# Structural Characterization and Antioxidant Activity of Polysaccharides From Indigo Naturalis Ophiopogon japonicus: Effects of Processing and Extraction Methods

## TL;DR

This study examines how processing and extraction methods affect the structure and antioxidant activity of polysaccharides in a traditional herbal medicine.

## Contribution

The study reveals how traditional processing alters polysaccharide structure and enhances specific antioxidant properties.

## Key findings

- IN processing increased polysaccharide and uronic acid content and introduced high-molecular-weight fractions.
- Reflux-extracted processed samples showed strongest DPPH radical scavenging activity (IC50 = 3.19 mg/mL).
- Structural changes enhanced DPPH and superoxide anion scavenging but reduced hydroxyl radical scavenging.

## Abstract

Indigo naturalis‐processed
Ophiopogon japonicus
(IN‐OJ) is a common processing method in the Menghe medical school to enhance its heat‐clearing and detoxifying effects. However, its impact on the structure and bioactivity of the main active component—polysaccharides—remains unclear. In this study, polysaccharides were extracted from both raw and IN‐processed
O. japonicus
using hot reflux and ultrasound‐assisted methods. Their structural features were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), gel permeation chromatography (GPC), high‐performance liquid chromatography (HPLC), and Fourier‐transform infrared spectroscopy (FT‐IR). Monosaccharide composition, molecular weight, and microscopic morphology were systematically analyzed. Antioxidant activities were evaluated in vitro through DPPH, superoxide anion, and hydroxyl radical scavenging assays, using Vitamin C (Vc) as a positive control. The results indicated that IN processing significantly increased the polysaccharide and uronic acid contents. Molecular weight profiles revealed an additional high‐molecular‐weight fraction in the processed samples (U‐IP: 520,049 kDa; W‐IP: 356,167 kDa). Monosaccharide analysis showed notable increases in arabinose and fucose. Microscopic observations indicated a transition from a loose, honeycomb‐like structure to a denser, aggregated morphology, while FT‐IR spectroscopy confirmed the introduction of additional carboxyl groups. Furthermore, antioxidant assays demonstrated that IN processing significantly enhanced scavenging activities against DPPH and superoxide anion radicals, with W‐IP exhibiting the strongest DPPH radical scavenging capacity (IC50 = 3.19 mg/mL). However, hydroxyl radical scavenging activity decreased post‐processing, likely due to steric hindrance within the aggregated structures. The positive control Vc exhibited markedly superior scavenging activity across all assays. These findings suggest that IN processing enhances specific antioxidant capacities of OJ polysaccharides through structural modification, with reflux extraction yielding the most active fractions, thus providing a scientific basis for optimizing this traditional method in functional food applications.

Indigo naturalis processing significantly increased polysaccharide and uronic acid content, altered monosaccharide composition, and introduced high‐molecular‐weight fractions. Structural changes enhanced DPPH and superoxide anion scavenging activities, especially in reflux‐extracted processed samples (W‐IP, IC50 = 3.19 mg/mL). The study validates the traditional “Ban‐Zhi” method and provides a basis for developing functional foods.