Abstract

Cistanche deserticola Ma, commonly known as "desert ginseng", is a precious parasitic medicinal plant widely distributed in north‑western China. Its polysaccharide components represent one of the most vital bioactive substances with anti‑aging, immunomodulatory and antioxidant functions. In this study, three extraction approaches including hot‑water extraction, enzymatic extraction and ultrasonic‑assisted extraction were compared for polysaccharide yield from Cistanche deserticola. Single‑factor experiments combined with Box‑Behnken response‑surface methodology were applied to optimise ultrasonic‑assisted extraction parameters, with solid‑liquid ratio, ultrasonic extraction time and ultrasonic temperature as independent variables. In‑vitro antioxidant capacity of obtained polysaccharides was characterised by DPPH and ABTS radical‑scavenging assays, using vitamin C (Vc) as positive control. The results demonstrated that ultrasonic‑assisted extraction delivered the highest polysaccharide yield among three tested techniques. The optimal conditions were solid‑liquid ratio 1:100 (5.0 g raw powder in 500 mL distilled water), ultrasonic extraction time 40 min and extraction temperature 48 ℃, yielding a polysaccharide extraction rate of 7.49 %. Under these optimised conditions, DPPH radical‑scavenging activity of Cistanche deserticola polysaccharides reached 84.27 % of Vc activity, and ABTS radical‑scavenging capacity accounted for 73.95 % relative to Vc. This research provides experimental reference for large‑scale production and application of Cistanche deserticola polysaccharides in functional food, nutraceutical and health‑care industries.

Keywords: Cistanche deserticola; desert ginseng; plant polysaccharide; ultrasonic‑assisted extraction; response‑surface methodology; DPPH; ABTS; antioxidant activity; natural functional ingredient

1. Introduction

Cistanche deserticola Ma is a perennial holoparasite herb thriving in desert regions of Xinjiang, Inner Mongolia and Gansu Province of China. For thousands of years in traditional Chinese medicine, it has been reputed as "desert ginseng" for tonifying kidney‑essence, relaxing bowel function and delaying senescenceWiley. In modern nutritional and pharmacological research, bioactive constituents from Cistanche deserticola, particularly polysaccharides, phenolic compounds, flavonoids and alkaloids, have attracted increasing global research interest in natural antioxidant raw‑materials for functional‑food development.

Polysaccharides are high‑molecular‑weight macromolecules polymerised from monosaccharide units connected via glycosidic bonds. Cistanche deserticola polysaccharides possess broad molecular‑weight distribution ranging from tens of thousands to millions Daltons, and consist of glucose, galactose, mannose and other monosaccharide residues. Complex branching structures contribute to their unique physicochemical features and biological performance. Apart from polysaccharides, the raw herb also contains abundant essential amino acids, unsaturated fatty acids and trace elements such as zinc, selenium and copper, supporting human physiological metabolism and immune homeostasis. Endogenous antioxidant compounds in Cistanche deserticola include catechin, gallic acid, quercetin, kaempferol, betaine and cistanche alkaloids. These substances eliminate reactive oxygen species and restrain lipid peroxidation to mitigate oxidative damage. It is noteworthy that antioxidant efficacy is heavily affected by extraction workflow, existing forms of bio‑molecules and compound interaction effects among different phytochemicals.

Extraction technology is a decisive step to recover plant‑derived active compounds. Common industrial‑lab scale extraction strategies for botanical polysaccharides contain hot‑water extraction, alcoholic extraction, ultrasonic‑assisted extraction, microwave‑assisted extraction and supercritical‑fluid extraction. Each method has distinct strengths and drawbacks. Hot‑water extraction is low‑cost and operation‑friendly for mass manufacturing, yet usually gives moderate yield and co‑extracts considerable impurities. Alcohol‑based protocols favour lipophilic metabolites but are unsuitable for water‑soluble polysaccharides and carry organic‑solvent risks. Ultrasonic‑assisted extraction utilises mechanical cavitation effects to disrupt plant cell walls, accelerating mass transfer of intracellular substances into aqueous solvent. It features shorter processing duration and improved extraction yield; nevertheless, high ultrasonic intensity or excessive temperature may degrade heat‑sensitive bio‑components and requires higher capital investment for equipment. Supercritical‑fluid extraction achieves solvent‑free extracts with high purity, but suffers complicated operation thresholds and expensive instrument investment.

Although multiple investigations have addressed Cistanche deserticola polysaccharide extraction and bioactivity, systematic comparative assessment among hot‑water, enzymatic and ultrasonic‑assisted workflows remains limited. This work was designed to compare polysaccharide yields from three representative extraction protocols, optimise ultrasonic‑assisted conditions by single‑factor screening and response‑surface modelling, and quantify in‑vitro antioxidant profiles of purified polysaccharide products. The objective was to establish practical processing parameters to maximise polysaccharide recovery while retaining antioxidant potency. Findings can support further exploitation of Cistanche deserticola as edible‑medicinal resource for global functional‑food markets and offer methodological reference for natural‑product process optimisation.

2. Materials and Methods

2.1 Raw Materials and Chemical Reagents

Dried tuber blocks of Cistanche deserticola were purchased from local Chinese‑herb supplier in Baise city. Compound cellulase‑pectinase preparation and phosphate buffer solution were obtained from Guangzhou Kangzhongyang Biotechnology Co., Ltd. Analytical grade 1,1‑diphenyl‑2‑picrylhydrazyl (DPPH), 2,2′‑azino‑bis‑(3‑ethylbenzothiazoline‑6‑sulfonic acid) diammonium salt (ABTS) were supplied by Shanghai Dongfulong Bio‑Reagent Co., Ltd. Other chemicals including phenol, concentrated sulfuric acid, 95 % ethanol were analytical purity grade.

2.2 Instruments and Apparatus

CG‑01 analytical balance, 6800 high‑speed pulveriser, RQ‑100 slicing machine, TWS‑1000 thermostatic water‑bath; WH50 rotary evaporator, SG30‑01A ultrasonic extractor (350 W, 40 kHz); UAR‑2000 UV‑Vis spectrophotometer, THQ‑03 centrifuge, BTS‑31E freeze‑dryer.

2.3 Sample Preparation

Dried Cistanche deserticola tubers were washed with ultrapure water and oven‑dried at 40 ℃ until surface moisture removed. Material was sliced to 0.3 mm thickness, pulverised and sifted through 100‑mesh sieve to obtain homogeneous powder for subsequent extraction tests.

2.4 Three Polysaccharide Extraction Protocols

2.4.1 Hot‑Water Extraction

5.0 g Cistanche deserticola powder was transferred to 1000 mL beaker mixed with 500 mL distilled water. Water‑bath extraction was maintained for 2 h under 100 ℃. Manual stirring was performed every 100 min to suspend precipitates and facilitate polysaccharide dissolution. The mixture was cooled to ambient temperature, centrifuged at 4000 r/min for 10 min to collect supernatant fluid. Supernatant was concentrated under reduced pressure at 40 ℃ by rotary evaporator down to 20 mL volume. 60 mL of 95 % ethanol was added for alcohol precipitation and stood overnight. Precipitate was gathered after centrifugation (3000 r/min,10 min), lyophilised by freeze‑dryer to acquire crude polysaccharide powder.

2.4.2 Enzymatic Extraction

5.0 g sample powder was dispersed in 500 mL compound cellulase‑pectinase buffer solution (pH 5.0). Enzymatic hydrolysis proceeded in 45 ℃ water bath for 5 h. Reaction termination was realised by heating the solution up to 100 ℃ for 10 min to inactivate enzyme protein. After cooling, liquid was centrifuged (4000 r/min, 10 min). Supernatant was vacuum‑concentrated to 20 mL, mixed with 95 % ethanol for overnight precipitation. Centrifugation and freeze‑drying steps were identical to hot‑water procedure to harvest crude polysaccharide.

2.4.3 Ultrasonic‑Assisted Extraction

5.0 g Cistanche deserticola powder was blended with 500 mL distilled water. Homogeneous suspension was placed inside ultrasonic extractor set as power 350 W and frequency 40 kHz. After scheduled ultrasonic treatment, sample liquid cooled down naturally. Centrifugation, rotary evaporation, ethanol precipitation and lyophilisation steps followed consistent workflow mentioned above to yield crude polysaccharide extracts.
 

2.5 Determination of Polysaccharide Extraction Yield

Total carbohydrate content was quantified by phenol‑sulfuric‑acid assay referring to modified method described in published literature. Glucose standard solutions were configured at serial concentration gradients. 1 mL 10 % phenol reagent plus 3 mL concentrated sulfuric acid were mixed with standard solutions. Reaction was incubated under 85 ℃ for 15 min, absorbance value was read at 540 nm wavelength to plot calibration curve. Regression equation: \(y=9.6959x+0.0077\), coefficient of determination \(R^2=0.999\).

Crude polysaccharide samples (0.1 g) were dissolved in 100 mL distilled water. Aliquot was analysed under identical phenol‑sulfuric‑acid reaction. Polysaccharide yield (%) was calculated by formula (1):

\(Yield(\%)=\frac{C\times V\times n}{M}\times100\% \tag{1}\)

Where: C=polysaccharide concentration (g/mL); V=volume of sample solution; n=dilution multiple; M=weight of raw Cistanche deserticola powder (g).

2.6 Single‑Factor Experimental Design for Ultrasonic‑Assisted Extraction

Three major influencing variables were investigated: solid‑liquid ratio, ultrasonic extraction duration and ultrasonic temperature. Polysaccharide yield acted as evaluation indicator. Solid‑liquid ratio gradient: 1:400, 1:200, 1:100, 1:50; ultrasonic‑time gradient: 10 min, 20 min, 30 min, 40 min, 50 min; temperature gradient: 37 ℃, 43 ℃, 48 ℃, 53 ℃. Each group was performed in triplicate.

2.7 Response‑Surface Methodology (Box‑Behnken Design)

Based on single‑factor test outputs, three‑factor‑three‑level Box‑Behnken response‑surface scheme was constructed. Factor A represented solid‑liquid ratio; Factor B stood for ultrasonic extraction time; Factor C represented ultrasonic temperature. Polysaccharide extraction yield was set as response value Y. Regression‑model fitting and variance analysis were completed to screen optimal extraction parameters.

2.8 Antioxidant‑Activity Assessment In‑vitro

Crude polysaccharide sample solutions were prepared within concentration range: 0.1 mg/mL‑0.7 mg/mL. Vitamin C (Vc) solution served as positive antioxidant benchmark control group.

2.8.1 DPPH Radical‑Scavenging Activity Measurement

2 mL polysaccharide sample solution reacted with 1.5 mL DPPH anhydrous‑ethanol working solution. Mixed liquid was incubated under dark environment for 40 min. Absorbance \(A_i\) was detected at 517 nm wavelength. \(A_j\) denoted absorbance of control group with Vc; \(A_0\) represented blank‑group absorbance when distilled water substituted sample liquid. DPPH radical‑scavenging rate (%) was computed via formula (2):

\(DPPH\ radical\ scavenging\ rate(\%)=\frac{A_0-(A_i-A_j)}{A_0}\times100\% \tag{2}\)

2.8.2 ABTS Radical‑Scavenging Activity Assay

ABTS working liquid was freshly prepared. 2 mL polysaccharide sample solution mixed with 4 mL ABTS reagent, incubated in darkness for 15 min. Absorbance reading was recorded at 752 nm. \(A_i\), \(A_j\), \(A_0\) correspond to sample group, positive‑control group and blank‑control group respectively. ABTS radical‑scavenging rate (%) was calculated by formula (3):

\(ABTS\ radical\ scavenging\ rate(\%)=\frac{A_0-(A_i-A_j)}{A_0}\times100\% \tag{3}\)

2.9 Statistical Analysis

All experimental trials were repeated three times. Data processing, regression analysis and variance analysis were conducted by statistical software. Graph plotting was performed for visual comparison of experimental groups.

3. Results and Discussion

3.1 Comparison of Polysaccharide Extraction Yield for Three Extraction Technologies

Hot‑water extraction, enzymatic extraction and ultrasonic‑assisted extraction were implemented under respective standard workflows. Significant differences existed in final polysaccharide yields. Hot‑water extraction delivered the lowest yield value; enzymatic extraction produced moderate recovery; ultrasonic‑assisted extraction achieved highest yield of (7.27±0.04) %. Since post‑extraction concentration, precipitation and lyophilisation steps were identical for three batches, yield divergence directly originated from extraction mechanism distinctions.

Hot‑water extraction only relies on thermal aqueous dissolution. Certain high‑molecular‑weight polysaccharide fractions exhibit limited water solubility under atmospheric‑pressure heating condition, leading to incomplete release from plant tissue. Enzymatic extraction degrades cellulose and pectin constituents in cell walls with compound hydrolase to facilitate active‑substance liberation. However, the complex enzyme system displays limited hydrolysis efficiency for other cell‑wall matrix components, constraining total polysaccharide output. Ultrasonic cavitation force continuously shocks and fractures Cistanche deserticola cell wall structure, enlarging solvent‑solid contact area and accelerating mass‑transfer efficiency of intracellular polysaccharide molecules into aqueous phase. Consequently, ultrasonic‑assisted extraction remarkably improved polysaccharide recovery efficiency. According to this comparative outcome, ultrasonic‑assisted extraction was selected for subsequent process‑optimisation investigation.

3.2 Single‑Factor Test Results for Ultrasonic‑Assisted Extraction

3.2.1 Effect of Solid‑Liquid Ratio on Polysaccharide Yield

Solid‑liquid ratio determines concentration gradient driving‑force between plant‑cell interior and external aqueous solvent. With increasing liquid proportion, polysaccharide extraction yield first ascended and then declined. At solid‑liquid ratio 1:100 (5 g powder added to 500 mL distilled water), yield peaked at 7.46 %. When solvent volume was insufficient (solid‑liquid ratio 1:50), solvent capacity became saturated, limiting polysaccharide dissolution. Excessively high solvent ratio (1:200,1:400) would dilute solute concentration and raise workload for later vacuum‑concentration without further yield improvement. Thus, solid‑liquid ratio 1:100 was chosen as the central level for response‑surface design.

3.2.2 Influence of Ultrasonic Extraction Time on Extraction Performance

Polysaccharide yield continuously rose with extension of ultrasonic treatment time within 30 min. After exceeding 30 min, yield‑growth tendency flattened. Short ultrasonic duration cannot supply adequate mechanical cavitation energy to fully break cell‑wall barriers. Once cell‑wall disruption achieved saturation, prolonging ultrasonic processing time could hardly promote polysaccharide release. Excessively long ultrasonic exposure may potentially trigger partial degradation of polysaccharide macromolecular chains. Hence, 30 min was selected as reference central level for response‑surface design.

3.2.3 Impact of Ultrasonic Extraction Temperature

Polysaccharide extraction yield climbed as temperature increased up to 53 ℃. Once temperature surpassed 55 ℃, yield dropped sharply. Moderate heating enhances molecular diffusion rate and water solubility of polysaccharide. Nevertheless, high thermal condition will trigger thermal degradation of polysaccharide macromolecules, breaking glycosidic bonds and destroying target components, resulting in decreased measurable yield. Based on single‑factor response trend, 48‑53 ℃ temperature interval was adopted for further multi‑factor optimisation.

3.3 Response‑Surface Optimisation Analysis

Box‑Behnken response‑surface experimental design was executed taking solid‑liquid ratio (A), ultrasonic time (B), ultrasonic temperature (C) as independent variables and polysaccharide yield as response Y. Multiple quadratic regression equation was fitted as:

\(Y=7.120-0.065A+0.280B-0.210C+0.390AB+0.04AC+0.072BC-0.190A^2-0.860B^2-0.075C^2\)

Variance‑analysis outcomes illustrated that overall regression model reached extremely significant level (\(P=0.0003\)). The lack‑of‑fit term showed non‑significant difference (\(P=0.0592>0.05\)), demonstrating good fitting degree between regression equation and actual experimental data. Adjusted determination coefficient \(R^2_{Adj}=0.9232\), proving more than 92 % variation of polysaccharide yield can be interpreted by this mathematical model.

Factor significance comparison: ultrasonic extraction time (B) exerted extremely significant influence on yield (\(P=0.0035\)); ultrasonic temperature (C) produced significant impact (\(P=0.0256\)); solid‑liquid ratio (A) showed non‑significant effect (\(P=0.3938\)). Factor‑influence priority order: ultrasonic extraction time > ultrasonic extraction temperature > solid‑liquid ratio. Interaction term AB (solid‑liquid ratio × ultrasonic time) achieved extremely‑significant interaction effect.

From experimental runs of response‑surface matrix, maximum measured polysaccharide yield 7.49 % appeared under conditions: solid‑liquid ratio 1:100, ultrasonic extraction time 40 min, ultrasonic extraction temperature 48 ℃. This set of parameters was confirmed as optimal ultrasonic‑assisted extraction condition for Cistanche deserticola polysaccharides.

3.4 Antioxidant‑Activity Evaluation of Optimally‑Extracted Polysaccharides

Under optimal ultrasonic‑assisted extraction parameters, in‑vitro radical‑scavenging capacity of Cistanche deserticola polysaccharides was measured with Vc as positive benchmark reference. The DPPH radical‑scavenging capacity of polysaccharide sample reached 84.27 % relative to Vc scavenging performance; its ABTS radical‑scavenging activity accounted for 73.95 % compared with Vc standard. DPPH and ABTS are two classic complementary in‑vitro antioxidant evaluation systems, reflecting free‑radical elimination capacity towards different radical species. The test data verified that Cistanche deserticola polysaccharide possesses prominent antioxidant potential.

The antioxidant mechanism of plant polysaccharides is generally associated with hydroxyl groups distributed on sugar‑chain branches. Hydroxyl moieties donate hydrogen protons to neutralise free radicals and terminate radical‑chain reaction. Additionally, residual co‑extracted polyphenolic and flavonoid impurities in crude polysaccharide powder may generate synergistic antioxidant effect. It should be noted that extraction technological parameters could alter polysaccharide molecular‑weight distribution and co‑existing minor‑component content, further modifying final antioxidant potency. Optimised ultrasonic‑assisted workflow not only maximised polysaccharide yield but also reserved relatively high antioxidant activity for extracts.

3.5 Comprehensive Discussion

Compared with conventional hot‑water and enzymatic extraction, ultrasonic‑assisted extraction combines mechanical cavitation physical disruption with aqueous solvent dissolution. It realises higher polysaccharide yield without large consumption of enzyme preparation. According to variance‑analysis results, ultrasonic processing time was the most dominant variable. Insufficient ultrasonic duration restrains cell‑wall fragmentation; excessive time may risk bio‑molecule degradation. Temperature must be strictly controlled below thermal‑degradation threshold of polysaccharide substances. Solid‑liquid ratio mainly affects dissolution equilibrium and post‑processing energy cost; within reasonable interval its influence is relatively weaker than time and temperature.

The obtained crude polysaccharide exhibits strong in‑vitro free‑radical‑removal capability, which supports its development prospect as natural antioxidant raw‑material substituting partial synthetic antioxidants in functional‑food formulas. This study only assessed crude polysaccharide. Future investigation can focus on polysaccharide purification, monosaccharide‑composition analysis, molecular‑weight characterisation, and cell‑level or animal‑model antioxidant validation. Besides, further research can explore trade‑off relationship between extraction yield and biological activity; certain harsh high‑yield extraction parameters might impair bio‑activity even though increasing crude‑mass output.

4. Conclusion

In this research, three extraction workflows for Cistanche deserticola polysaccharides were systematically compared. Ultrasonic‑assisted extraction outperformed hot‑water extraction and enzymatic extraction regarding polysaccharide recovery rate. Single‑factor screening combined with Box‑Behnken response‑surface methodology determined optimal ultrasonic‑assisted extraction parameters: solid‑liquid ratio 1:100 (5.0 g Cistanche deserticola powder mixed with 500 mL distilled water), ultrasonic extraction time 40 min, extraction temperature 48 ℃. Under these optimised settings, polysaccharide extraction yield achieved 7.49 %. Antioxidant‑activity detection indicated that DPPH radical‑scavenging activity of polysaccharide extracts reached 84.27 % of vitamin‑C performance; ABTS radical‑scavenging capacity was equivalent to 73.95 % of Vc.

The present study supplies practical processing parameters for ultrasonic‑assisted production of Cistanche deserticola polysaccharides. Experimental outcomes validate that polysaccharides from Cistanche deserticola possess considerable antioxidant bioactivity. The findings lay experimental foundation for further utilisation of Cistanche deserticola as natural functional raw‑material in global nutraceutical, dietary‑supplement and functional‑food sectors.

References

[1] Liu Z X, Li B B, Zhao Y, et al. Preparation and stability of resveratrol Pickering emulsion stabilised by walnut protein / Cistanche deserticola polysaccharide[J]. Food Science, 2024, 45(23): 2328‑2334. [2] Ma J C, Li X P, Gong Y F. Optimisation of compound beverage processing with rose jam and Cistanche deserticola by response‑surface methodology[J]. China Condiment, 2024, 49(11):102‑109. [3] Shu W L, Ye Y, Meng W D. Preparation optimisation and component analysis of original Cistanche deserticola oral liquid[J]. Food Research and Development, 2024,45(19):132‑139. [4] Liu W H. Study on extraction and antioxidant activity of volatile oil from Cistanche deserticola by ultrasonic‑assisted salting‑out steam‑distillation[J]. Guangzhou Chemical Industry, 2024,52(15):46‑50,58. [5] Xiang S Q. Nutritional‑component analysis and antioxidant‑activity research of Cistanche deserticola[J]. Journal of Food Safety Review, 2024(12): 96‑98. [6] Chen Y, Song Z P, Wu H. Extraction‑process optimisation and antioxidant‑activity analysis of polysaccharides from Peristrophe bivalvis[J]. Science and Technology of Food Industry, 2023,44(5):166‑173.