Effects of Extraction Methods on the Physicochemical Properties and Biological Activities of heteroglycans from Hibiscus sabdariffa calyx

The hot-water extraction method has been employed to enhance the diffusion rate of heteroglycans, thereby augmenting the extraction efficiency. This study investigates the antioxidant activities and immunological properties of water-soluble polysaccharides derived from the Hibiscus sabdariffa calyx (HWPF and LWPF). These polysaccharides were isolated through two distinct extraction methods involving hot water followed by ethanol precipitation. The physicochemical characterization was conducted through a series of experiments encompassing monosaccharide composition, ultraviolet-visible spectrum analysis, and Fourier-transform infrared (FT-IR) spectroscopy. The evaluation of antioxidative properties employed various techniques, including DPPH and ABTS radical scavenging activities, Ferric Reducing Antioxidant Power, ferrous chelating power, total reducing power, and protection against DNA damage. The assessment of anti-inflammatory potential involved the inhibition of hemolytic activity induced by a hypotonic solution and heat. Furthermore, the impact of the polysaccharides on the production of pro-inflammatory cytokines was analyzed through Luminex assay and RTPCR assay in PBMCs and LPS-stimulated RAW 264.7 cells, respectively. The findings revealed that heteroglycans (LWPF and HWPF) exhibit a content of Ara-Man-Rha-GluA-GalA (41.2:17.2:15.7:9.2:8.8) and Ara-Glu-Rha-Xyl-GluA-Man (31.5:23.8:12.16:10.4:7:6) with molecular weights of approximately 4.5 KDa and 41.3 KDa, respectively. HWPF demonstrated robust metal chelating and scavenging activities. Additionally, each fraction exhibited substantial protection of DNA against damage induced by H2O2. LWPF was observed to significantly down-regulate the expression of pro-inflammatory cytokines IL-6 and COX-2 at both transcriptional and translational levels, along with inhibiting NO production. In summary, these results indicate that the choice of extraction methods influences both the structure and biological activities of heteroglycans, providing valuable insights into the structure-activity relationship. Keywords: Hibiscus sabdariffa, heteroglycans, hot water extraction, physicochemical properties, Antioxidant activity, Pro-inflammatory cytokines.