Maryna van de Venter

Antidiabetic screening and scoring of 11 plants traditionally used in South Africa

AIM To investigate the traditional antidiabetic uses of indigenous or naturalised South African plants using an optimised screening and scoring method. MATERIALS AND METHODS Eleven plant species were screened against Chang liver, 3T3-L1 adipose and C2C12 muscle cells measuring glucose utilisation in all three cell lines and toxicity in the hepatocytes and adipocytes only. A scoring system was devised to aid interpretation of results. RESULTS Catharanthus roseus results correlated with previously reported in vivo results, with best stimulation of glucose utilisation in hepatocytes. Momordica foetida and Momordica balsamina extracts were active in myocytes but only the latter stimulated glucose utilisation in hepatocytes. Brachylaena discolor gave the best overall results, with all plant parts giving high activity scores and negligible toxicity. In vitro toxicity results for Catharanthus roseus, Vinca major, Momordica balsamina and some Sclerocarya birrea extracts raise concern for chronic use. CONCLUSION This screening system increases the likelihood of identifying drug candidates using in vitro antidiabetic screening of crude plant extracts, whilst the scoring system aids data interpretation. ETHNOPHARMACOLOGICAL RELEVANCE The multitude of metabolic steps affected by Type II diabetes offer many drug targets but they complicate in vitro screening to validate traditional uses or find new drug leads from plants.

Antiinflammatory, analgesic and antioxidant activities of Cyathula prostrata (Linn.) Blume (Amaranthaceae).

ETHNOPHARMACOLOGICAL RELEVANCE Cyathula prostrata (Linn) Blume (Amaranthaceae) is an annual herb widely used traditionally in the treatment of various inflammatory and pain related health disorders in Nigeria. The aim of this study is to evaluate the anti-inflammatory, analgesic and antioxidant activities of the methanolic extract of Cyathula prostrata (Linn) Blume. MATERIALS AND METHODS The anti-inflammatory (phorbol 12-myristate 13-acetate (PMA)-induced reactive oxygen species (ROS), lipopolysaccharide (LPS) induced nitric oxide production in U937 macrophages, LPS-induced COX-2 expression, carrageenan-induced rat paw oedema, arachidonic acid-induced ear oedema and xylene-induced ear oedema), analgesic (acetic acid-induced writhing and hot plate tests) and antioxidant activities (DPPH [1,1-diphenyl-2-picrylhydrazyl] and lipid peroxidation assays) activities of the plant extract were investigated. RESULTS The methanolic extract of Cyathula prostrata did not show inhibitory activity in the in vitro PMA-induced reactive oxygen species, LPS-induced nitric oxide production and LPS-induced COX-2 expression assays. In the in vivo anti-inflammatory assays, the extract (50, 100 and 200mg/kg) showed a significant (P<0.05) dose-dependent inhibition in the carrageenan, arachidonic acid and xylene-induced tests. Cyathula prostrata produced a significant (P<0.05, 0.001) dose-dependent inhibition in the acetic acid and hot plate analgesic tests respectively. The plant extract did not exhibit any antioxidant activity in the DPPH and lipid peroxidation assays. CONCLUSION The results suggest that the methanolic extract of Cyathula prostrata possesses anti-inflammatory and analgesic activities and this authenticates the use of the plant in the traditional treatment of ailments associated with inflammation and pain.

Quantitative and qualitative analysis of sterols/sterolins and hypoxoside contents of three Hypoxis (African potato) spp.

The glycoside, hypoxoside, identified and isolated from the corms of the African potato (Hypoxis hemerocallidea) has shown promising anticancer activities. The African potato is used as an African traditional medicine for its nutritional and medicinal properties. Most research has been carried out on H. hemerocallidea (formerly known as H. rooperi), with very little or nothing on other Hypoxis spp. Thin layer chromatography (TLC) was used to confirm the presence of sterols/sterolins, whereas a GC method was developed to identify and quantify sterols (especially β-sitosterol) in chloroform extracts of H. hemerocallidea, H. stellipilis and H. sobolifera var. sobolifera. High performance liquid chromatography (HPLC) was used to identify and quantify hypoxoside content in these Hypoxis spp. TLC results showed that H. sobolifera var. sobolifera contained the most sterols and sterolins compared to the other two Hypoxis spp. Gas chromatography (GC) results show that β-sitosterol and campesterol were the two main phytosterols present in the Hypoxis extracts. H. sobolifera var. sobolifera and H. hemerocallidea contained the most β-sitosterol and hypoxoside, respectively. H. sobolifera and H. hemerocallidea contained 74.69 µg of β-sitosterol and 12.27 µg of hypoxoside per 5 mg of chloroform extracts, respectively. These results show a significant difference in the sterol/sterolin and hypoxoside contents between species of the genus Hypoxis, which may influence their degree of biological activities.

Antiproliferative and apoptosis inducing activity of Markhamia tomentosa leaf extract on HeLa cells

ETHNOPHARMACOLOGICAL RELEVANCE Markhamia tomentosa (Benth) K. Schum ex. Engl. (Bignoniaceae), a tree widely dispersed in West Tropical Africa, is used traditionally to treat various diseases as it possesses antimicrobial, antioxidant, analgesic, anticancer and anti-inflammatory activities. MATERIALS AND METHODS This study evaluates the cytotoxic effect and underlying mechanisms of the ethanolic extract of Markhamia tomentosa on HeLa and MCF-7 cancer cell lines and non-cancerous Vero cell line. Brine shrimp lethality test was used for preliminary screening. Cytotoxicity was determined using the MTT assay and IC50 was calculated. Effect of Markhamia tomentosa on the cell cycle was monitored by flow cytometry and the apoptosis-induction capability confirmed by exposure of phosphatidylserine to the outer leaflet of the plasma membrane. Loss of mitochondrial membrane potential was analysed by flow cytometry using JC-1. RESULTS Markhamia tomentosa was toxic to brine shrimps with LD50 of 31.62µg/ml. Cell viability and growth of HeLa cells was inhibited by the extract with an IC50 of 189.1±1.76µg/ml at 24h post treatment. However, no cytotoxic effect was observed in MCF-7 and Vero cell lines. The extract induced cell cycle arrest in HeLa cells in the G0/G1 phase resulting in cell death after 24h exposure. Induction of apoptosis in HeLa cells was substantiated by Annexin V-FITC/PI double staining showing phosphatidylserine translocation and depolarisation of the mitochondrial membrane potential by flow cytometry of JC-1 stained cells. CONCLUSION The ethanolic extract of Markhamia tomentosa induces G0/G1 in HeLa cells followed by induction of the intrinsic pathway of apoptosis.

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. Copyright

Rooperol as an antioxidant and its role in the innate immune system: An in vitro study

ETHNOPHARMACOLOGICAL RELEVANCE Biologically active rooperol is formed when the glucose subunits of the nontoxic glycoside, hypoxoside, are cleaved by β-glucosidase. Hypoxoside is isolated from Hypoxis, a medicinal plant genus frequently used by the indigenous people of South Africa as an immune system booster. The aim of this study was to investigate rooperols antioxidant and anti-inflammatory properties using the ferric reducing ability of plasma (FRAP) assay, NO and ROS production, and phagocytosis. MATERIALS AND METHODS Differentiation of human promonocytic U937 leukemia cells to monocyte-macrophages was induced using 10-100 nM 1,25(OH)(2)D(3) and PMA over 72 h. Differentiation was confirmed by light microscopy and flow cytometry. Undifferentiated and/or differentiated cells were treated with DMSO (0.25 v/v%, vehicle control), hypoxoside (50 μg/mL), rooperol (20 μg/mL) or PMA (10/20 nM, positive control). ROS production was measured in undifferentiated and differentiated monocyte-macrophages using DCFH-DA and flow cytometry. Phagocytosis of pHrodo™ Escherichia coli BioParticles(®) was measured using pre-treated monocyte-macrophage differentiated U937 cells. NO production was measured in monocyte-macrophage differentiated U937 cells using DAF-2 DA and flow cytometry. RESULTS Rooperol was shown to have similar or greater antioxidant potential than ascorbic acid. Differentiation of human promonocytic U937 leukemia cells to monocyte-macrophages were confirmed morphologically (cell attachment, clump- and pseudopodia-formation) and biochemically (CD11b and CD14 cell surface marker expression). Rooperol significantly increased ROS and NO production, and phagocytosis in undifferentiated and/or differentiated human promonocytic U937 leukemia cells. Hypoxoside had no or very little effect on ROS and NO production, and phagocytosis. CONCLUSION This study confirms previous reports that hypoxoside has to be converted to rooperol to be biologically active. The FRAP assay confirms the antioxidant capacity of rooperol seen in previous studies, whereas rooperols induction of ROS and NO production, and phagocytosis constitute novel findings. Possible mode(s) of action for the in vitro anti-inflammatory activities of rooperol may be explained by ROS and NO production, and phagocytosis.

Cell survival or apoptosis: rooperol's role as anticancer agent.

Nontoxic hypoxoside, isolated from Hypoxis, is converted to cytotoxic rooperol in the presence of beta-glucosidase. In this study, we investigated rooperols mechanism of action. IC50 values of hypoxoside and rooperol were determined against the HeLa, HT-29, and MCF-7 cancer cell lines, and peripheral blood mononuclear cells. DNA cell cycle arrest occurred in late G1 and/or early S phases, associated with increased p21(Waf1/Cip1) levels. Apoptosis was shown by caspase-3 and/or caspase-7 activation, phosphatidylserine translocation, DNA fragmentation, cell blebbing, and apoptotic body formation. Increased phospho-Akt, phospho-Bcl-2, and p21(Waf1/Cip1) proteins, and cell size correspond to cell survival strategies (associated with endoreduplication).

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. Copyright

The apoptotic and autophagic properties of two natural occurring prodrugs, hyperoside and hypoxoside, against pancreatic cancer cell lines

Pancreatic cancer is only the 12th most common cancer, but the fourth leading cause of cancer-related deaths in the world. This is due to late prognosis, poor response to chemotherapy and early metastases. Natural prodrugs may play an important role in the treatment of pancreatic cancer. The main aim of this study was to determine the cytotoxicity of five natural prodrugs, namely harpagoside, hyperoside, hypoxoside, oleuropein and polydatin, by investigating apoptosis and autophagy as possible mechanism/s of action. Hypoxoside and hyperoside have shown selective cytotoxicity at IC50 values of ∼25 and 50μM against INS-1 and MIA PaCa-2 pancreatic cancer cells, respectively. Hypoxoside and hyperoside induced G2/M phase arrest and caspase-3 activation in INS-1 and MIA PaCa-2 cells, respectively. Hoechst/phalloidin staining confirmed morphological changes, including condensed chromatin multinucleation, membrane blebbing and loss of cytoskeletal arrangement in INS-1 and MIA PaCa-2 cells. Acridine orange staining was absent in INS-1 (hypoxoside) and MIA PaCa-2 (hyperoside) treated cells, whereas LC3B expression was not significantly increased. INS-1 and MIA PaCa-2 treated cells favour the cell death pathway, apoptosis, over the cell survival pathway, autophagy.

Cytotoxicity of syringin and 4-methoxycinnamyl alcohol isolated from Foeniculum vulgare on selected human cell lines

This study was carried out to determine the cytotoxic effect of seven plant extracts and the isolated compounds – syringin and 4-methoxycinnamyl alcohol – on cancerous and non-cancerous cells. The ethanol extract of Foeniculum vulgare was found to exhibit the most significant toxicity with an IC50 value of 19.97 μg/mL on HeLa cells. Bioassay-guided fractionation led to the isolation of two compounds, syringin (1) and 4-methoxycinnamyl alcohol (2). Both compounds showed toxicity against MCF-7, HeLa and DU145 cancer cell line. The results showed that compound 2 showed high toxicity against all the cancer cell lines with IC50 values of 14.24, 7.82 and 22.10 μg/mL, respectively. 4-Methoxycinnamyl alcohol also showed no apoptotic effect in cell cycle analysis after 48 h at a concentration of 10 μg/mL. However, DNA fragmentation study revealed that necrosis took place at a concentration of 10 μg/mL after 48 h exposure.