Structural characterization of a low-molecular-weight heteropolysaccharide (glucomannan) isolated from Artemisia sphaerocephala Krasch

doi:10.1016/j.carres.2011.10.020

Carbohydrate Research

Volume 350, 1 March 2012, Pages 31-39

https://doi.org/10.1016/j.carres.2011.10.020 Get rights and content

Abstract

Using 60% (w/v) ammonium sulfate precipitation, a heteropolysaccharide (designated 60S), with relatively low molecular weight (38.7 kDa), was isolated from the seeds of Artemisia sphaerocephala Krasch. The structural properties of 60S were elucidated by partial acid hydrolysis, methylation analysis, 1D and 2D NMR spectroscopy, and MALDI-TOF-MS. The results of the partial acid hydrolysis and methylation analysis indicated that the main chain of 60S consisted of (1→4)-linked d-Manp and (1→4)-linked d-Glcp in a molar ratio of 1:1.3. Over half of the glucosyl residues in the main chain were branched at the O-6 position. The terminal sugar residues were mainly composed of T-Araf, T-Arap, T-Galp, T-GlcpA, and T-Glcp. Besides, 3-Araf and 2-Galp were also observed in comparable amounts. Based on all the aforementioned results and the data obtained by 1D and 2D NMR spectroscopy as well as MALDI-TOF-MS, a structure of 60S is proposed as follows:

R could be one or some of -(3-α-Araf)_n-(A), T-α-Galp (B), T-α-Glcp (C), T-Araf (H) or T-Arap.

Graphical abstract

Introduction

A crude water-extractable polysaccharide from Artemisia sphaerocephala Krasch (ASK) seed has been used in the food industry in China as a thickener, a water-holding agent, and a stabilizer since the 1980s. Recent reports showed that the crude polysaccharide from ASK was able to alleviate hyperglycemia, hyperlipemia, and insulin resistance in streptozotocin-induced type 2 diabetic rats,¹ and the polysaccharide showed significant antioxidant effects on diabetic rats.² It was also reported that the ASK polysaccharide could decrease the blood glucose levels in alloxan-induced type 1 diabetic rats.³ However, limited information on the structural properties of the ASK polysaccharide is available.

In our previous study,⁴ two polysaccharide fractions were isolated from the water-extractable ASK polysaccharide using 60% (w/v) ammonium sulfate precipitation. The higher molecular weight polysaccharide (551.3 kDa) was collected from the precipitate and designated 60P, while the fraction with relatively low molecular weight (38.7 kDa) was recovered from the supernatant and named as 60S. Substantial differences were observed between 60P and 60S in terms of physicochemical properties: 60P was comprised of 55.4% (w/w) neutral sugar, 25.8% (w/w) uronic acid, and 24.1% (w/w) protein, while in 60S, the percentage of neutral sugar and uronic acid was 87.1% (w/w) and 10.4% (w/w), respectively, with no protein detected. In addition, monosaccharide composition analysis showed that 60S was composed of 38.3% (w/w) glucose, 28.1% (w/w) mannose, 24.2% (w/w) galactose, 9.4% (w/w) arabinose, and a trace amount of rhamnose. The proposed structure of 60P was reported previously: it has a (1→4)-linked xylopyranose backbone branched at every other xylopyranose at the O-2 position with mostly T-α-GlcpA-4→OMe, and occasionally 4-α-d-GalpA, T-Araf, T-Arap or 3-β-Araf.⁵

The objective of the current study was to elucidate the fine structure of 60S, employing partial acid hydrolysis, methylation analysis, MALDI-TOF-MS, and 1D and 2D NMR spectroscopy including homonuclear ¹H–¹H correlations spectroscopy (COSY, TOCSY), heteronuclear ¹³C–¹H multiple-quantum coherence spectroscopy (HMQC), and heteronuclear multiple bond correlation spectroscopy (HMBC).

Section snippets

Isolation and fractionation

The 60S fraction was isolated from A. sphaerocephala Krasch seed according to the method described previously.⁴ Briefly, the seeds were soaked in water (1:400, w/v) at 70 °C for 24 h under constant stirring, followed by precipitation in 60% (w/v) ammonium sulfate at room temperature. After centrifugation, the polysaccharide in the supernatant was collected, freeze-dried, and ball-milled to get 60S powder.⁴

Partial acid hydrolysis

Sample of 60S (40 mg) was hydrolyzed with 0.1 M trifluoroacetic acid (TFA) (10 mL) at 100 °C for

Partial acid hydrolysis and monosaccharide composition

Due to the structural complexity of the polysaccharide, partial acid hydrolysis was employed in the present study for characterization of the 60S sample. Figure 1 is the HPSEC elution profiles of the 60S fraction and its hydrolysates (before ethanol precipitation). The molecular weights of the hydrolysates decreased with the increase in hydrolysis time. Two peaks at the retention volumes of 20.30 mL and 21.70 mL were derived from small molecular weight fragments of 60S which were generated after

Discussion

In the present study, using partial acid hydrolysis, methylation analysis, NMR spectroscopy, and MALDI-TOF-MS, the structure of the 60S fraction was deduced to be (galacto)glucomannan (GGM). GGMs occur primarily in the lignified secondary walls of gymnosperms and also in the walls of the endosperm of some seeds.¹⁶ Some of the glucomannan, such as conjac mannan, is less water soluble compared with the 60S fraction, probably because of the rigid linear back chain (less branched).²³ Some

Acknowledgments

The authors wish to thank Dr. Ying Wu, Ms. Cathy Wang, Miss Keying Qian, and Mr. John Nikiforuk of Agriculture and Agri-Food Canada for technical assistance and insightful discussion.

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Structural characterization of a low-molecular-weight heteropolysaccharide (glucomannan) isolated from Artemisia sphaerocephala Krasch

Abstract

Graphical abstract

Introduction

Section snippets

Isolation and fractionation

Partial acid hydrolysis

Partial acid hydrolysis and monosaccharide composition

Discussion

Acknowledgments

J. Ethnopharmacol.

Food Hydrocolloids

Carbohydr. Polym.

Carbohydr. Polym.

Carbohydr. Polym.

Methods Enzymol.

Carbohydr. Res.

Carbohydr. Res.

Phytochemistry

Carbohydr. Res.

Food Hydrocolloids

Carbohydr. Res.