered across numerous journals making the information difficult to compare and integrate. To explore whether any potential antidiabetic mechanisms for A. linearis have been neglected and to compare the suitability of extracts of green and "fermented" A. linearis as potential antidiabetic treatment strategies, this study utilised a comprehensive in vitro antidiabetic target-directed screening platform in combination with high content screening and analysis/cellomics. The antidiabetic screening platform consisted of 20 different screening assays that incorporated 5 well-characterised antidiabetic targets i.e. the intestine, liver, skeletal muscle, adipose tissue/obesity and pancreatic ß-cells. Both the green and fermented extracts of A. linearis demonstrated very broad antidiabetic mechanisms as they revealed several promising activities that could be useful in combatting insulin resistance, inflammation, oxidative stress, protein glycation and pancreatic ß-cell dysfunction and death - with a strong tendency to attenuate postprandial hyperglycaemia and the subsequent metabolic dysfunction which arises as a result of poor glycaemic control. The green extract was more successful at combatting oxidative stress in INS-1 pancreatic ß-cells and enhancing intracellular calcium levels in the absence of glucose. Conversely, the fermented extract demonstrated a greater ability to inhibit α-glucosidase activity as well as palmitic acid-induced free fatty acid accumulation in C3A hepatocytes and differentiated L6 myotubes, however, further studies are required to clarify the potentially toxic and pro-inflammatory nature of the fermented extract.

Wild-Type Zebrafish (Danio rerio) Larvae as a Vertebrate Model for Diabetes and Comorbidities: A Review.

Zebrafish have become a popular alternative to higher animals in biomedical and pharmaceutical research. The development of stable mutant lines to model target specific aspects of many diseases, including diabetes, is well reported. However, these mutant lines are much more costly and challenging to maintain than wild-type zebrafish and are simply not an option for many research facilities. As an alternative to address the disadvantages of advanced mutant lines, wild-type larvae may represent a suitable option. In this review, we evaluate organ development in zebrafish larvae and discuss established methods that use wild-type zebrafish larvae up to seven days post fertilization to test for potential drug candidates for diabetes and its commonly associated conditions of oxidative stress and inflammation. This provides an up to date overview of the relevance of wild-type zebrafish larvae as a vertebrate antidiabetic model and confidence as an alternative tool for preclinical studies. We highlight the advantages and disadvantages of established methods and suggest recommendations for future developments to promote the use of zebrafish, specifically larvae, rather than higher animals in the early phase of antidiabetic drug discovery.

In Vitro Evaluation of the Phytopharmacological Potential of Sargassum incisifolium for the Treatment of Inflammatory Bowel Diseases.

Background: Comprised of Crohn's disease and ulcerative colitis, inflammatory bowel diseases (IBD) are characterized by chronic inflammation of the gastro-intestinal tract, which often results in severe damage to the intestinal mucosa. This study investigated metabolites from the South African endemic alga, Sargassum incisifolium, as potential treatments for IBD. Phytochemical evaluation of S. incisifolium yielded prenylated toluhydroquinones and toluquinones, from which semi-synthetic analogs were derived, and a carotenoid metabolite. The bioactivities of S. incisifolium fractions, natural products, and semi-synthetic derivatives were evaluated using various in vitro assays. Methods: Sargahydroquinoic acid isolated from S. incisifolium was converted to several structural derivatives by semi-synthetic modification. Potential modulation of IBD by S. incisifolium crude fractions, natural compounds, and sargahydroquinoic acid analogs was evaluated through in vitro anti-inflammatory activity, anti-oxidant activity, cytotoxicity against HT-29 and Caco-2 colorectal cancer cells, and PPAR-γ activation. Results: Sargahydroquinoic acid acts on various therapeutic targets relevant to IBD treatment. Conclusions: Conversion of sargahydroquinoic acid to sarganaphthoquinoic acid increases peroxisome proliferator activated receptor gamma (PPAR-γ) activity, compromises anti-oxidant activity, and has no effect on cytotoxicity against the tested cell lines.

In Vitro Antidiabetic Activity and Mechanism of Action of Brachylaena elliptica (Thunb.) DC.

In South Africa, the number of people suffering from diabetes is believed to be rising steadily and the current antidiabetic therapies are frequently reported to have adverse side effects. Ethnomedicinal plant use has shown promise for the development of cheaper, cost-effective antidiabetic agents with fewer side effects. The aim of this study was to investigate the antidiabetic activity and mechanism of action of aqueous leaf extract prepared from Brachylaena elliptica. The potential of the extract for cytotoxicity was evaluated using MTT assay in HepG2 cells. The effects of the plant extract on glucose utilization in HepG2 cells and L6 myotubes, triglyceride accumulation in 3T3-L1, INS-1 proliferation, glucose metabolism in INS-1 cells, and NO production in RAW macrophages were also investigated using cell culture procedures. The inhibitory effects of the extract on the activities of different enzymes including alpha-amylase, alpha-glucosidase, pancreatic lipase, dipeptidyl peptidase IV (DPP-IV), collagenase, and CYP3A4 enzymes were evaluated. The extract also tested against protein glycation using standard published procedure. The plant extract displayed low level of toxicity, where both concentrations tested did not induce 50% cell death. The extract caused a significant increase in glucose uptake in HepG2 liver cells, with efficacy significantly higher than the positive control, berberine. The crude extract also displayed no significant effect on muscle glucose uptake, triglyceride accumulation in 3T3-L1, glucose metabolism in INS-1 cells, alpha-amylase, alpha-glucosidase, DPP-IV, lipase, protein glycation, and collagenase compared to the respective positive controls. The extract displayed a proliferative effect on INS-1 cells at 25 µg/ml when compared to the negative control. The plant also produced a concentration-dependent reduction in NO production in RAW macrophages and also demonstrated weak significant inhibition on CYP3A4 activity. The findings provide evidence that B. elliptica possess antidiabetic activity and appear to exact its hypoglycemic effect independent of insulin.

Gelatin as a convenient surrogate protein to model the in vitro effects of advanced glycation end-product formation.

Protein glycation has been implicated in skin ageing and several other disease states; however, the slow rate of glycation end-product formation makes in vitro studies challenging and often impractical. Gelatin, a denatured form of collagen, was identified as a convenient glycation surrogate amenable to cell culture conditions. The suitability of glycated gelatin to model the effects of AGE formation was verified using RAW 264.7 macrophages which revealed a remarkable correlation to previously documented effects. Effects of glycated gelatin on the central role of NF-ĸB and its downstream consequences (COX-2 and CD86) confirmed the pro-inflammatory nature of advanced glycation end-products. Together, these findings provide confidence that this model could prove a valuable tool to study the poorly understood mechanisms characterizing cellular dysfunction in response to AGE accumulation.

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.

In Vitro Investigation of Potential Anti-Diabetic Activity of the Corm Extract of Hypoxis Argentea Harv. Ex Baker.

The corms of Hypoxis argentea are widely used as a traditional remedy for diabetes mellitus in South Africa. In this study, we investigated the effects of non-toxic concentrations (12.5-100 µg mL-1) of the aqueous extract of H. argentea (HAA) corms on glucose uptake, pancreatic beta cell proliferation, and adipocyte differentiation. HAA stimulated glucose uptake in HepG2 cells up to 19.6 % and 17.0 % in L6 myotubes. Live-cell imaging microscopy revealed significant increases (p < 0.001) in total INS-1 cell numbers exposed to HAA, although no effect was observed on adipogenesis in 3T3-L1 pre-adipocytes. HAA produced weak to moderate inhibition of porcine pancreatic α-amylase, α-glucosidase, porcine pancreatic lipase, dipeptidyl peptidase IV (DPP IV) activities, as well as protein glycation. Our results suggest that the acclaimed anti-diabetic effects of H. argentea could be mediated by its promotion of glucose utilization and preservation of pancreatic beta cell populations while preventing fat accumulation in adipocytes.

Antifungal and antiproliferative activities of endophytic fungi isolated from the leaves of Markhamia tomentosa.

CONTEXT: Plants harbor endophytes with potential bioactivity. Markhamia tomentosa (Benth) K. Schum ex. Engl. (Bignoniaceae) is reported to possess antioxidant, anti-inflammatory and anticancer activities. OBJECTIVE: The antifungal and antiproliferative properties of endophytic fungi extracts and fractions from M. tomentosa were evaluated. MATERIAL AND METHODS: Endophytic fungi were isolated from the leaves of M. tomentosa and identified by ITS-rDNA sequence analysis. The antagonistic effect of the fungal strains was investigated against pathogenic fungi viz, Fusarium oxysporum, Sclerotinia sclerotiorium, Rhizoctonia solani, and Botrytis cinerea using the dual culture assay for 5-7 days. Antiproliferative effect of the fungal extracts and fractions (3.91-250 µg/mL) on HeLa cancer cell line was tested and IC50 was calculated. Poisoning food assay and antifeedant activity against the pathogenic fungi and Spodoptera litura larvae, for 7 days and 2 h, respectively, was also tested at concentrations of 250, 500 and 1000 µg/mL. RESULTS: Fungal endophytes Trichoderma longibrachiatum and Syncephalastrum racemosum were isolated from the leaves of M. tomentosa. Isolated endophytic fungal strains and solvent extracts showed MIC value of 1000 µg/mL against tested pathogenic fungi in the dual culture and poisoning food assays. Methanol fraction of S. racemosum isolate showed the most effective antiproliferative activity with IC50 of 43.56 µg/mL. Minimal feeding deterrent activity against S. litura larvae was also observed. DISCUSSION AND CONCLUSION: These findings showed that the leaves of Markhamia tomentosa harbor strains of endophytic fungi with promising health benefits, and suggest their antifungal and antiproliferative effects against pathogenic fungi and HeLa cancer cell line.

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.

Effect of Sutherlandia frutescens on the lipid metabolism in an insulin resistant rat model and 3T3-L1 adipocytes.

High fat diet induced insulin resistance correlates with dyslipidaemia and ectopic fat deposits in skeletal muscle and liver. The effects of Sutherlandia frutescens, an antidiabetic medicinal plant, on lipid metabolism were evaluated in an insulin resistant (IR) rat model and in 3 T3-preadipocytes. Wistar rats received normal diet (ND) or high fat diet (HFD). After the onset of IR in the HFD group, the rats were subdivided into two subgroups, which either continued with HFD or were treated with 50 mg S. frutescens/kg BW/day and HFD (HFD + SF). After 4 weeks, the HFD + SF rats had a significantly lower body weight than the HFD rats (p < 0.05). Blood plasma analysis showed a decrease in insulin, free fatty acids and triglycerides. Related changes in lipid parameters were observed in the liver, skeletal muscle and adipose tissue. To investigate the effects of S. frutescens on adipose tissue, 3 T3-L1 cells were used as a model. Treatment with S. frutescens led to a decrease in triglyceride accumulation, whilst glucose consumption and lactate production were increased (p < 0.05). These results indicate that S. frutescens directly affects mitochondrial activity and lipid biosynthesis in adipose tissue and provide a mechanism by which S. frutescens can restore insulin sensitivity by modulating fatty acid biosynthesis.

Sutherlandia frutescens limits the development of insulin resistance by decreasing plasma free fatty acid levels.

Intake of high caloric food induces raised plasma free fatty acids, culminating in insulin resistance (IR) and Diabetes mellitus type 2 (DMT2). The present study has shown for the first time that Sutherlandia frutescens reduces plasma free fatty acid levels in rats fed a high fat diet, thereby preventing the development of insulin resistance. A commercially available S. frutescens extract was administered to rats to examine its effects on the progression of high fat diet induced IR. In comparison to rats fed high fat diet only (positive control for IR), levels of plasma free fatty acids (FFA) were significantly reduced after one week (p < 0.025). Twelve weeks of treatment with S. frutescens reduced the level of plasma free fatty acids below that of rats fed a normal diet (negative control) (p < 0.025). QUICKI and HOMA-IR index confirmed that S. frutescens treated rats did not develop IR when fed a high fat diet for twelve weeks. In addition to preventing IR and reducing plasma FFA, chronic medication over twelve weeks decreased total cholesterol levels and the LDL/HDL ratio. We propose that S. frutescens is an effective medicinal remedy to prevent elevated plasma free fatty acids and IR, and therefore DMT2.