Structural characterisation and structure-antioxidant activity relationship of polysaccharides from Dendrobium catenatum Lindl

Abstract Dendrobium catenatum Lindl. has been long used in China as a functional food and traditional Chinese medicine and polysaccharides from Dendrobium catenatum Lindl. (DOP) exhibited extensive bioactivities. However, studies on the structure-activity relationship of DOP are rarely reported. Here, two polysaccharides named DOP-1 and DOP-2 were obtained, which differed in the ratio of monosaccharide composition and molecular weight. Structural characteristics were elucidated by spectral and chemical analysis. The main structures of DOPs were the linkage of β-(1→4)-D-Manp, with some attached 2-O- or 3-O-acetylated groups. Additionally, the DPPH, hydroxyl and superoxide radicals scavenging assays of DOP-1 and DOP-2 showed that DOP-2 exhibited the higher antioxidant activity, which might be related to its lower molecular weight, higher mannose proportion and lower degree of acetylation, and higher phenolic content. Our results provide a more accurate basis for the application of DOPs in the pharmaceutical and food industries. Graphical Abstract


Introduction
Dendrobium catenatum Lindl. (D. catenatum), also known as Denrobium officinale Kimura & Migo, has been long used in traditional Chinese medicine for the unparalleled effects (Huang et al. 2015). Polysaccharides as an important active ingredient from D. catenatum focused more and more attention. It has been reported that D. catenatum polysaccharide (DOP) exhibited a large range of bioactivities including hypoglycemic , anti-inflammatory (Liang et al. 2018), and antioxidant activities (Luo et al. 2016).
Using modern methods to explore the structure and efficacy of the active ingredients of traditional Chinese medicine is an important field of study. It has been widely confirmed that many plant polysaccharides possess effective antioxidant activity (Jiao et al. 2016). The antioxidant activities of polysaccharides were related to their structural properties, including monosaccharide composition, molecular weight (Mw), and the type of glycosidic linkage (Chen et al. 2009). Polysaccharides with lower Mw always have a higher antioxidant activity than those of higher Mw with the same composition. In addition, the type and position of other functional groups like sulfate, amine and carboxyl could also affect their antioxidant activity (Chen et al. 2009). However, previous reports about DOP have only focused on the structural characterisation, in vitro antioxidant capacity, or other activities (Kuang et al. 2020;Liu et al. 2020). There are few reports on the relationship between their structural properties and bioactivities.
In this study, we provided two kinds of simple and high efficient isolation methods to obtain two purified DOPs named as DOP-1 and DOP-2. The structural characteristics of the DOPs were investigated by the combination of chemical (monosaccharide composition, periodate oxidation, Smith-degradation and methylation) and spectral (FT-IR and NMR) analysis, with Mw analysis by gel permeation chromatography. In addition, in vitro radical scavenging effects of DOPs and their structure-antioxidant activity relationships were investigated.

Isolation and structural analysis
The yield of crude polysaccharide of D. catenatum was 17.2%. Freeze-thawing and ultrafiltration were performed to obtain DOP-1, and the process of oxidation decolorisation and dialysis were to obtain DOP-2, with yields of 10.3% and 7.0%, respectively. The Mws of DOPs were calculated as 395 kDa and 33 kDa, respectively ( Figure S1A). The total sugar contents of DOP-1 and DOP-2 were determined as 99.47% and 98.46%, respectively. No UV/vis absorption peaks of DOP-1 or DOP-2 were observed at 260-280 nm ( Figure S2A), indicating that there was no protein in DOPs. Total phenolic contents of DOP-1 and DOP-2 were detected as 147.25 and 955.71 lg/g, respectively (Table S1). As compared with HPLC profile of standard monosaccharides ( Figure S1B The bands around 1720 cm À1 , 1373 cm À1 and 1245 cm À1 , respectively, corresponded to C ¼ O stretching, ÀCH vibration and C À O vibration in O-acetyl groups ( Figure S2) (Xing et al. 2015). The bands around 870 cm À1 and 810 cm À1 were attributed to the mannose pyranose ring .
Because of the higher solubility, the 1 D, HSQC, and HMBC NMR spectrum of DOP-2 were in the higher response to identify its structure. The major 1 H and 13 C NMR chemical shifts signals of individual glycosidic linkages of DOP-2 were assigned (Table S4) according to published 2 D NMR data (Ghosh et al. 2008;Hsieh et al. 2008;Luo et al. 2009;Zhang et al. 2019). In the 1 H NMR spectrum of DOP-2, a complex group of signals were exhibited in the anomeric regions at d 4.70 À 5.40. Four anomeric proton signals were confirmed by combining the information from HSQC spectra, which were labeled as A, B, D, and E, respectively. The HSQC spectrum displayed five anomeric signals at  Table S4 based on HSQC spectra and literature data.
In the HMBC spectrum ( Figure  A cross-peak of the H-1 signals of residue E with C-4 of residue C was identified. A very weak cross peak between the C-6 of residue E with H-1 of residue A was also observed. Combining all the information of structural analysis, the compositions of DOP-2 included non-O-, 2-O-, and 3-O-acetylated b-1,4-D-Manp, a-1,4-D-Glcp, and a-1,6-D-Glcp in a ratio of 8.77:3.60:1.84:1.82:1.00. Taken together, the main repeated unit was proposed in Figure 1. In previous reports (Xing et al. 2015;Wei et al. 2016), O-acetyl-glucomannan was also found in D. catenatum. However, it seems that it is the unique O-acetyl-glucomannan from D. catenatum which contain the a-D-1,4-Glcp and a-D-1,6-Glcp linkages.

Antioxidant activity
DPPH, hydroxyl and superoxide radical scavenging assays were used to evaluate the antioxidant activity of DOP-1 and DOP-2. Figure S4A showed that the DPPH radical scavenging efficiencies of the DOPs increased with increasing concentrations. As shown in Figure S4B, the hydroxyl radical scavenging ability of DOP-1 almost kept invariant with the concentrations. However, that of DOP-2 was 51.14% at 8 mg/mL, indicating a better radical scavenger than DOP-1. As shown in Figure S4C, the superoxide anion radical scavenging efficiencies of DOP-2 were slightly higher than that of DOP-1, when the concentrations were higher than 3 mg/mL. The scavenging effect was dose-dependent for DOP-2, but there was no dose dependency for DOP-1.
Previous reports found that complex carbohydrates exhibit varying antioxidant activity correlated with their different structural characterisation. For example, polysaccharide extracted from red algae was found to show antioxidant behavior, which possibly due to its high content of phenolic compounds and high amount of mannose (El-Sayed et al. 2015). Other polysaccharides with higher amount of mannose have also been reported to be better radical scavengers (Xiang et al. 2012). The difference of structural properties between DOP-1 and DOP-2 could influence their antioxidant activity to some extent.
DOP-2 exhibited stronger hydroxyl and superoxide anion radical scavenging ability than DOP-1 at high concentration, which was partly attribute to its higher molar proportion of mannose and lower Mw. Relative higher antioxidant activity of DOP-2 (31.59% acetylated) in aqueous solution might correlate with the degree of acetylation for the more hydroxyl groups in DOP-2 than that in 50% acetylated DOP-1 (Chen et al. 2014). In addition, DOP-2 contained higher phenolic, which acted as electron donors to scavenge the free radicals (El-Sayed et al. 2015). DPPH radical scavenging ability of DOP-1 was slightly enhanced at concentrations above 2 mg/mL. This result was probably due to the changed solubility in methanol solution for DPPH radical scavenging assays (Xiang et al. 2012). Because polysaccharides have complicated conformation, further studies should be carried out to elucidate the relations between antioxidant activity and structure.

Conclusions
In this study, various strategies were used to isolate and purify D. catenatum polysaccharides. The structures of DOPs were characterised by spectroscopy analysis and chemical analysis. DOP-2 showed lower Mw, higher mannose proportion and lower degree of acetylation than DOP-1. DOP-2 displayed higher antioxidant activity in vitro than DOP-1. Altogether, DOPs might be considered as a natural antioxidant to be applied as medicines and functional foods. And their antioxidant activities are closely related to Mw, mannose proportion, degree of acetylation and phenolic content.