The antidiabetic and antioxidant properties of Athrixia phylicoides aqueous extract : an in vitro and ex vivo assessment

Chellan, Nireshni (Stellenbosch : University of Stellenbosch, 2011-03)

Thesis

ENGLISH ABSTRACT: Introduction: Athrixia phylicoides is an aromatic, indigenous shrub with high antioxidant content and numerous indigenous medicinal properties inferred by ingestion of an herbal brew of the plant. Commercialization of “bush tea” (derived from A. phylicoides) holds economic and developmental potential for indigenous communities provided the safety and efficacy of the herbal tea is established. Recently A. phylicoides has been shown by McGaw et al. (2007) to have similar antioxidant activity to Rooibos tea, and a unique, new flavonol (i.e. a polyphenolic antioxidant plant metabolite) 5-hydroxy-6,7,8,3′,4′,5′-hexamethoxyflavon-3-ol, unique to A. phylicoides, was isolated by Mashimbye et al. in 2006. With changes in the socio-economic climate and a new trend in merging Western lifestyle with traditional practices, new interest has been shown in herbal/natural remedies. Study Aim: The aim of this study was to firstly, determine the in vitro effect of A. phylicoides aqueous extract on glucose metabolism in cell lines that mimic the three key organs implicated in glucose homeostasis. Secondly, the study aimed to determine the potential ex vivo antioxidant and anti-inflammatory effect of the extract in pancreatic β-cells and peripheral mononuclear cells respectively. Methods: Leaves and fine twigs of A. phylicoides were processed into an aqueous extract. C2C12, Chang and 3T3-L1 cells were cultured under standard conditions and acutely exposed to increasing concentrations of extract and water vehicle, as well as 1 μM insulin and metformin as positive controls. Glucose uptake from 8 mM glucose culture media was determined using a fluorimetric oxidase method. Radioactive 14C-glucose oxidation to 14CO2 and determination of glycogen content of cells were used to assess the fate of intracellular glucose. RT-PCR was used to assess the extract effect on insulin-signalling gene expression. The antioxidative effect of A. phylicoides extract in pancreatic β-cells isolated from Wistar rats was determined by measuring nitric oxide (NO) production in response to hyperglycemic conditions. NO was labelled with diaminofluorocein diacetate and fluorescence was measured using flow cytometry. Insulin secretion of pancreatic β- cells was measured using radio-immuno assay. The anti-oxidative effect of the extract in lipopolysaccharide-stimulated peripheral mononuclear cells isolated from Wistar rats was determined by measuring the production of TNF-α using an ELISA kit. Results: C2C12 myocytes showed maximal increased glucose uptake at the 0.05 μg/μl extract concentration (228.3% ± 66.2, p<0.001). In Chang cells, A. phylicoides extract maximally increased the amount of glucose taken up at the 0.05 μg/μl concentration (134.5% ± 2.5, p<0.05). In 3T3-L1 cells, the extract maximally increased the amount of glucose taken up at the 0.025 μg/μl concentration (143.5% ± 10.3, p<0.001). An extract-induced increase in insulin receptor and glucose transporter four expression was seen in C2C12 myocytes. The oxidation of 14C-glucose to 14CO2 by C2C12 myocytes was maximally increased following acute exposure to the extract at 0.1 μg/μl (2919.3 fmol/1x10^6 cells ± 428, p<0.01). The oxidation of 14C-glucose to 14CO2 by Chang cells was maximally increased following acute exposure to extract at 0.1 μg/μl (4476.7 fmol/1x10^6 cells ± 1620, p<0.05); as seen in the C2C12 cells. A. phylicoides extract increased glycogen storage at all three concentrations tested in Chang cells, but maximally at the 0.025 μg/μl concentration (13.6 μg/1x10^6 cells ± 0.7, p<0.05). A. phylicoides extract did not have any measurable effect on the oxidative status of β-cells or the anti-inflammatory status of peripheral mononuclear cells. The extract did show an increase in first phase insulin secretion of β-cells in hyperglycemic conditions, although it was not significant. Conclusion: Athrixia phylicoides aqueous extract stimulates in vitro glucose uptake and metabolism in an insulin-mimetic manner, suggesting that this extract could potentially be beneficial to type two diabetics as an adjunct therapy.

AFRIKAANSE OPSOMMING: Inleiding: Athrixia phylicoides is 'n aromatiese, inheemse struik met 'n hoë antioksidant inhoud. Vele tradisionele medisinale eienskappe is gekoppel aan die ingestie van 'n kruie brousel van die plant, wat ook bekend as “bostee” is. Kommersialisering van “bostee” hou ekonomiese en ontwikkelings potensiaal in vir inheemse gemeenskappe mits die veiligheid en effektiwiteit van die kruietee bevestig kan word. McGaw et al. (2007) het onlangs bevind dat A. phylicoides se antioksidant aktiwiteit vergelykbaar is met die van rooibostee. 'n Unieke nuwe flavonol ('n polifenoliese antioksidant plant metaboliet) 5-hydroksie-6,7,8,3′,4′,5′-hexamethoksieflavon-3-ol, eie aan A. phylicoides, is deur Mashimbye et al. in 2006 geïsoleer. Met veranderings in die sosio-ekonomiese klimaat en 'n nuwe tendens om die westerse lewenstyl met tradisionele gebruike aan te vul word nuwe belangstelling in kruie/natuurlike rate ondervind. Studie Doelwitte: Die doelwitte van hierdie studie was eerstens om die in vitro effek van A. phylicoides waterekstrak op die glukosemetabolisme van drie sellyne wat die sleutel organe naboots wat glukosehomeostase beheer, te bepaal. Tweedens, is die potensiële ex vivo antioksidant en anti-inflammatoriese effek van die ekstrak op pankreatiese β-selle en perifere mononuklêere-selle onderskeidelik ondersoek. Metodes: n Waterige ekstrak is van die blare en fyn takkies van A. phylicoides berei. C2C12, Chang and 3T3-L1 selle is gekultuur onder standaard kondisies en akuut blootgestel aan stygende ekstrakkonsentrasies. Water het as kontrole gedien, met 1 μM insulien en metformien as positiewe kontroles. Glukose opname vanuit 8 mM glukose kultuurmedia is bepaal deur 'n fluorimetriese oksidase metode. Radioaktiewe 14C-glukose-oksidasie na 14CO2 en die bepaling van die glukogeen inhoud van selle is gebruik om die lot van intrasellulêre glukose te bepaal. RT-PKR is gebruik om die effek van die ekstrak op die insulien-seinpad geen-uitdrukking te ondersoek. Die antioksidant effek van A. phylicoides ekstrak in pankreatiese β-selle geïsoleer van Wistar rotte, is bepaal deur stikstofoksied (NO) produksie na aanleiding van hiperglukemiese kondisies. NO is met diaminofluorosien diasetaat gemerk en die fluoresensie gemeet deur vloeisitometrie. Insulien afskeiding deur die pankreatiese β-selle is deur radio-immuno metode bepaal. Die anti-oksidatiewe effek van die ekstrak op lipopolisakkaried-gestimuleerde perifere mononuklêere-selle afkomstig van Wistar rotte is bepaal deur die meting van TNF-α produksie met 'n ELISA kit. Resultate: C2C12 miosiete het 'n maksimale toename in glukoseopname by 'n 0.05 μg/μl ekstrakkonsentrasie (228.3% ± 66.2, p<0.001) gehad. Dieselfde ekstrakkonsentrasie het maksimale toename in glukoseopname in Chang selle (134.5% ± 2.5, p<0.05 getoon. In 3T3-L1 selle is maksimale toename in die glukoseopname by 'n konsentrasie van 0.025 μg/μl (143.5% ± 10.3, p<0.001) bereik. 'n Ekstrak-geinduseerde verhoging in die insulienreseptor en glukosetransporter vier ekspressie is in C2C12 miosiete waargeneem. Die oksidasie van 14C-glukose na 14CO2 deur C2C12 miosiete is maksimaal verhoog deur akute blootstelling aan die ekstrak by 'n konsentrasie van 0.1 μg/μl (2919.3 fmol/1x10^6 cells ± 428, p<0.01). Die oksidasie van 14C-glukose na 14CO2 deur Chang selle was maksimaal verhoog deur akute blootstelling aan die ekstrak by 'n konsentrasie van 0.1 μg/μl (4476.7 fmol/1x10^6 cells ± 1620, p<0.05) soos gevind in die C2C12 selle. Die ekstrak het glukogeenstoring verhoog teen al drie die konsentrasies waarteen getoets is in Chang selle, maar 'n maksimale effek is gevind by 'n konsentrasie van 0.025 (13.6 μg/1x10^6 cells ± 0.7, p<0.05). A. phylicoides ekstrak het nie 'n meetbare effek op die oksidatiewe status van β-selle of die anti-inflammatoriese status van perifere mononuklêere-selle gehad nie. Die ekstrak het wel 'n verhoging in die eerstefase insuliensekresie van β-selle in hyperglukemiese kondisies gehad, alhoewel die verhoging nie statisties betekenisvol was nie. Afleiding: Athrixia phylicoides waterekstrak stimuleer in vitro glukoseopname en metabolisme in 'n insulin-mimetiese manier, wat beteken dat die ekstrak potensiëel voordele vir tipe twee diabete kan inhou as aanvullingsterapie.

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