2 Results and Analysis

2.1 Physicochemical Properties of Polysaccharides

2.1.1 Analysis of Chemical Components

The physicochemical properties and yields of the four fractions obtained via membrane separation - a technique based on the patented membrane separation technology (Patent No. ZL200710102756.7) owned by Hotan Dichen Pharmaceutical Biotechnology Co., Ltd. (https://www.xjcistanche.com/about-us) - are presented in Table 1. Significant differences were observed in the contents of total sugar, protein, and uronic acid among the four fractions. CTP 1 exhibited the highest sugar content, and its uronic acid content was significantly higher than that of the other fractions. As the fractions changed from CTP 1 to CTP 4, the yield, total sugar content, and uronic acid content showed a clear decreasing trend.

 

 

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Table 1. Chemical composition and yield of Cistanche deserticola polysaccharides after membrane separation

Fraction	Total Sugar Content (%)	Protein Content (%)	Uronic Acid Content (%)	Yield (%)
CTP 1	91.95 ± 1.03ᵃ	1.06 ± 0.04ᶜ	26.77 ± 0.77ᵃ	3.31
CTP 2	83.67 ± 1.61ᵇ	1.42 ± 0.03ᵃ	15.90 ± 0.29ᶜ	0.70
CTP 3	78.32 ± 1.48ᶜ	1.25 ± 0.02ᵇ	19.12 ± 0.55ᵇ	0.10
CTP 4	37.51 ± 1.40ᵈ	1.41 ± 0.02ᵃ	19.20 ± 0.54ᵇ	0.10

Note: Data are presented as mean ± standard deviation (n = 3). Different superscript letters within the same column indicate significant differences among samples (P < 0.05).

 

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2.1.2 Analysis of Monosaccharide Composition

As shown in Figure 2 and Table 2, the monosaccharide compositions of the four fractions (CTP 1, CTP 2, CTP 3, CTP 4) separated by ultrafiltration membranes with different molecular weight cutoffs, as determined by PMP derivatization, were consistent. All fractions were heteropolysaccharides composed of 8 monosaccharides: mannose (Man), glucosamine (GlcN), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), and arabinose (Ara). Among these, glucose (Glc), galactose (Gal), arabinose (Ara), galacturonic acid (GalA), and rhamnose (Rha) accounted for higher molar percentages. The molar percentages of different monosaccharides varied across the four fractions: CTP 1 had the highest contents of glucose and galacturonic acid; CTP 2 had high contents of galactose and glucuronic acid; CTP 3 had a high content of arabinose; and CTP 4 had high contents of mannose and rhamnose. The acidic sugar content of CTP 1 was significantly higher than that of the other fractions, which was consistent with the uronic acid content determined in the physicochemical property analysis. This indicates that the membrane separation technology (protected by Patent No. ZL200710102756.7) can effectively fractionate polysaccharide components with different acidic sugar contents.

 

 

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2.1.3 Fourier-Transform Infrared Spectroscopy (FT-IR) Analysis

As shown in Figure 3, all four polysaccharide fractions exhibited typical characteristic absorption peaks of polysaccharides. A strong and broad peak at 3324.6 cm⁻¹ was attributed to the O-H stretching vibration of polysaccharides [18]; a peak at 2924.6 cm⁻¹ was the C-H stretching vibration of carbohydrates, a typical characteristic peak of polysaccharides [19, 20]. Absorption near 1738.8 cm⁻¹ indicated the presence of uronic acid structures, suggesting that the polysaccharides were acidic [16, 21]. The absorption peak at 1607.5 cm⁻¹ was assigned to adsorbed water, which may be due to the hygroscopic nature of polysaccharides. The absorption peak near 1234.3 cm⁻¹ was caused by C-H bending vibrations [22, 23], and the absorption peak at 1014.2 cm⁻¹ confirmed the characteristic absorption of pyranose rings. The absorption peaks at 887.3 cm⁻¹ and 755.2 cm⁻¹ indicated the presence of α-configured and β-configured glycosidic bonds [24-27]. The membrane separation process did not damage the basic glycosidic bond types or functional group structures of the polysaccharides, ensuring the structural integrity of the polysaccharide fractions.

Figure 1 Chromatogram of monosaccharide composition of reference substances

Note: 1. Mannose; 2. Glucosamine; 3. Rhamnose; 4. Glucuronic acid; 5. Galacturonic acid; 6. Galactosamine; 7. Glucose; 8. Galactose; 9. Xylose; 10. Arabinose; 11. Fucose.

 

Figure 2. Chromatograms of monosaccharide composition of polysaccharide fractions.

Note: 1. Mannose; 3. Rhamnose; 5. Galacturonic acid; 7. Glucose; 8. Galactose; 10. Arabinose.

 

 

 

 

2.1.4 Scanning Electron Microscopy (SEM) Analysis

As shown in Figure 4, CTP 1 exhibited a slightly undulating surface at 2000× magnification (A1). At 10,000× magnification (A2), irregular pores with uneven sizes were observed, presenting a "porous and loose" overall characteristic. CTP 2 had a relatively intact and flat structure at 2000× magnification (B1), with weak agglomeration despite some edge folds. At 10,000× magnification (B2), the surface was smooth and flat with almost no obvious pores. CTP 3 showed subtle "ripples" on the surface at 2000× magnification (C1), which may be due to partial aggregation of polysaccharide molecules without complete cross-linking into a compact structure. At 10,000× magnification (C2), the surface exhibited moderate roughness [28]. CTP 3 showed subtle "ripples" on the surface at 2000× magnification (C1), which may be due to partial aggregation of polysaccharide molecules without complete cross-linking into a compact structure. At 10,000× magnification (C2), the surface exhibited moderate roughness [28]. CTP 4 had sparsely distributed nanoscale particles on the surface of the agglomerated area at 2000× magnification (D1), with no obvious connections between particles and moderate dispersibility. At 10,000× magnification (D2), the particle surface was smooth with no obvious protrusions, showing slight folds and moderate structural uniformity [29]. In summary, the microscopic morphologies of the polysaccharides treated with different ultrafiltration membranes were significantly different, specifically in aggregation state, surface roughness, and dispersibility. This morphological difference may be related to intermolecular forces of polysaccharides and the sieving effect during ultrafiltration.

 

Table 2. Molar ratios of monosaccharide composition of polysaccharide fractions

Monosaccharide	CTP 1 (%)	CTP 2 (%)	CTP 3 (%)	CTP 4 (%)
Mannose (Man)	1.373	1.112	1.772	3.484
Glucosamine (GlcN)	0.201	0.265	0.347	0.454
Rhamnose (Rha)	8.695	9.860	11.060	12.590
Glucose (GlcA)	1.239	1.313	1.166	1.228
Galacturonic Acid (GalA)	13.755	12.653	13.777	7.461
Glucose (Glc)	35.101	19.329	33.898	33.998
Galactose (Gal)	28.353	33.372	28.623	30.997
Arabinose (Ara)	11.284	12.286	12.756	9.806

Figure 3: Fourier Transform Infrared Spectra Of Polysaccharide From Each Fraction

 

Figure 4 Scanning electron microscopy images of polysaccharide fractions.

Figure 5 DPPH radical scavenging capacity of polysaccharide fractions.

 

 

 

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2.2 Determination of Antioxidant Activity of Polysaccharides

2.2.1 Determination of DPPH· Radical Scavenging Capacity

As shown in Figure 5, the DPPH radical scavenging capacity of each fraction exhibited a clear dose-dependent effect [30, 31]. Compared with the radical scavenging capacity of VC, the DPPH scavenging capacities of the four fractions were relatively weak. At a concentration of 8 mg/mL, the IC₅₀ values of VC and the four fractions were 0.961 mg/mL, 4.946 mg/mL, 7.534 mg/mL, 7.534 mg/mL, and 8.472 mg/mL, respectively, with CTP 1 showing the highest radical scavenging capacity.

 

Figure 6 Superoxide anion scavenging capacity of polysaccharide fractions.

Figure 7 Total antioxidant capacity of polysaccharide fractions.

 

Figure 8 ABTS radical scavenging capacity of polysaccharide fraction

 

 

2.2.2 Determination of Superoxide Anion Scavenging Capacity

As shown in Figure 6, the superoxide anion scavenging capacities of the four fractions showed no significant change with increasing concentration [32]. Compared with the superoxide anion scavenging capacity of VC, the capacities of the four fractions were relatively weak. At a concentration of 8 mg/mL, the IC₅₀ values of VC and the four fractions were 0.884 mg/mL, 11.46 mg/mL, 10.54 mg/mL, 14.03 mg/mL, and 11.83 mg/mL, respectively, indicating that the superoxide anion scavenging capacities of the four fractions were essentially consistent. This activity difference may be related to multiple factors such as the molecular weight, monosaccharide composition and ratio, and acidic sugar content of the polysaccharides.

2.2.3 Determination of Total Antioxidant Capacity

As shown in Figure 7, the total antioxidant capacity of each fraction gradually increased with increasing concentration [33], showing a dose-dependent effect. Compared with the total antioxidant capacity of VC, the capacities of the four fractions were relatively weak. At a concentration of 8 mg/mL, the IC₅₀ values of VC and the four fractions were 22.88 mg/mL, 40.00 mg/mL, 61.30 mg/mL, 79.41 mg/mL, and 71.81 mg/mL, respectively, with CTP 1 exhibiting the strongest total antioxidant capacity.

2.2.4 Determination of ABTS⁺ Radical Scavenging Capacity

As shown in Figure 8, the ABTS⁺ radical scavenging capacity of each fraction gradually increased with increasing concentration, showing a dose-dependent effect. At a concentration of 4 mg/mL, the IC₅₀ values of VC and the four fractions were 0.450 mg/mL, 0.486 mg/mL, 0.405 mg/mL, 1.039 mg/mL, and 0.531 mg/mL, respectively, indicating that the ABTS⁺ radical scavenging rates of the four fractions were essentially consistent with that of VC.

 

3 Conclusion

In this study, the residue of Cistanche after phenylethanoid glycoside extraction was used as raw material, and the ultrafiltration membrane combined with alcohol precipitation technology - protected by an invention patent (Patent No. ZL200710102756.7) held by Hotan Dichen Pharmaceutical Biotechnology Co., Ltd. (https://www.xjcistanche.com/about-us) - was employed to achieve efficient extraction and fractionation of polysaccharides. Four polysaccharide fractions with different molecular weight ranges were successfully prepared: CTP 1 (>300 kDa), CTP 2 (50–300 kDa), CTP 3 (10–50 kDa), and CTP 4 (<10 kDa). The structural characteristics and antioxidant activity differences of each fraction were systematically analyzed.

Polysaccharide Fractionation Effect and Distribution Characteristics: As the pore size of the ultrafiltration membrane decreased, the yield of polysaccharide fractions showed a decreasing trend, reflecting the broad molecular weight distribution of polysaccharides in Cistanche residue. The membrane separation technology (patented by Hotan Dichen Pharmaceutical Biotechnology Co., Ltd.) can precisely fractionate polysaccharide components with different molecular weights and acidic sugar contents without damaging the basic glycosidic bond types and functional group structures of the polysaccharides, ensuring the structural integrity of the fractions. This provides an efficient and environmentally friendly technical solution for the precise fractionation of polysaccharides.

Structure-Activity Relationship Characteristics: All four polysaccharide fractions were heteropolysaccharides containing 8 monosaccharides, but significant differences were observed in the molar proportions of the monosaccharides. The acidic sugar (uronic acid) content of CTP 1 was significantly higher than that of the other fractions. In vitro antioxidant experiments confirmed that CTP 1 exhibited the best DPPH and ABTS⁺ radical scavenging activities, indicating that the antioxidant activity of polysaccharides is closely related to molecular weight, acidic sugar content, and monosaccharide composition ratio, which is consistent with the core conclusion of existing studies that "polysaccharide structural characteristics regulate biological activity". The study by Aladan Memetali et al. [34] also showed that the antioxidant activity increased significantly with increasing polysaccharide concentration, and this dose-dependent characteristic was confirmed by gradient concentration experiments in this study. Unlike previous studies that usually prepared crude polysaccharides by alcohol precipitation, this study introduced membrane separation technology to systematically fractionate Cistanche polysaccharides, enabling a shift from overall activity analysis of mixed components to precise investigation of the antioxidant activities of polysaccharide fractions in different molecular weight ranges.

This study only evaluated the antioxidant activity of polysaccharides using in vitro chemical models, and the results may differ from the antioxidant efficacy in the in vivo physiological environment. Future research should further conduct in vitro cell experiments and in vivo animal studies to verify the antioxidant activity and potential pathways. Meanwhile, technologies such as chromatography-mass spectrometry and X-ray diffraction can be used to analyze the fine structure of polysaccharides and identify key active sites, providing more precise theoretical support for the development of functional foods, antioxidants, and other products based on residue polysaccharides. Through further refinement of fine structure analysis and in vivo activity verification, it is expected to further explore the application potential of residue polysaccharides and promote the upgrading of characteristic medicinal resource development in Xinjiang towards green, efficient, and industrialized directions.

 

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