Physicochemical properties, immunostimulatory and antioxidant activities of a novel polysaccharide isolated from Mirabilis himalaica (Edgew) Heim

Abstract

Herbal medicines often contain bioactive polysaccharides. However, many medicinal herbs have not been explored for any active saccharides that may play key roles in their bioactivities. Herein, we extracted a novel polysaccharide from Mirabilis himalaica (Edgew) heim (denoted MHHP), a popular medicinal ingredient in traditional medicines. The structural and morphological characteristics of MHHP were measured and elucidated by high-performance gel permeation chromatography, gas chromatography connected with mass spectrometry, Fourier transform infrared and nuclear magnetic resonance spectroscopy as well as scanning electron microscopy. MHHP was homogeneous with a molecular weight of 16.1 kDa, M _w/M _n = 1.33, containing mainly α-d-glucan residues with (1→4)-linkage. The biological activities of MHHP upon proliferation of splenic lymphocyte, activation of related cytokine and production of nitric oxide (NO) in RAW264.7 cells were investigated in vitro. MHHP induced proliferation of mouse spleen lymphocytes and significantly promoted the secretion in TNF-α, IL-6 and NO production in RAW264.7 cells. Meanwhile, MHHP exhibited relatively low antioxidant abilities. Our data suggested that MHHP may have potential immunoregulatory and anti-inflammatory activity, with a moderate antioxidant activity.

Fig. 1. Elution curve of the water-extractable (MHHP) on a Sepharose Fast Flow column.

Fig. 2. Determination of molecular weight of MHHP via high-performance gel permeation chromatography. (A) The standard curve of dextrin; (B) GPC profile of dextrin; (C) GPC chromatograph of MHHP polysaccharide.

Fig. 3. GC profile of MHHP with acid hydrolysis and acetylation. (a) GC of standard monosaccharides; (b) acid hydrolyzed and acetylated monosaccharides of MHHP polysaccharide.

Fig. 4. UV spectrum (A) and Fourier transform infrared (FT-IR) spectrum (B) of MHHP polysaccharide.

Fig. 5. Analysis of structure of HMMP by NMR. (A) ¹H NMR spectrum; (B) ¹³C NMR spectrum; (C) COSY spectrum; (D)–(E) HSQC spectrum; (F) determined structure of MHHP polysaccharide. Solvent for NMR: D₂O. Temperature: 40 °C.

Fig. 6. Digital SEM photographs of MHHP polysaccharide in freeze-dry powder (A0–A3) and in ethanol (B0–B3) detected at magnifications of 1k, 5k, 20k and 80k, respectively.

Fig. 7. Effect of MHHP on lymphocyte proliferation in vitro (A), on IFN-γ (B) and IL-2 (C) secretion by mouse spleen lymphocyte (x ± SD, n = 3), Con-A was used as a positive control. The mouse lymphocyte cells were cultured in the presence of MHHP at three different concentrations for 48 h. The culture supernatant collected were assayed the levels of IFN-γ. *p < 0.05, **p < 0.01 versus cell control.

Fig. 8. Immunomodulatory activity of MHHP polysaccharide on RAW 264.7 cell. RAW 264.7 cells were cultured in the presence of MHHP at the three different concentrations for 48 h. The culture supernatant collected were assayed for the levels of TNF-α (a), IL-6 (b), and NO contents (c). LPS was used as a positive control, *p < 0.05, **p < 0.01 versus cell control.

Fig. 9. Antioxidant activity assay of MHHP. (A) DPPH radical scavenging assay; (B) superoxide anion-scavenging activity assay; (C) hydroxyl radical scavenging ability assay; values are means x ± SD, n = 3.