Preparation, Physicochemical Properties, and Anti-inflammatory Analysis of Liubao Tea Polysaccharides

Introduction: Liubao tea, a post-fermented tea from China, contains bioactive polysaccharides with potential health benefits. However, the relationship between physicochemical properties and biological activities of Liubao tea polysaccharides (LTP) remains unclear. materials and methods: Materials and reagents Liubao tea (LBT) was provided by Guangxi Wuzhou ShengyuanTea Co., Ltd, located in Wuzhou, China. The DMEM culture medium was acquired from HyClone Co., based in Los Angeles, USA. Nitric oxide (NO), Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and tumor necrosis factor-α(TNF-α) assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Polyclonal antibodies for RelA (p65), p-p65, NF-κB (IκB), p-IκB, and toll-like receptors 4 (TLR4), were secured from Abcam Co., CA, USA. Analytical-grade chemicals and solvents were used throughout. LTP preparation and purification The preparation of LTP was carried out according to the methods described in our previous report [11]. LTP was extracted with distilled water at 80 °C for 2 h with a solid-to-liquid ratio of 1:20 (w/v), and the extraction was repeated three times. Then, the extract was concentrated and precipitated with ethanol (final concentration 80%, v/v) at 4 °C overnight. Polysaccharide purification was performed as described by Zhu et al. [12]. Using a gradient of sodium chloride solutions at concentrations of 0, 0.2, 0.5, and 1.0 M, a DEAE-Sepharose Fast Flow chromatography column measuring 2.6×40 cm was administered at a steady flow rate of 1 mL/min, resulting in optimal separation of the polysaccharide fractions based on the ionic properties of the polysaccharides. Chemical composition. The total sugar content was measured using the phenol-sulphuric acid method, as described by Zhu et al. [13], and the uronic acid levels were assessed through the meta-hydroxyphenyl methodology [13]. Protein quantities were determined via the established Bradford method [14]. Monosaccharide composition The composition analysis of monosaccharides in the purified LTP followed a modified procedure as detailed in the report by Zhu et al. [14]. Mw distribution Dextran standards were employed to create a standard curve. The weight-average Mw was determined using high-performance gel permeation chromatography (HPGPC) (Waters1515, Waters, Milford, MA, USA) [15]. Fourier-transform infrared (FT-IR) spectroscopy For FT-IR spectroscopy, the LTP sample, combined with 100 mg of KBr powder, underwent a comprehensive scan via an infrared spectrometer (PE-1730, based in Massachusetts, USA). This analysis covered a spectral range from 4000 to 400 cm-1. X-ray diffraction (XRD) X-ray diffraction analysis was performed using a D8 ADVANCE X-ray diffractometer (Bruker, Billerica, Massachusetts, USA), employing a tube voltage of 35 kV and a tube current of 100 mA. The diffraction angles were scanned from 5° to 70° with a step size of 0.05°. Scanning electron microscopy (SEM) The sample’s surface morphology was analyzed with a HITACHI SU8010 scanning electron microscope (produced by JEOL in Tokyo, Japan) at 1000× and 10000× magnifications. Before the imaging process, 2 mg of the sample was mounted on a metal block using a conductive adhesive, followed by the deposition of gold onto its surface. Nuclear magnetic resonance (NMR) spectroscopy A total of 20 mg of LTP was dissolved in D2O and underwent lyophilization three times. The resultant substance was then re-dissolved in 0.5 mL of D2O. 1H spectroscopy data were acquired using the Avance III HD spectrometer (Bruker, Billerica, MA, USA). Evaluation of anti-inflammatory activity Cell culture RAW264.7 cells were cultured in DMEM high glucose medium supplemented with 10 Methods: LTP was extracted and purified using DEAE-Sepharose chromatography. Chemical composition, monosaccharide profile, molecular weight distribution, and structural characteristics were analyzed using spectroscopic techniques (FT-IR, XRD, SEM, NMR). Anti-inflammatory activities were evaluated in LPS-stimulated RAW264.7 macrophages by measuring nitric oxide (NO), proinflammatory cytokines (TNF-α, IL-1β, IL-6), and key signaling molecules. Results: LTP consisted primarily of galacturonic acid (0.404), galactose (0.218), and mannose (0.141), with two major fractions: LTP-1 (13121 Da) and LTP-2 (2056 Da). Structural analysis revealed a pyranose ring backbone with β-glycosidic linkages. LTP dose-dependently inhibited NO production and suppressed TNF-α, IL-1β, and IL-6 secretion in LPS-stimulated macrophages. Mechanistic studies demonstrated that LTP downregulated COX-2 and iNOS expression by inhibiting the TLR4/NF-κB signaling pathway, reducing p65 and IκBα phosphorylation. Conclusion: This study establishes the structure-activity relationship of LTP, demonstrating its antiinflammatory effects through TLR4/NF-κB pathway modulation. These findings provide scientific evidence for developing LTP-based functional foods and nutraceuticals with immunomodulatory properties.