Evaluation of the therapeutic potential of green rooibos (Aspalathus linearis) extract in neurological disease
Date
2021-04
Authors
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Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: The intricate relationship between oxidative stress and neuroinflammation has been demonstrated in the aetiology of most neurodegenerative diseases. Current pharmacological treatments of these diseases have limitations and side effects. This has led to extensive investigations of novel therapeutic approaches to modify the course of these conditions. Globally, phytochemicals, especially polyphenols, have been investigated in vitro and in vivo for their antioxidant and anti-inflammatory properties. Aspalathus linearis (Rooibos) is one such plant. Although unfermented (green) rooibos is well known for its antioxidant capacity, no information is available on its neuroprotective capacity.
Aim: This study aimed to establish the neuroprotection potential of unfermented rooibos extract in neuronal cells and astrocytes. In addition, an evaluation of the antioxidant and anti-inflammatory capacity, as well as potential mechanism of action of rooibos in neuroprotection was done in vivo in a zebrafish larval model. Method: Antioxidant properties of unfermented rooibos were quantified using cell-free systems (DPPH, ORAC and xanthine/xanthine oxidase). Green rooibos extract (Green oxithinTM) neuroprotective capacity was assessed in mouse neuroblastoma (Neuro-2a) cells exposed to different concentrations of hydrogen peroxide (250 or 125 μM) after a 24-hour treatment with different rooibos extract concentrations (12.5, 25, 50 and 100 μg/mL), as well as in human astrocytes treated with varying concentrations of rooibos extract (800, 400, 200, 100 and 50 μg/ml) and exposed to 20 μg/mL of E.coli lipopolysaccharide (LPS). MCP-1 secretion by astrocytes were determined by ELISA and XTT assay was used to assess cell viability. Intracellular ROS was quantified as a parameter of cellular redox status and MAO-A inhibition as further indicators of neuroprotection. Moreover, an in vivo PTZ-induced seizure model was employed to probe GABA receptor modulation as potential mechanism of action of rooibos.
Results: Green rooibos scavenged free radicals of both exogenous and endogenous origin in a dose dependent manner with a similar profile activity to vitamins C or E. Green rooibos extract protected N2a cells, predominantly at the assault of a lethal concentration (250 μM) of H2O2 where up to 15% (p<0.0001) cell survival was observed. Intracellular ROS levels were significantly reduced (p<0.0001) at both 250 and 125 μM H2O2. In a dose dependent way, MCP-1 production decreased significantly in cells pre-treated with rooibos (p<0.0024). Finally, current data generated in zebrafish do not support a GABA receptor-associated mechanism of action rooibos.
Conclusion: Green rooibos extract modulated the production of MCP-1 by astrocytes and mildly inhibited MAO-A.
AFRIKAANSE OPSOMMING: Die komplekse verhouding tussen oksidatiewe stress en neuroinflammasie word geïllustreer deur die etiologie van meeste neurodegeneratiewe siektes. Huidige farmakologiese behandeling van hierdie siektes het beperkings en newe-effekte. Daarom word uitgebreide ondersoeke gedoen na nuwe terapeutiese aanslae om hierdie toestande aan te spreek. Fitochemikalieë en veral polifenole word wyd ondersoek, beide in vitro en in vivo, vir hul anti-oksidant en anti-inflammatoriese effekte. Aspalathus linearis (rooibos) is een so ‘n plant. Alhoewel ongegisde (groen) rooibos bekend is vir sy anti-oksidant aktiwiteit, is geen inligting bekend oor die neurobeskermende kapasiteit van rooibos nie. Doel: Ons doel was om die neurobeskermende eienskappe van groen rooibos te bepaal, in neurone en astrosiete. Die anti-oksidant en ensiem inhiberende eienskappe van rooibos is ook ondersoek. Laastens is ‘n evaluering van ‘n moontlike meganisme van aksie van rooibos in ‘n in vivo zebravis larwe model gedoen. Metodes: Anti-oksidant effek van groen rooibos is gekwantifiseer deur gebruik te maak van selvrye sisteme (DPPH, ORAC en xanthine/xanthine oksidase). Die neurobeskermende effek van groen rooibosekstrak (Green oxithinTM) is bepaal in muis neuroblastoomselle (Neuro-2a), deur die selle bloot te stel aan 250 of 125 µM waterstofperoksied, na ‘n 24-uur kondisionering met verskillende rooibosekstrakkonsentrasies (12.5, 25, 50 en 100 µg/mL). Anti-inflammatoriese effekte van rooibos is ook bepaal in menslike astrosiete wat behandel is met verskillende konsentrasies van rooibos (800, 400, 200, 100 en 50 µg/ml) asook 20 μg/mL E.coli lipopolisakkaried (LPS). MCP-1 sekresie is bepaal met ELISA. Sel oorlewing (XTT) en intersellulêre RSS vlakke is ook bepaal, asook die vermoë van rooibos om MAO-A te inhibeer. Laastens in ‘n in vivo zebravis PTZ model gebuik om te ondersoek of die kalmerende effekte van rooibos via GABA reseptors plaasvind. Resultate: Groen rooibos het vrye radikale, beide eksogeen en endogen in oorsprong – op ‘n dosis-afhanklike manier geabsorbeer, soortgelyk aan die effek van vitamien C en E. In die teenwoordigheid van 250 µM H2O2 het groen rooibos effektief seldood verminder (p<0.0001), terwyl intrasellulêre RSS verlaag (p<0.0001) is by beide 250 en 125 µM H2O2. MCP-1 sekresie deur astrosiete was ook beduidend laer (p<0.0024) in selle wat met rooibos behandel is. Laastens het data in zebravisse nie op ‘n GABA reseptor-geassosieerde meganisme van rooibos gedui nie.
AFRIKAANSE OPSOMMING: Die komplekse verhouding tussen oksidatiewe stress en neuroinflammasie word geïllustreer deur die etiologie van meeste neurodegeneratiewe siektes. Huidige farmakologiese behandeling van hierdie siektes het beperkings en newe-effekte. Daarom word uitgebreide ondersoeke gedoen na nuwe terapeutiese aanslae om hierdie toestande aan te spreek. Fitochemikalieë en veral polifenole word wyd ondersoek, beide in vitro en in vivo, vir hul anti-oksidant en anti-inflammatoriese effekte. Aspalathus linearis (rooibos) is een so ‘n plant. Alhoewel ongegisde (groen) rooibos bekend is vir sy anti-oksidant aktiwiteit, is geen inligting bekend oor die neurobeskermende kapasiteit van rooibos nie. Doel: Ons doel was om die neurobeskermende eienskappe van groen rooibos te bepaal, in neurone en astrosiete. Die anti-oksidant en ensiem inhiberende eienskappe van rooibos is ook ondersoek. Laastens is ‘n evaluering van ‘n moontlike meganisme van aksie van rooibos in ‘n in vivo zebravis larwe model gedoen. Metodes: Anti-oksidant effek van groen rooibos is gekwantifiseer deur gebruik te maak van selvrye sisteme (DPPH, ORAC en xanthine/xanthine oksidase). Die neurobeskermende effek van groen rooibosekstrak (Green oxithinTM) is bepaal in muis neuroblastoomselle (Neuro-2a), deur die selle bloot te stel aan 250 of 125 µM waterstofperoksied, na ‘n 24-uur kondisionering met verskillende rooibosekstrakkonsentrasies (12.5, 25, 50 en 100 µg/mL). Anti-inflammatoriese effekte van rooibos is ook bepaal in menslike astrosiete wat behandel is met verskillende konsentrasies van rooibos (800, 400, 200, 100 en 50 µg/ml) asook 20 μg/mL E.coli lipopolisakkaried (LPS). MCP-1 sekresie is bepaal met ELISA. Sel oorlewing (XTT) en intersellulêre RSS vlakke is ook bepaal, asook die vermoë van rooibos om MAO-A te inhibeer. Laastens in ‘n in vivo zebravis PTZ model gebuik om te ondersoek of die kalmerende effekte van rooibos via GABA reseptors plaasvind. Resultate: Groen rooibos het vrye radikale, beide eksogeen en endogen in oorsprong – op ‘n dosis-afhanklike manier geabsorbeer, soortgelyk aan die effek van vitamien C en E. In die teenwoordigheid van 250 µM H2O2 het groen rooibos effektief seldood verminder (p<0.0001), terwyl intrasellulêre RSS verlaag (p<0.0001) is by beide 250 en 125 µM H2O2. MCP-1 sekresie deur astrosiete was ook beduidend laer (p<0.0024) in selle wat met rooibos behandel is. Laastens het data in zebravisse nie op ‘n GABA reseptor-geassosieerde meganisme van rooibos gedui nie.
Description
Thesis (MSc)--Stellenbosch University, 2021.
Keywords
Rooibos tea (Aspalathus linearis) -- Health aspects, Neurodegenerative diseases, Novel therapeutic approaches, Unfermented rooibos extract, Antioxidants, Green rooibos -- Therapeutic use, Anti-inflammatory, UCTD