Evaluation of antioxidant, anti-inflammatory, and analgesic potentials of combined polyphenol-rich fractions from Ziziphus mauritiana and Ziziphus spina-christi leaves through modulation of inflammatory and oxidative stress markers in Sprague Dawley rats model.

The main objective of the present study was to combine the ethanol extracts of Ziziphus mauritiana and Ziziphus spina-christi (ZMZS) and fractioned with different solvents of increasing polarity for evaluation of antioxidant, anti-inflammatory and analgesic potentials. The ethanolic extracts of leaves were prepared using Soxhlet apparatus, mixed in ratio (1:1 w/w) and then fractionated into n-hexane, ethyl acetate and n-butanol. In vitro antioxidant activity of all fractions was assessed using total phenolic contents (TPC), total flavonoid contents (TFC), 2,2-dipehnyl, 1-picryl hydrazyl (DPPH) free radical scavenging ability, reducing power and bleachability of β-carotene in Linoleic acid system assays. Ethyl acetate fraction (EAF) showed the highest antioxidant activity in terms of the highest TPC (212.15 mg/g) and TFC (39.54 mg/g), better DPPH radical scavenging (IC 4.82 µg/ml), reducing power and β-carotene bleachability. The reverse-phase high-performance liquid chromatography (Rp-HPLC) was performed for the characterization of polyphenols in Ziziphus mauritiana and Ziziphus spina-christi extracts individually and ethyl acetate fraction of ZMZS. Results showed that EAF was enriched with phenolic acids, notable coumarin, gallic acid, salicylic acid and chlorogenic acid, whereas quercetin and kaempferol were the most abundant flavonoids. In vivo anti-inflammatory activity of most potent ethyl acetate fraction was performed using a carrageenan-induced model and analgesic activity using hot plate method in male Sprague Dawley rats. Further, the suppression of inflammatory biomarkers and the oxidative stress parameters were evaluated. Results indicate that EAF possesses significant anti-inflammatory and anti-analgesic properties at 500 mg/kg BW as evidenced by their ability to reduce paw edema, percentage inhibition and prolonged latency time and proinflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and C-reactive protein (CRP). The EAF also normalized the oxidative stress levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GP), reduced glutathione (GSH) and catalase (CAT) when compared to the standard drug indomethacin. The present study concluded that ethyl acetate fraction (EAF) of combined ZMZS extract has remarkable antioxidant as well as anti-inflammatory and analgesic potentials possibly due to interactions of total phenolic and total flavonoid contents.