An inulin-type fructan (AMP1-1) from Atractylodes macrocephala with anti-weightlessness bone loss activity

Highlights

• An inulin-type fructan (AMP1-1) from Atractylodes macrocephala was first isolated.

• AMP1-1 has a linear backbone consist of α-d-Glcp-1→(2-β-d-Fruf-1)₇.

• AMP1-1 has anti-weightless bone loss activity.

• AMP1-1 could promote osteogenic activity and inhibit osteoclastic activity.

Abstract

Atractylodes macrocephalon polysaccharides of alleviating weightlessness-induced bone loss (WIBL) are rarely reported. In this study, a neutral polysaccharide fragment named AMP1-1 was isolated and purified. Monosaccharide composition and gel permeation chromatography analysis indicated that AMP1-1 was composed of glucose and fructose with molecular weight of 1.433 kDa. Based on data of gas chromatography–mass spectrometer (GCMS), a linear backbone consisted of α-d-Glcp-(1→ and →1)-β-d-Fruf-2→ was discovered. Combining results from nuclear magnetic resonance (NMR), the inulin-type fructan AMP1-1 was identified as α-d-Glcp-1→(2-β-d-Fruf-1)₇. Anti-WIBL activity of AMP1-1 was evaluated though analyzation of mechanical properties, BALP and TRAP 5b activities on femur. In vitro mRNA expression indicated that anti-WIBL activity of AMP1-1 was achieved by promote bone formation and inhibit bone resorption in primary osteoblasts and RAW264.7 cell lines under simulated weightlessness. In conclusion, the inulin-type fructan AMP1-1 with α-d-Glcp-1→(2-β-d-Fruf-1)₇ had anti-WIBL activity via remodeling bone homeostasis.

Introduction

Weightlessness-induced bone loss (WIBL) is a kind of bone mass loss caused by long-term spaceflight (Amin, 2010; Chen et al., 2019). Unlike bone loss on ground, the WIBL does not only induce metabolic disorders for astronauts (Grimm et al., 2016), but also cause damage of bone such as poor recovery after returning to the ground (Sibonga et al., 2007; Sibonga, 2013). Therefore, WIBL has severely affected space missions and the health of astronauts (Sibonga et al., 2007; Sibonga, 2013). At present, prevention of WIBL mainly focuses on resistance training and drug intervention (Diao, Chen, Wei, & Wang, 2018). However, resistance training for alleviating WIBL is less effective. Although chemicals could effectively alleviate WIBL, they have serious side effects on human body (Diao et al., 2018). Consequently, finding safe bone protectants with high biological activity, non-toxic and insignificant side effects from natural products could be a possible way (Wang et al., 2018).

Polysaccharides, as natural products with high biological activities and a variety of characteristics (Azam et al., 2020; Hwang et al., 2019), have gradually drawn the attention of scholars in the field of WIBL prevention (Ou et al., 2021; Qi et al., 2012). Ou found that polysaccharides of Rehmannia glutinosa could significantly increase the bone mineralization deposition rate (Ou et al., 2021). Qi revealed that polysaccharides of Cordyceps sinensis could increase body weights, mechanical strength and bone mineral density (Qi et al., 2012). However, the mechanism and structure-activity relationship of polysaccharides were not involved.

There have been few studies of the structure-activity relationship about WIBL protecting polysaccharides, however, many studies focused on polysaccharides structure-activity relationship of osteoporosis under 1 g condition (Azam et al., 2020; Hwang et al., 2019; Huang et al., 2020). On the ground, structures of polysaccharides could affect bone homeostasis both on osteoblasts and osteoclasts. First, polysaccharides could induce differentiation of osteoblasts and promote osteogenesis (Hwang et al., 2020; Li, Xing, Gong, Wang & Sun, 2019). β-d-Fruf, α-d-Glcp, β-d-Galp and β-d-Manp residues in polysaccharides structure all showing promoting effect on proliferation, differentiation and activity of osteoblasts (Yan, Zhang, Wang, & Zhang, 2019; Zhang, Wang, Hou, & Yan, 2019; Yan, Huang, et al., 2019; Zhang, Zhang, Jiang, Li, & Yan, 2020). Among them, (2 → 1)-linked-β-d-Fruf as backbone showed alkaline phosphatase (ALP) activity elevation in osteoblasts (Zhang et al., 2019; Yan, Huang, et al., 2019; Zhang, Zhang, Jiang, Li, & Yan, 2020). In addition, glucose and fructose residues in polysaccharides terminal linking could promote osteoblasts differentiation (Zhang et al., 2019; Yan, Huang, et al., 2019). Second, polysaccharides mainly function in inhibiting differentiation and activity on osteoclasts thought down-regulating NFATc1, MMP9, Ctsk, and TRAP (Chen et al., 2005; Du et al., 2016; Song et al., 2018). However, the connection between structures of polysaccharides and their anti-WIBL effect has not been established.

In our preliminary work, the potential of polysaccharides from Atractylodes macrocephala with anti-WIBL activity was evaluated. These data revealed that total crude polysaccharides fraction of Atractylodes macrocephala (AMP) could promote osteoblast proliferation. Moreover, the ALP activity, which was decreased under simulated weightlessness, was significantly increased by AMP treatments. The data mentioned above indicated that AMP had anti-WIBL activity. However, the accurate active fragment(s) of anti-WIBL in Atractylodes macrocephalon polysaccharides (AMP) was(were) still unknown. Therefore, AMP1-1 with anti-WIBL activity was isolated and purified from AMP in this paper. Gel permeation chromatography (GPC), monosaccharide composition detection, methylation analysis, nuclear magnetic resonance (NMR) and other methods were used to identify AMP1-1, managing to search for the relationship between structure and activity of anti-WIBL on AMP1-1.