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Chronic kidney disease (CKD) is one of the deadliest diseases that can be induced by factors such as having high ectopic lipids and glucose build-up in proximal tubule cells. This leads to an imbalance of antioxidant and pro-oxidants in the kidney, causing excessive reactive oxidative stress (ROS). ROS contributes to renal dysfunction by promoting inflammation, apoptosis and fibrosis. In severe conditions, CKD may advance to end-stage renal disease (ESRD).
Boerhaavia diffusa (BD) is known for its immunomodulatory, antioxidant and anti-inflammatory properties. It is widely consumed as a dietary supplement for general health maintenance. It is also a common remedy used for managing hepatic and urinary disorders such as urinary tract infections and kidney stones. Here, we hypothesized that the administration of BD extract may exert renoprotective effects and potentially alleviate CKD progression through its antioxidant properties.
BD root powder (Bixa Botanical, India) was extracted using 60% methanol and water at a ratio of 1:25 (w/v) for 48h continuous stirring at room temperature. Subsequently, the solvents were removed using a freeze-dryer, resulting in the production of dried extracts.
The antioxidant properties of BD were evaluated by 2,2-diphenwheyl-1-picrylhydrazyl (DPPH) and Ferric Reducing Antioxidant Power (FRAP) assays. These assays provided insights into the free radical scavenging (FRS) and metal-chelating activities (MCA) of BD, respectively.
Human kidney proximal tubule cells (HK-2) were cultured in low glucose (LG, 5.5 mM) DMEM and treated with varying concentrations of BD extracts for 24 h. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the percentage of cell proliferation following the addition of BD extracts was also determined.
We assessed the BD extracts on their FRS activity in varying concentrations (Figure 1 and Table 1). Our results revealed that both extracts had FRS activity in a dose-dependent manner. The methanolic extract displayed a significantly lower half maximal inhibitory concentration (IC50 value) than the water extract, indicating a higher radical scavenging potency.
Similarly, the MCA of the extracts showed that the methanol extract exhibited higher FRAP activity compared to the water extract, measuring 0.657 and 0.358 µmol/mL of FeSO4 equivalents per mg extract, respectively (Figure 2 and Table 1).
We assessed the effect of BD extracts on cell viability at varying. The methanolic extract revealed a significantly increased cell proliferation rate after 24h of incubation for 250 and 500 µg/mL concentrations by approximately 10% (Figure 3a). Conversely, the water extract did not significantly alter cell viability, which remained between 90-100%.
Cytotoxicity evaluation of BD extracts at concentrations of 0-1000 µg/mL (selected based on the DPPH IC50 values) on HK-2 cells using LG-DMEM for 24 hours. The methanolic extract revealed a significantly increased cell proliferation rate at 250 and 500 µg/mL concentrations by approximately 15% (p < 0.001) (Figure 3a). Conversely, the water extract did not significantly alter cell viability, which remained between 90 – 100% (Figure 3b).
Conclusions
Our results affirm BD’s antioxidant properties, highlighting its potential as a therapeutic option for protecting cells against ROS-induced damage to ameliorate CKD.