Method Article

Comparative Study on the Polysaccharide Contents and Antioxidant Activities of Hippophae rhamnoides subsp. sinensis and Hippophae gyantsensis

DOI:

10.3791/68855

⸱

August 15th, 2025

 ,  ,  ,  ,  ,  ,  , 
1State Key Laboratory of Southwestern Chinese Medicine Resources, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, 2School of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine

In This Article

Summary

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This study compared the polysaccharide contents of Hippophae rhamnoides subsp. sinensis Rousi and Hippophae gyantsensis (Rousi) Y. S. Lian obtained through hot water extraction and quantified through phenol-sulfuric acid colorimetry. We also investigated in vitro antioxidant activities of polysaccharides from these two Hippophae species through 2,2-diphenyl-1-picrylhydrazyl free radical scavenging experiments.

Abstract

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This paper reports a well-designed and in-depth comparative study on the polysaccharide yields, contents, and antioxidant activities of two Hippophae species of great research value, namely, Hippophae rhamnoides subsp. sinensis Rousi and Hippophae gyantsensis (Rousi) Y. S. Lian. The total polysaccharides of H.rhamnoides subsp. sinensis and H. gyantsensis were obtained through hot water extraction. Yields were compared, and polysaccharide contents were accurately determined through phenol-sulfuric acid colorimetry and compared. Meanwhile, the antioxidant activity of the polysaccharides was evaluated through 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging. Results showed that the polysaccharide yield (percentage of crude extract relative to the raw material) of H.rhamnoides subsp. sinensis (1.18% ± 0.02%) was significantly lower than that of H. gyantsensis (3.12% ± 0.06%) (P < 0.01). The polysaccharide content (defined as pure polysaccharides in the extract) of H.rhamnoides subsp. sinensis (352.97±1.07 mg/g) was significantly higher than that of H. gyantsensis (300.21 ± 1.49 mg/g) (P < 0.01). The half-inhibitory concentration of the DPPH free radical scavenging activity of H.rhamnoides subsp. sinensis polysaccharides (0.026 ± 0.004 mg/mL) was slightly lower than that of H. gyantsensis polysaccharides (0.021 ± 0.004 mg/mL). However, no significant difference was found between the two Hippophae species (P > 0.05). This experiment clarified the differences in polysaccharide yield, content, and antioxidant activity between H.rhamnoides subsp. sinensis and H. gyantsensis. This work can provide a scientific basis for the quality evaluation of sea buckthorn and the further development of polysaccharide active components.

Introduction

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Sea buckthorn is the dried ripe fruit of plants in the Elaeagnaceae family. It is a commonly used medicinal and edible material in China, as well as an important source of dietary supplements and health care products in India, Russia, Finland, and other European countries because of its properties of invigorating the spleen to aid digestion, relieving cough and expelling phlegm, promoting blood circulation, and dissipating blood stasis1. Sea buckthorn contains a rich array of bioactive components, such as flavonoids, phenolic compounds, polysaccharides, glycosides, and organic acids, and has a long-standing history of application in traditional Chinese medicine2,3.Generally, flavonoids are considered the most important bioactive compounds of sea buckthorn. However, polysaccharides have been recently recognized to also possess excellent physiological and pharmacological activities and have therefore attracted the attention of scientific researchers. Sea buckthorn polysaccharides have been demonstrated to have activities in regulating the immune system, protecting the liver, and regulating lipid metabolism disorders and intestinal microbiota, as well as antioxidant, anti-inflammatory, antitumor, and hypoglycemic effects4,5,6. They can act as natural antioxidants, thus enabling the development of novel functional foods and pharmaceuticals. Meanwhile, sea buckthorn polysaccharides are also liable to decomposition by gastric acid, which is beneficial to the absorption and utilization of nutrients in the human body7,8.

Hot water extraction (HWE), microwave-assisted extraction, and ultrasonic-assisted extraction are the commonly used methods for the extraction of sea buckthorn polysaccharides9. The structures of polysaccharides obtained through different extraction methods vary10in terms of monosaccharide composition, molecular weight (Mw), branching degree, uronic acid content, glycosidic linkage types, and trihelix conformation11,12. Generally, high-Mw polysaccharide solutions possess high viscosity and low solubility, which may affect their biological activities and applications13. HWE is the most commonly employed technique for polysaccharide extraction in laboratory and industrial settings owing to its advantage of simplicity14,15. Moreover, it is one of the most commonly used extraction methods for sea buckthorn polysaccharides9. Therefore, in this study, polysaccharides were extracted through HWE. The primary methods for determining polysaccharide content include the phenol-sulfuric acid and anthrone-sulfuric acid methods16,17,18,19. The phenol-sulfuric acid method is extensively applied because of its simple detection procedure, good stability, and high sensitivity20. Wen et al. determined the content of polysaccharides extracted from sea buckthorn through the phenol-sulfuric acid method21. Therefore, in this experiment, the content of polysaccharides was determined by using the phenol-sulfuric acid method. The depth of the orange compound solution formed after the phenol-sulfuric acid reaction is proportional to the content of polysaccharides and can be determined at a wavelength of 490 nm22.

Natural polysaccharides exhibit remarkable antioxidant activities in vivo and in vitro. Raspberry polysaccharides may alleviate oxidative stress in high-fat diet models by regulating the gut microbiota23. Polysaccharides are the main active components in Pueraria lobata and exhibit diverse biological activities, including antioxidation24. Dou et al. found that blackberry polysaccharides have physiological functions, such as antioxidation25. The antioxidant activity of polysaccharides is usually assessed on the basis of their ability to scavenge various free radicals, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, and superoxide anion radicals, as well as their ABTS radical cation scavenging capacity26. DPPH analysis is widely used because it measures the ability of antioxidants to reduce violet DPPH radicals into pale-yellow, stable molecules through hydrogen atom transfer27. The remaining violet DPPH radicals are quantified by using a ultraviolet (UV)-visible (Vis) spectrophotometer at approximately 515-520 nm to determine antioxidant activity28,29.

Previous studies have investigated the content and antioxidant activity of sea buckthorn polysaccharides21. However, the vast majority of these studies were limited to Hippophae rhamnoides subsp. sinensis. It is the most frequently utilized and widely distributed Hippophae species in China and is officially recorded in the Chinese Pharmacopoeia. Studies on Hipphophae gyantsensis, a species endemic to Xizang, China, that is frequently employed in Tibetan medicine and documented in the Sichuan Provincial Catalogue of Tibetan Medicinal Materials, have rarely been reported. In addition, no research has compared the contents and antioxidant activities of polysaccharides from different Hippophae species, namely, H. rhamnoides subsp. sinensis and H. gyantsensis. Therefore, this study first compares the polysaccharide contents of H. rhamnoides subsp. sinensis and H. gyantsensis extracted through HWE and quantified through phenol-sulfuric acid colorimetry. The antioxidant activities of polysaccharides from these two Hippophae species were further investigated through DPPH free radical scavenging assays. The present study is expected to provide scientific data for the quality evaluation and further development of the active polysaccharide components of the above two Hippophae species.

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Protocol

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1. Samples and materials

  1. Collect sea buckthorn samples of different Hippophae from the Qinghai-Xizang Plateau.
    NOTE: The samples were identified by Associate Professor Yue Liu from Chengdu University of Traditional Chinese Medicine. Voucher specimens were deposited in the School of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine. Sampling information is shown in Table 1. The details of the reagents and equipment used in this study are listed in the Table of Materials.
  2. Dry the samples in an oven at 40 °C for 72 h.
  3. Grind 20 g of each sea buckthorn sample into powder and pass it through a No. 3 sieve. Process each sample with three parallel replicates. Continue to crush the samples that did not pass through the sieve, then sieve them until all have been sieved.
  4. Store the samples at −20 °C for follow-up studies.

2. Extraction of sea buckthorn polysaccharides

NOTE: This study adopted the previously established extraction method for sea buckthorn with some modifications to obtain polysaccharides30.

  1. Weigh 5.0 g of sea buckthorn sample powder and place it in a 250 mL round-bottomed flask. Add 50 mL of anhydrous ethanol to the powder and soak it at room temperature (RT; 25 °C) for 24 h to remove lipophilic components. Process each sample with three parallel replicates.
  2. Filter the sample powder, which has been soaked overnight, by using a Buchner funnel, then discard the filtrate to obtain the filter residue. Wash the filter residue three times with 50 mL of deionized water and dry it to a constant weight at 40 °C.
  3. Transfer the defatted filter residue into a dried 250 mL round-bottomed flask, add 150 mL of deionized water at a solid: liquid ratio of 1:30 (g/mL), and perform reflux extraction in a water bath at 80 °C twice for 2 h each time. Separate the filtrate to obtain the crude sea buckthorn polysaccharide solution.
  4. Concentrate the extract using a rotary evaporator at 50 °Cfor 1.5 h. Store the concentrated solution in a refrigerator at 4 °C for later use.
  5. Add Sevage reagent (CHCl3:C4H9OH = 4:1, v:v) to the crude polysaccharide solution at a Sevage reagent: crude polysaccharide solution volume ratio of 1:4. Stir the mixture with a magnetic stirrer at a speed of 1050 rpm for 20 min.
  6. Transfer the liquid into a 50 mL centrifuge tube and centrifuge it at 4000 × g for 15 min, then collect the supernatant. Repeat the above operation three times until no white flocculent precipitate appears at the interface between the two phases.
  7. Concentrate the sea buckthorn polysaccharideextract after protein removal. Add anhydrous ethanol at a volume ratio 1:3 for anhydrous ethanol precipitation and place the mixture at 4 °C overnight.
  8. Remove the upper layer of anhydrous ethanol, transfer the mixture into a 50 mL centrifuge tube, and centrifuge it at 4000 × g for 15 min to obtain the precipitate. Freeze-dry the precipitate to obtain total polysaccharides.
  9. Calculate the total polysaccharide yields by using Equation (1) below:
    Polysaccharide yield calculation formula: m/M × 100%; equation for scientific analysis.
    NOTE: In Equation (1), m represents the weight (g) of sea buckthorn polysaccharides, and M represents the mass (g) of the sea buckthorn sample.

3. Polysaccharide content determination

  1. Solution preparation
    1. Preparation of phenol reagent solution
      1. Accurately weigh 3 g of phenol and transfer it into a brown volumetric flask. Add 50 mL of deionized water to dissolve phenol.
      2. Shake the mixture thoroughly until it is homogeneous. Label the flask and store it for later use.
    2. Preparation of glucose standard solution
      1. Accurately weigh 10 mg of anhydrous glucose and transfer it into a 10 mL volumetric flask. Add deionized water to dissolve glucose and shake the mixture well.
      2. Dilute the mixture to the desired volume with deionized water to obtain a glucose standard solution.
    3. Preparation of test sample solution.
      1. Accurately weigh 5 mg of freeze-dried sea buckthorn polysaccharides (ZG1-ZG5 and JZ1-JZ5) into volumetric flasks (n = 10). Add deionized water to each flask to a final volume of 50 mL.
      2. Sonicate the mixture for 20 min until polysaccharides have completely dissolved. Process each sample with three parallel replicates.
  2. Construction of the standard curve
    NOTE: In the phenol-sulfuric acid method, polysaccharides react with concentrated sulfuric acid to generate furfural derivatives, which then condense with phenol to form an orange-yellow compound. The maximum absorption wavelength of this compound is approximately 490 nm. Therefore, the detection wavelength for this experiment was set at 490 nm22.
    1. Dilute the standard glucose solution with deionized water to a concentration of 0.1 mg/mL. By using a micropipette, carefully transfer 200, 300, 400, 500, 600, and 700 µL of the glucose standard solution into six separate clean test tubes. Add pure water to a total volume of 1.0 mL, then gently mix each solution to ensure uniform dilution.
    2. Add 1.0 mL of 6% phenol solution and 5.0 mL of sulfuric acid to the test tubes mentioned above. Mix well, cool, and then let stand at RT for 30 min.
      NOTE: Phenol is a toxic chemical compound, and concentrated sulfuric acid is corrosive. Gloves should be worn when preparing phenol and sulfuric acid solutions, and the procedure should be conducted inside a fume hood.
    3. Transfer the aforementioned samples into cuvettes and use pure water as a blank instead of the sample.Measure their absorbance at 490 nm with a UV-Vis spectrophotometer and three replicates.
    4. Plot the standard curve with glucose concentrations as the x-axis and absorbance values as the y-axis. Obtain the regression equation and calculate the correlation coefficient (R²).
      NOTE: The UV-Vis spectrophotometer must be preheated for half an hour in advance. When measuring the absorbance in this step, the absorbance must be between 0.2 and 0.8. Therefore, the concentration of the sample needs to be quantified.
  3. Methodology study
    1. Precision test:
      1. Take JZ2 and prepare the test solution in accordance with the method described in step 3.1.3. Perform phenol-sulfuric acid colorimetry by following the method described in step 3.2.2.
      2. Subsequently, determine the absorbance in accordance with the procedure outlined in step 3.2.3. Repeat this procedure six times and calculate the relative standard deviation (RSD).
    2. Stability test:
      1. Prepare the JZ2 sample solution by following the method described in step 3.1.3. At 0, 2, 4, 6, 8, and 10 h after sample preparation, perform phenol-sulfuric acid colorimetry in accordance with the method specified in step 3.2.2.
      2. Next, determine the absorbance by following the procedure outlined in step 3.2.3 and calculate the RSD.
    3. Repeatability test:
      1. Prepare the JZ2 sample solution by following the method described in step 3.1.3. Prepare six replicates in parallel. Conduct phenol-sulfuric acid colorimetry in accordance with the method specified in step 3.2.2.
      2. Afterward, determine the absorbance by following the procedure outlined in step 3.2.3 and calculate the RSD.
    4. Recovery test:
      1. Take JZ2 and prepare the test solution in accordance with the method described in step 3.1.3. Prepare nine replicates in parallel. Add the reference solutions equivalent to 80%, 100%, and 120% of the total polysaccharide content in the sample and prepare three parallel samples.
      2. Perform phenol-sulfuric acid colorimetry in accordance with the method specified in step 3.2.2. Subsequently, determine the absorbance by following the procedure outlined in step 3.2.3 and calculate the recovery rate by using Equation (2).
        Recovery rate equation: (C-A)/B × 100%; mathematical formula, efficiency calculation.
        NOTE: In Equation (2), A represents the amount of glucose in the sea buckthorn sample solution, B represents the amount of glucose added, and C represents the measured value of the solution containing glucose and the sea buckthorn sample solution.
  4. Content determination
    1. Prepare the sample solution as described in step 3.1.3 and perform phenol-sulfuric acid colorimetry in accordance with the method specified in step 3.2.2.
    2. Determine the absorbance by following the procedure outlined in step 3.2.3 and calculate the polysaccharide content using the standard curve method. Perform these operations in parallel three times for each sample.

4. DPPH radical scavenging activity31,32

  1. Accurately weigh 2.7 mg of DPPH into an empty 50 mL volumetric flask. Dissolve it in 50 mL of anhydrous ethanol to prepare a DPPH stock solution with a concentration of 0.054 mg/mL and obtain the DPPH working solution.
    NOTE: The DPPH solution should be stored in the dark.
  2. Accurately weigh 5 mg of vitamin C (Vc) into an empty 10 mL volumetric flask and dissolve it in 10 mL of deionized water to prepare a Vc solution with a concentration of 0.5 mg/mL. Dilute it successively to prepare Vc test solutions with concentrations of 0.5, 0.125, 0.03125, 0.0078125, and 0.001953 mg/mL.
  3. Accurately weigh 5 mg of each sea buckthorn polysaccharide (ZG1-ZG5 and JZ1-JZ5). Dissolve each sample in 10 mL of deionized water to prepare sea buckthorn polysaccharide sample solutions with a concentration of 0.5 mg/mL. Dilute the above solutions successively to prepare sea buckthorn polysaccharide test solutions with concentrations of 0.5, 0.125, 0.03125, 0.0078125, and 0.001953 mg/mL.
  4. Using the DPPH solution as the substrate, prepare the following groups in a 96-well plate: negative control group A0 (150 µL of water and 50 µL of DPPH solution), sample group A1 (150 µL of sample or Vc solution and 50 µL of DPPH solution), and sample control group A2 (150 µL of sample and 50 µL of anhydrous ethanol).Perform this procedure in triplicate.
  5. Allow the reaction to proceed for 30 min in the dark. Next, measure the absorbance at 517 nm. Repeat the experiment three times and calculate the DPPH free radical scavenging rate using Equation (3).
    NOTE: The absorbance of the DPPH solution should be maintained at 0.7 ± 0.02. Excessively high and low concentrations can lead to inaccurate absorbance readings, thus affecting the final experimental results.
    Scavenging rate formula, equation showing \( \text{Scavenging rate} = 1 - \frac{A_1 - A_2}{A_0} \).
    ​In Equation (3), A0 is the absorbance of deionized water and DPPH solution, A1 is the absorbance of the sample or Vc and DPPH solution, and A2 is the absorbance of the sample and anhydrous ethanol.
  6. Calculation of the half-inhibitory concentration (Ic50)
    1. Generate a visually organized data table illustrating the correlation between reaction test compound concentrations (0.5, 0.125, 0.03125, 0.0078125, and 0.001953 mg/mL) and their corresponding percentage reaction values.
    2. Use nonlinear curve fitting (with GraphPad Prism) to determine the IC50 and standard error.

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Results

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Yield of crude polysaccharides
Polysaccharides were extracted through HWE. The water bath reflux is shown in Supplementary Figure 1. The concentration of the polysaccharide extract solution is illustrated in Supplementary Figure 2. The centrifugation stratification effect after deproteination is presented in Supplementary Figure 3. The five batches of H. rhamnoides subsp. sinensis have polysaccharide yields of 0.89% ± 0.01%, 0.78% ±...

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Discussion

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Sea buckthorn is a traditional Tibetan medicine. Polysaccharides are important components of sea buckthorn. Many studies have been conducted on the determination of polysaccharide content and antioxidant activity21. However, only a few have compared the polysaccharide contents and antioxidant activities of different Hippophae species, such as the medicinal species H. rhamnoides subsp. sinensis and H. gyantsensis.Therefore, this work determined the polysaccharide...

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Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have no conflicts of interest to disclose.

Acknowledgements

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This work was financially supported by the National Natural Science Foundation of China (No. 82374145), Natural Science Foundation of Sichuan Province (No. 2025ZNSFSC0608), and National Natural Science Foundation of China (No. U23A20520)

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Analytical balance (with a precision of 0.0001 g)Sartorius Scientific Instruments (Beijing) Co., Ltd.BSA224S-CW
Analytical balance (with a precision of 0.00001 g)Sartorius Scientific Instruments (Beijing) Co., Ltd.CPA225D
Benchtop low-speed centrifugeShanghai Zigui Instrument Co., Ltd.TD4K-Z
DPPHShanghai Yuanye Bio-Technology Co., Ltd.CAS 1898-66-4
EthanolChengdu Kelong Chemical Products Co., Ltd.CAS 64-17-5
EP tubeHunan BKMAK Holding Co., Ltd.BS-20-M2 mL
Freeze dryerLabconco CorporationLABCONCO
Glucose anhydrousChengdu Kelong Chemical Products Co., Ltd.CAS 50-99-7Reference Standard
GraphPad  Prim 9.5.0 GraphPad SoftwareN/ADetermine the IC50 value and the P value.
Hot Air Circulation Drying OvenShanghai Yiheng Technology Instrument Co., Ltd.NB-DGG-9070A
L-Ascorbic AcidBeijing Labgic Technology Co., Ltd.CAS 50-81-7positive control drug
Microplate ReaderShanghai Shanpu Biotechnology Co., Ltd.Read Max 1500
96-well plateBeijing Labgic Technology Co., Ltd.11512
OriginPro 2025OriginLabN/ACreate a clearance rate line chart
PIPETTE TIPSBeijing Labgic Technology Co., Ltd.BS-200-T200 μL
PIPETTE TIPSBeijing Labgic Technology Co., Ltd.T-1000-B1000 μL
PhenolChengdu Kelong Chemical Products Co., Ltd.CAS 108-95-2
Rotary Evaporator BUCHI Laboratory Equipment Trading (Shanghai) Ltd.Buchi R-100
Standard Reagent Type Pure Water SystemQingdao Flom Technology Co., Ltd.FBZ2001-up-p
Thermostatic digital display water bathGuan'an Scientific Instruments (Zhejiang) Co., Ltd.WB100-6F
UV-Visible SpectrophotometerShanghai Mapada Instruments Co., Ltd. V-1100D
Ultrasonic cleanerSheng Zheng ChaoJie Industrial Co.,Ltd.CJ-060SDUltrasonic cleaner 360 W 50 Hz

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Tags

Polysaccharide ContentAntioxidant ActivitySea BuckthornHot Water ExtractionPhenol Sulfuric AcidDPPH AssayFree Radical ScavengingPolysaccharide Yield

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