Abstract

To achieve high-value utilization of polysaccharides from Cistanche deserticola residues, this study used the residue remaining after the extraction of phenylethanoid glycosides (an industrial process based on the patented membrane separation technology, Patent No. ZL200710102756.7, held by Hotan Dichen Pharmaceutical Biotechnology Co., Ltd., https://www.xjcistanche.com/about-us) as the raw material. We systematically investigated the differences in chemical structural characteristics and antioxidant activities among various polysaccharide fractions obtained through membrane separation. The results indicated significant variations in chemical composition and activity among polysaccharide fractions with different molecular weights. Utilising ultrafiltration membranes of varying molecular weight cutoffs (300 kDa, 50 kDa, 10 kDa) combined with alcohol precipitation, four distinct polysaccharide fractions were successfully prepared: >300 kDa (named CTP 1), 50–300 kDa (CTP 2), 10–50 kDa (CTP 3), and <10 kDa (CTP 4). Monosaccharide composition analysis indicated that all four fractions primarily contained mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose, but their proportional ratios differed. Fourier-transform infrared spectroscopy confirmed that all fractions exhibited typical characteristic polysaccharide absorption peaks. Scanning electron microscopy results demonstrated that ultrafiltration membranes with different pore sizes significantly altered the aggregation state and surface roughness of the polysaccharides by retaining components of varying molecular weights. This morphological difference may be related to intermolecular interactions and the sieving effect during ultrafiltration. In antioxidant activity assays, CTP 1 showed the optimal performance, achieving DPPH radical scavenging rates exceeding 61% and ABTS⁺ radical scavenging rates surpassing 95% at 8 mg/mL. All four fractions demonstrated appreciable antioxidant activity. This study confirms that membrane separation technology can effectively fractionate polysaccharides from Cistanche deserticola residue. The research provides a theoretical foundation and data supporting the resource recycling of herbal medicine residues and the high-value development of polysaccharide components.

Keywords: Cistanche deserticola; polysaccharides; antioxidant activity; membrane separation technology; structural analysis

 

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Introduction

Cistanches Herba (Cistanche), the dried fleshy stem with scale leaves of Cistanche deserticola Y. C. Ma (desert cistanche) or Cistanche tubulosa (Schenk) Wight (CTP), is first recorded in Shennong Bencao Jing (Shennong's Classic of Materia Medica). As a typical representative of traditional tonic Chinese medicines in China and a food-medicine homologous substance, it is mainly produced in northwest desert and desert areas such as Xinjiang and Inner Mongolia, earning it the reputation of "Desert Ginseng". It has the effects of tonifying kidney yang, nourishing kidney yin, benefiting essence and blood, and moistening the intestines to relieve constipation [2-3]. The phenylethanoid glycosides contained in it have significant biological activities and are the main substances extracted in current industrial production. However, a large amount of utilizable substances remain in the residue after extraction, which is discarded as waste, not only causing resource waste but also potentially imposing an environmental burden. Therefore, achieving high-value recycling of the residue is of great significance for the efficient development of Chinese medicinal resources and environmentally friendly development.

Polysaccharides, as major active components in Cistanche, exhibit various pharmacological activities such as antioxidant, anti-aging, hepatoprotective, anti-inflammatory, immunomodulatory, and anti-tumor effects [4-7], and can be widely used in drug development, as well as in food and health products. Previous studies have extensively optimized the extraction process and conducted preliminary activity evaluations of Cistanche polysaccharides, but systematic research on polysaccharides in the residue after phenylethanoid glycoside extraction remains scarce, particularly lacking in-depth analysis of efficient separation and fractionation technologies for residue polysaccharides and activity differences among different fractions, which greatly restricts the improvement of the comprehensive utilization value of Cistanche resources.

In the field of polysaccharide research, ungraded crude polysaccharides have problems such as mixed components, unclear activity contributions, and ambiguous targets, making it difficult to accurately reveal the material basis for their antioxidant, immunomodulatory, and other functions. Meanwhile, impurities such as proteins, pigments, and inorganic salts in crude polysaccharides also interfere with their activity evaluation and application development. Traditional methods such as column chromatography have high resolution but are difficult to scale up, while alcohol precipitation fractionation is rough and has environmental risks. Membrane separation technology, with its advantages of high efficiency, environmental friendliness, and precise fractionation, shows great potential in the separation and purification of polysaccharides. It enables precise fractionation of polysaccharide components through membranes with different molecular weight cutoffs, providing an ideal technical support for revealing the "molecular weight-activity" structure-activity relationship of polysaccharides [8]. Tang et al. [9] successfully purified three polysaccharide components directly from Lentinus edodes water extracts using a combination of 2.5 kDa, 5 kDa, and 10 kDa ultrafiltration membranes. Notably, the membrane separation technology employed in this study is protected by an invention patent (Patent No. ZL200710102756.7) held by Hotan Dichen Pharmaceutical Biotechnology Co., Ltd. (https://www.xjcistanche.com/about-us), which focuses on the production of phenylethanoid glycoside-containing raw materials from Cistanche using membrane separation, providing a robust industrial foundation for our research. The products developed by our research group based on systematic studies of phenylethanoid glycosides from Cistanche tubulosa [10] have entered the pilot production stage. Based on this, this study uses the residue of Cistanche after phenylethanoid glycoside extraction as raw material, employs ultrafiltration membranes combined with alcohol precipitation to fractionate and purify polysaccharides in the residue, and systematically analyzes the differences in chemical structural characteristics and antioxidant activities of polysaccharide fractions with different molecular weights. The aim is to open up new pathways for the resource recycling of Cistanche residue, improve the industrial chain, and lay a theoretical and experimental foundation for the development of new functional foods or antioxidants based on natural polysaccharides.

 

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1 Materials and Methods

1.1 Materials and Instruments

Cistanche deserticola residue was provided by Hotan Dichen Pharmaceutical Biotechnology Co., Ltd. (Hotan, China), generated from the industrial extraction of phenylethanoid glycosides via the patented membrane separation technology (Patent No. ZL200710102756.7; https://www.xjcistanche.com/about-us). Glucose standard was purchased from Tianjin Guangfu Fine Chemical Research Institute. m-Hydroxybiphenyl (98%), Coomassie Brilliant Blue G-250, dialysis bags (MD44), DPPH radical scavenging capacity assay kit, ABTS radical scavenging capacity assay kit, superoxide anion scavenging capacity assay kit, and total antioxidant capacity assay kit were all purchased from Beijing Solarbio Science & Technology Co., Ltd. Galacturonic acid (98%), bovine serum albumin (98%), and ascorbic acid (Vc) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Other chemical reagents were of analytical grade.

UV-19001 UV-Vis spectrophotometer (Shimadzu Instruments Co., Ltd.); YP10002 electronic balance (Shanghai Hengji Scientific Instruments Co., Ltd.); SQP electronic balance (Sartorius Scientific Instruments Co., Ltd.); EYELA water bath (SB-1200); CA-1111 cooling water circulation device (Shanghai Ailang Instruments Co., Ltd.); SHZ-D circulating water vacuum pump (Shanghai Jinfu Experimental Instrument Equipment Co., Ltd.); N-1100 rotary evaporator; XCLD-10FE freeze dryer (Shanghai Xingchuang Scientific Instrument Equipment Co., Ltd.); BONA-GM-18C organic membrane separation experimental machine; 300 kDa, 50 kDa, 10 kDa ultrafiltration membranes (polyethersulfone, Jinan Bona Co., Ltd., Shandong); TGL-16A centrifuge (Changsha Pingfan Instruments); BT25S analytical balance (Sartorius Scientific Instruments); Dionex U3000 high-performance liquid chromatograph (Thermo Fisher Scientific, USA); LC-DCY-12GK nitrogen blower (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.); UPH-I-10T ultrapure water system (Chengdu Ultra Pure Technology Co., Ltd.); DF-101S infrared spectrometer (Shimadzu Instruments Co., Ltd.); Zeiss Supra55VP scanning electron microscope (Carl Zeiss (Shanghai) Management Co., Ltd.).

 

Cistanche Specs

1.2 Experimental Methods

1.2.1 Extraction of Crude Cistanche Polysaccharides

According to the extraction process obtained from preliminary pre-tests, 150 g of Cistanche deserticola residue was weighed, extracted at 100 °C for 3 h with a solid-liquid ratio of 1:20 g/mL, centrifuged at 4000 r/min for 10 min, and the supernatant (polysaccharide content: 4.4 mg/mL) was collected to obtain the polysaccharide extract.

1.2.2 Fractionation and Separation of Cistanche Polysaccharides

The aqueous extract of Cistanche polysaccharides was subjected to ultrafiltration fractionation using 300 kDa, 50 kDa, and 10 kDa ultrafiltration membranes under conditions of 0.18 MPa pressure and 40 °C temperature. The retentates and permeates were collected separately, concentrated by rotary evaporation, and 95% ethanol was added to achieve a final ethanol concentration of 80%. The mixture was placed at 4 °C for 24 h, then centrifuged at 4000 rpm for 10 min, and the supernatant was discarded to obtain the precipitate. The precipitate was redissolved in water, dialyzed against a 3500 Da dialysis bag for 48 h, and freeze-dried to obtain Cistanche polysaccharides. Four fractions were obtained by collecting retentates and permeates: >300 kDa (named CTP 1), 50–300 kDa (CTP 2), 10–50 kDa (CTP 3), and <10 kDa (CTP 4).

1.2.3 Determination of Polysaccharide Chemical Components

1.2.3.1 Determination of Total Sugar Content

The phenol-sulfuric acid method was used to determine the polysaccharide content in the samples [11]. A standard curve was prepared using glucose, with a 0.1 mg/mL glucose standard solution. Precise volumes of 0.4 mL, 0.8 mL, 1.2 mL, 1.6 mL, and 2.0 mL of the standard solution were transferred to 10 mL stoppered test tubes, and water was added to a total volume of 2.0 mL. Then, 1.0 mL of freshly prepared 6% phenol solution was added, mixed well, and 5.0 mL of concentrated sulfuric acid was rapidly added along the tube wall. The mixture was shaken, heated in a 90 °C water bath for 15 min, then removed and cooled to room temperature. Using distilled water as a blank, the absorbance was measured at 490 nm with a UV-Vis spectrophotometer. The standard curve obtained was y=16.196x+0.0834, with R2=0.999. A 0.1 mg/mL polysaccharide solution was prepared, and the absorbance of each polysaccharide fraction was measured using the same method. The total sugar content of each fraction was calculated by substituting into the standard curve.

1.2.3.2 Determination of Protein Content

The Coomassie Brilliant Blue method was used to determine the protein content in the samples [12]. A standard curve was prepared using bovine serum albumin, with a 0.1 mg/mL standard protein solution. The standard protein solution was diluted to concentrations of 0.02, 0.03, 0.05, 0.07, and 0.09 mg/mL. Then, 1 mL of each concentration of the standard protein solution was added to test tubes, followed by 5.0 mL of Coomassie Brilliant Blue chromogenic reagent, and mixed by shaking. Using distilled water as a blank, the absorbance was measured at 595 nm after 10 min of reaction. The standard curve obtained was y=0.0034x+0.7673, with R2=0.996. The absorbance of each polysaccharide fraction was measured using the same method, and the protein content of each fraction was calculated by substituting into the standard curve.

1.2.3.3 Determination of Uronic Acid Content

The m-hydroxybiphenyl method was used to determine the uronic acid content in the samples [13]. A standard curve was prepared using galacturonic acid, with a 0.1 mg/mL galacturonic acid standard solution. The standard solution was diluted to concentrations of 0.01, 0.02, 0.03, 0.05, and 0.1 mg/mL. Then, 1 mL of each concentration of the standard solution was added to test tubes, and 6 mL of sodium tetraborate-sulfuric acid solution was added under ice-water bath conditions, mixed by shaking, heated in boiling water for 5 min, cooled to room temperature, and 0.1 mL of m-hydroxybiphenyl reagent was added, mixed thoroughly, and allowed to stand at room temperature for 30 min. Using distilled water as a blank, the absorbance was measured at 525 nm. The standard curve obtained was y=7.5339x+0.0494, with R2=0.995. A 0.1 mg/mL polysaccharide solution was prepared, and the absorbance of each polysaccharide fraction was measured using the same method. The uronic acid content of each fraction was calculated by substituting into the standard curve.

 

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1.2.4 Determination of Monosaccharide Composition

Chromatographic conditions: Thermo U3000 liquid chromatography system, equipped with an Agilent ZORBAX Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase was acetonitrile: phosphate buffer (12 g/L potassium dihydrogen phosphate, pH adjusted to 6.8 with 2 mol/L NaOH) at a volume ratio of 17:83, with isocratic elution. The flow rate was 0.8 mL/min, column temperature was 30 °C, detection wavelength was 250 nm, and injection volume was 10 μL.

Preparation of standard solution: 5 mg each of rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid, glucosamine hydrochloride, and galactosamine hydrochloride, and 10 mg of fucose were weighed sequentially, dissolved, and made up to 10 mL in a volumetric flask to prepare the standard stock solution.

Preparation of sample solution: 5 mg of each of the four polysaccharide fractions was weighed, added to 1 mL of 2 mol/L TFA solution, and heated at 121 °C for 2 h. Nitrogen was purged to dry the solution. Then, 3 mL of methanol was added for washing, and the solution was dried again; this methanol washing step was repeated 2–3 times. The residue was dissolved in 1 mL of sterile water and transferred to a chromatographic vial for testing. Then, 0.2 mL of the polysaccharide hydrolysate was added to a stoppered conical centrifuge tube, followed by 0.2 mL of 0.5 mol/L sodium hydroxide solution and 0.5 mL of 0.5 mol/L PMP methanol solution. The mixture was vortexed and reacted in a 70 °C water bath for 1 h. After the reaction, 0.2 mL of 0.5 mol/L hydrochloric acid was added to neutralize the sodium hydroxide, 1 mL of chloroform was added, and the mixture was vortexed for extraction three times to remove excess PMP. The chloroform layer was discarded, 0.3 mL of the aqueous layer was taken, and water was added to make up to 1 mL. Then, 0.2 mL of each of the 11 monosaccharide standards was derivatized using the same method [14-15].

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

An appropriate amount of each of the four polysaccharide fractions was placed on the sample stage of the ATR accessory of the infrared spectrometer, and FT-IR measurements were performed using a Fourier-transform infrared spectrophotometer in the frequency range of 4000–400 cm⁻¹ [16].

1.2.6 Scanning Electron Microscopy (SEM) Analysis

The morphology of the four polysaccharide fractions was analyzed using SEM [17]. A small amount of each polysaccharide fraction was uniformly fixed on a thin copper plate, and a layer of gold powder was evaporated on the sample surface using an ion sputter. The surface morphology was observed at 5.00 kV.

1.2.7 Determination of In Vitro Antioxidant Activity

1.2.7.1 Determination of DPPH· Radical Scavenging Capacity

The concentrations of the four polysaccharide fractions were set to 1, 2, 3, 4, and 8 mg/mL, and the positive control group (Vc) was also set to the same concentrations. The determination was performed according to the instructions of the DPPH· radical scavenging capacity assay kit.

1.2.7.2 Determination of Superoxide Anion Scavenging Capacity

The concentrations of the four polysaccharide fractions were set to 0.5, 1, 2, 4, and 8 mg/mL, and the positive control group (Vc) was also set to the same concentrations. The determination was performed according to the instructions of the superoxide anion scavenging capacity assay kit.

1.2.7.3 Determination of Total Antioxidant Capacity

The concentrations of the four polysaccharide fractions were set to 0.5, 1, 2, 4, and 8 mg/mL, and the positive control group (Vc) was also set to the same concentrations. The determination was performed according to the instructions of the total antioxidant capacity assay kit.

1.2.7.4 Determination of ABTS⁺ Radical Scavenging Capacity

The concentrations of the four polysaccharide fractions were set to 0.5, 1, 2, 4, and 8 mg/mL, and the positive control group (Vc) was also set to the same concentrations. The determination was performed according to the instructions of the ABTS radical scavenging capacity assay kit.

1.3 Data Processing

Each experiment was repeated three times under the same conditions, and the results were expressed as mean ± standard deviation. SPSS 21.0 software was used for significance analysis, with one-way ANOVA for single-factor analysis; P < 0.05 was considered statistically significant. Origin 2024 and GraphPad Prism 10.1.2 were used for graphing.

 

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References

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[10] Ainur Memetiturxun, GAO L, HUO S-X, et al. Optimization of Centrifugal Extraction Process for Verbascoside Using Box-Behnken Response Surface Methodology [J]. Chinese Journal of Modern Applied Pharmacy, 2022, 39(23): 3086-3091.

[11] JIN W, ZHOU H, ZHAO H, et al. Isolation, In Vitro Antioxidant Capacity, Hypoglycemic Activity and Immunoactivity Evaluation of Polysaccharides from Coriandrum sativum L [J/OL]. Antioxidants, 2025, 14(2): 149. https://doi.org/10.3390/antiox14020149

[12] SPLITTGERBER A G, SOHL J. Nonlinearity in protein assays by the Coomassie blue dye-binding method [J]. Analytical Biochemistry, 1989, 179(1): 198-201.

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[14] GUO Y, WANG L, LIU K, et al. A Rapid and Accurate UHPLC Method for Determination of Monosaccharides in Polysaccharides of Different Sources of Radix Astragali and Its Immune Activity Analysis [J/OL]. Molecules, 2024, 29(10): 2287. https://doi.org/10.3390/molecules29102287

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