Structural characterization, physicochemical property, and antioxidant activity of polysaccharide components from Eucommia ulmoides leaves
Chemical and Biological Technologies in Agriculture volume 10, Article number: 122 (2023)
Eucommia ulmoides (E. ulmoides) leaves are identified as a new resource of medicine and food homology. In this study, the structural characterization, physicochemical properties, and antioxidant activity of E. ulmoides polysaccharides (EUP) were studied.
Three components with different molecular weights of 1.51 × 105 Da (EUP1), 3.05 × 104 Da (EUP2) and 1.17 × 105 Da (EUP3) were purified from E. ulmoides leaves. They were composed of l-rhamnose (Rha), d-arabinose (Ara), d-mannose (Man), d-glucose (Glu) and d-galactose (Gal), while EUP2 also contained small amounts of d-xylose (Xyl). Three components all had typical polysaccharides absorption peaks, which may be polysaccharides with β configuration of pyranose structure, and amorphous structure of acid polysaccharides with good thermal stability below 270 ℃. However, the molecular weight, monosaccharide composition and apparent morphology of the three components were different, resulting in a stronger scavenging ability of EUP2 and EUP3 against DPPH and OH free radicals.
The results will provide a theoretical reference for developing EUP-related foods and drugs.
Eucommia ulmoides is a rare traditional Chinese medicine. The earliest understanding and utilization of E. ulmoides can be traced back to Sheng Nong’s herbal classic, which was published 2000 years ago . Traditionally, E. ulmoides bark is mainly used because it has various pharmacological effects such as lowering blood pressure, increasing body immunity, antibacterial, antiviral, and so on . Recently, the flowers, seeds and leaves of E. ulmoides have also been included in the homology list of medicine and food in the Chinese National Health Commission. Studies have shown that E. ulmoides leaves contain similar active ingredients as bark, which also has good medicinal and edible value. At present, more than 100 active substances have been isolated from E. ulmoides leaves, mainly including polysaccharides, flavonoids, chlorogenic acid, geniposidic acid, etc. .
Natural polysaccharides obtained from plants have been proven to have high activity, low toxicity, good biocompatibility and biodegradability . In recent years, numerous plant polysaccharides have been isolated and extracted from plants, particularly medicinal and edible homologous plant resources, such as Lycium barbarum, Mentha haplocalyx, Ziziphus jujuba [5,6,7,8]. Eucommia ulmoides leaves are a new medicinal and food homologous plant resources. Studies have found that polysaccharides isolated from E. ulmoides leaves possess an important antioxidant capacity with effective scavenging activities on radicals . Our previous study also confirmed this conclusion . Moreover, E. ulmoides polysaccharides (EUP) have been proved to be beneficial in regulating the immune behavior of macrophages and promoting the enhancement of immune capacity . More importantly, EUP also has anti-inflammatory effects and bone immunomodulatory function [12, 13].
Although EUP has significant antioxidant, anti-inflammatory, immunity enhancement and bone strengthening effects, few studies have focused on the structural characteristics and activity differences of EUP components. In this research, polysaccharide components from E. ulmoides leaves were prepared and purified by microwave-assisted extraction combined anion exchange column method, and their molecular weight, monosaccharide composition and structure were studied by size-exclusion chromatography (SEC)-multi-angle laser light scattering (MALLS) gel chromatography, gas chromatography-mass spectrometry (GC–MS), Fourier transform infrared spectrometer (FT-IR), X-ray diffraction (XRD), nuclear magnetic resonance (NMR) and field emission scanning electron microscopy (FE-SEM). The physicochemical property of EUP components were analyzed by zeta potential, particle size and differential scanning calorimetry (DSC), and the antioxidant activity of EUP components was researched by electron spin resonance (ESR). The results will provide a theoretical reference for developing EUP-related foods and drugs.
Materials and methods
E. ulmoides (Huazhong 2) leaves were collected from the E. ulmoides Research Base in Yuanyang County, Xinxiang City, Chinese Academy of Forestry in September 2021. After picking, the samples were dried at 50 ℃ in the oven until constant weight, crushed and sieved to obtain 250 µm powder, and stored for analysis.
Crude polysaccharides preparation
The polysaccharides of E. ulmoides leaves were extracted according to the previous method with minor modifications . The E. ulmoides leaves powder and distilled water were mixed at a ratio of 1:30 (g: mL), and heated in a water bath at 40 ℃ for 2 h. Then it was put in a microwave oven (M1-L213, Midea Group Co., Ltd, Guangdong province, China), and extracted at 230 W for 1.5 min. The extract was centrifuged at 4000g × 10 min, and concentrated. Anhydrous ethanol was added to reach 80% of the total concentration of the solution, and reacted at 4 ℃ for 24 h. After centrifugation, the precipitate was freeze-dried to obtain crude polysaccharides from E. ulmoides leaves. The formula for calculating the extraction rate of crude polysaccharides was shown in Eq. (1):
m: The weight of crude EUP. M: The weight of pretreated sample.
Crude polysaccharides purification
The method of crude polysaccharides purification was based on a previous work with some modifications . The crude polysaccharides solution (200 mg/mL, 20 mL) was added to macroporous resin AB-8 column (1.6 cm × 30 cm), eluted with distilled water and concentrated. The concentrated solution was put into a dialysis bag for 24 h (retained molecular weight = 3500 Da), and EUP was obtained by freeze-dried.
The EUP was dissolved in distilled water (10 mg/mL, 20 mL) and then added to a DEAE-52 column (1.6 cm × 40 cm) through 0.45 μm filter membrane. Then, 0.1 mol/mL, 0.2 mol/mL, and 0.3 mol/mL NaCl solutions were successively eluted at a flow rate of 1 mL/min. An automatic collector (DBS-100, Shanghai Huxi Analytical Instrument Co., Ltd., Shanghai, China) was used to collect, and phenol–sulfuric acid method was used to monitor at 490 nm. According to the elution curve, three components were obtained, collected, concentrated, dialysis, freeze-dried, and named EUP1, EUP2 and EUP3, respectively.
Structural characterization of polysaccharides
Molecular weight distribution
The detection method of molecular weight distribution of EUP1, EUP2 and EUP3 was assayed as described previous study with minor changes . The EUP1, EUP2 and EUP3 samples were prepared into 1 mg/mL solution and filtered through 0.45 μm membrane. A mixture of 50 mmol/L NaNO3 and 0.02% NaN3 was used as the mobile phase. The molecular weight of polysaccharides was determined by SEC–MALLS gel chromatography system (Wyatt Technology Co., California, USA) with an injection volume of 1 μL and a flow rate of 0.45 mL/min.
The monosaccharide composition of EUP1, EUP2 and EUP3 was based on the procedure of a previous study with slight modifications . 3 mL 2 mol/L trifluoroacetic acid was added to 3 mg EUP1, EUP2 and EUP3 samples, respectively, reacted at 120 ℃ for 4 h, evaporated, and washed with methanol 5 times. The treated samples were added to 10 mg hydroxylamine hydrochloride and 0.5 mL pyridine and reacted at 90 ℃ for 30 min, and cooled. Then, 0.5 mL acetic anhydride was added and reacted at 90 ℃ for 30 min, evaporated, and dried. The dried samples were dissolved in methanol and reduced to 5 mL, then filtered through 0.22 μm membrane for GC–MS (Agilent Technologies 7890B, Palo Alto, California, USA) equipped with an HP-5 capillary column (60 m × 0.25 mm × 0.25 μm). Standard monosaccharides: l-rhamnose (Rha), d-arabinose (Ara), d-mannose (Man), d-glucose (Glu), d-galactose (Gal) and d-xylose (Xyl) were derived by the same method to determine the retention time and standard curve.
The temperature of a detector was 250 ℃ during operation and mobile phase was helium. The scheme for temperature control of the column was as follows: initial temperature was 100 ℃, rising to 200 ℃ at 5 ℃/min for 1 min, and then rising to 250 ℃ at 10 ℃/min for 5 min. The carrier gas flow rate was 1.0 mL/min, and 1 μL was injected regardless of the flow rate. The conditions of mass spectrometry were interface temperature of 280 ℃, quadrupole temperature of 150 ℃, electron bombardment energy of 70 eV, solvent extension time of 5 min and full scanning range m/z of 50–800.
The infrared spectroscopy analysis of EUP1, EUP2, and EUP3 was performed using the method of the previous study with some changes . The samples (3 mg) and KBr solid powder (300 mg) were mixed and extruded. A FT-IR spectrometer (Vertex 70, Bruker Instruments, Billerica, Germany) was used to analyze the characteristic spectra of EUP1, EUP2 and EUP3, with a scanning wave number range of 4000–400 cm−1, resolution of 4 cm−1, and cumulative scanning of 64 times.
Based on the reported method , the crystal structure of the polysaccharides was determined using a XRD powder diffratometer (D8 Advance, Bruker Instruments, Billerica, Germany) with a diffraction Angle (2ϴ) of 10° to 80°, step size of 0.05° (2ϴ) and time of 1 s/step.
NMR spectra of EUP1, EUP2, and EUP3 were determined using an AVANCE III spectrometer (Bruck, Germany). Each sample (10 mg) was dissolved in 0.55 mL of D2O. The fully dissolved samples were then transferred to NMR tubes, and 1H NMR and 13C NMR spectra were analyzed by NMR at room temperature.
The surface microstructures of EUP1, EUP2, and EUP3 were observed by FE-SEM (SU8100, Hitachi, Tokyo, Japan). Polysaccharides samples were sprayed with gold, and the images of sample powders at 10 K × multiples were observed under a high vacuum at 3 kV acceleration voltage.
Physicochemical property of polysaccharides
Zeta potential and particle size
The zeta potential and mean particle size of each polysaccharides solution (1 mg/mL) were measured at 25 ℃ using a nano-particle size potentiometer (Zetasizer Nano ZS90, Malvern Instruments, Malvern, UK).
The thermal stability of the polysaccharides was analyzed by DSC (DSCQ20, TA Instruments, New Castle De, USA) . 3 mg polysaccharides samples were placed into an aluminum crucible for analysis at a temperature range of 50–400 ℃, heating rate of 10 ℃/min, and nitrogen flow rate of 1 s/step.
DPPH radical scavenging activity
According to the reported method , DPPH radical scavenging ability of polysaccharides was determined by ESR (E-scan, Bruker Instruments, Billerica, Germany). The 50 µL polysaccharides solutions were evenly mixed with 0.2 mmol/L 50 µL DPPH solution, and dark reaction was performed at 25 ℃ for 2 h. The mixtures were then moved into a quartz capillary and placed into a resonant cavity for measurement. The ESR measurement conditions were frequency of 9.792069 GHZ, power of 5.0 mW, central magnetic field of 3487 Gauss modulation amplitude of 2.27 Gauss, modulation frequency of 86.00 KHZs, sweep time of 83.88 s. After the test, the DPPH radical scavenging ability was calculated according to the quadratic integral value, and the integral region was 3450–3525 Gauss. Methanol solution was used instead of the polysaccharides solution as the blank group. DPPH free radical clearance was calculated as follows Eq. (2):
A0: Quadratic integral value of the characteristic peaks of the blank group.
A: Quadratic integral value of the characteristic peaks of the sample group.
OH radical scavenging activity
The OH radical scavenging of EUP1, EUP2 and EUP3 was based on the procedure of a previous study with slight modifications . Since OH radicals exist for a short time in aqueous solution at room temperature and cannot be detected by ESR, DMPO (5,5-dimethyl-1-pyrroline n-oxide) was added as a hydroxyl radical capture agent in the reaction system to combine with the free radicals to form stable free radical adduct. 50 µL polysaccharides solution, 50 µL DMPO, 40 mmol/L, 50 µL FeSO4 solution and 40 mmol/L, 200 µL H2O2 were mixed in turn, and the dark reaction lasted for 10 min. The experimental conditions of ESR were the same as those of the DPPH radical, and only the scanning time was changed to 20.97 s. The calculation formula of OH radical scavenging was shown in Eq. (3):
B0: Quadratic integral value of the characteristic peaks of the blank group.
B: Quadratic integral value of the characteristic peaks of the sample group.
All determinations were performed in triplicate and the corresponding data were presented as the mean of three determinations ± SE (Standard error). Data analysis was performed using SPSS version 26.0 software (IBM SPSS, Armonk, NY, USA), in which p < 0.05 was considered statistically significant. All the figures were created with Origin 8.5 software (Origin Lab, Hampton, NH, USA).
Results and discussion
Analysis of structural characterization of EUP1, EUP2 and EUP3
Extraction, purification and molecular weight analysis
Microwave-assisted hot water was used to extract crude polysaccharides from E. ulmoides leaves. The extraction rate of crude polysaccharides was 6.5 ± 0.3%. The crude polysaccharides were separated and purified to obtain three components: EUP1, EUP2, and EUP3 (Fig. 1A).
Molecular weight is an important characteristic of polysaccharides, and a key factor affecting antioxidant, antibacterial, immune regulation and other biological activities of polysaccharides . The chromatographic diagram of SEC–MALLS showed that EUP1, EUP2 and EUP3 were all one main peak, indicating that the three components were homogeneous . The average molecular weights (Mw) of EUP1, EUP2 and EUP3 were 1.51 × 105 Da, 3.05 × 104 Da and 1.17 × 105 Da, respectively. Meanwhile, the Mw/Mn coefficients of EUP1, EUP2 and EUP3 were 1.24, 1.40 and 1.41, respectively, indicating that the three components were evenly distributed and concentrated (Fig. 1B).
Monosaccharide composition analysis
GC–MS chromatograms showed that EUP1, EUP2 and EUP3 were mainly composed of Rha, Ara, Man, Glu and Gal, among which the content of Gal and Ara was the highest. A small amount of Xyl (2.6 mol%) was also detected in EUP2. However, the molar percentage of monosaccharide was different among the three components. The highest content of Rha and Glu was EUP3, and the highest content of Ara and Gal was EUP1. Meanwhile, the highest content of Man was EUP2. The monosaccharide compositions of EUP1, EUP2 and EUP3 were significantly different (p < 0.05) (Table 1).
The FT-IR spectrum showed that EUP1, EUP2 and EUP3 had similar characteristic peaks. There was a strong and wide characteristic absorption peak at 3400 cm−1, which was caused by O–H stretching vibration of the polysaccharides hydroxyl and hydrogen bond. The weak absorption peak at 2930 cm−1 was C–H antisymmetric stretching vibration of CH3 and CH2 groups , and the peak at 1748 cm−1 was the absorption peak produced by the tensile vibration of lipid carbonyl C=O. Additionally, there were C=O tensile vibration of the carboxyl group at 1612 cm−1 and C–H/O–H bending vibration at 1410 cm−1, which indicated aldehyde acids existed, and EUP1, EUP2 and EUP3 were acidic polysaccharides . There was an absorption peak at 1262 cm−1, indicating that the polysaccharides contained acetyl groups, and the absorption peaks at 1078 cm−1 and 1020 cm−1 indicated that the polysaccharides had pyranoid sugar rings . Moreover, a small peak at 770 cm−1 showed the β configuration of the sugar unit. Therefore, the structures of EUP1, EUP2 and EUP3 were inferred to be a pyranose structure with β configuration (Fig. 1C).
Crystal structure analysis
XRD is an important method to quickly study the microstructure of crystals or some amorphous materials. The diffraction peaks of crystalline materials are narrow and sharp, while those of amorphous materials are scattered and wide . According to the XRD diffraction diagram, EUP1, EUP2 and EUP3 only showed a single bread-like peak at 2ɵ = 20°, which showed that the crystallization of three components was amorphous structure (Fig. 1D).
NMR spectroscopy is the most powerful technique for the structural analysis of complex polysaccharides, which can simplify the structural analysis of carbohydrates. In the 1H NMR spectra of the three components, most of the signals appeared in the range of δH3.4–5.2 ppm, and there was no signal at δH5.4 ppm, indicating that EUP1, EUP2, and EUP3 were pyrano polysaccharides (Fig. 2). The signal at δH5.1–5.2 ppm indicated α configuration in monosaccharide residues, and the strong peak signal near δC103–109 ppm and δH4.3 ppm showed β configuration in monosaccharide residues . The results were consistent with that of the FTIR spectrum analysis. Moreover, the δH1.2 ppm and δH1.1 ppm signals were derived from the methoxyl groups of O-2 and O-2, 4-linked Rha, respectively. The signal at δH1.9–2.3 ppm was assigned to the acetyl group, and δH3.42–3.86 ppm was the chemical shift of protons on polysaccharides C2–C6 . A massive signal at δH3.67 ppm was from the methoxy group bound to the carboxyl group of Gal . The proton signal of Ara residues was observed at δH5.0 ppm, δH4.5 ppm, δH4.03 ppm, δH4.1 ppm, and δH3.8 ppm. The enhanced degree shift at δH4.6 ppm can be attributed to the solvent D2O.
In the 13C NMR spectrum, signals near δC 16.6 ppm were confirmed to be associated with CH3 of Rha. The dense distribution of carbon signal between δC 60.0–75.1 ppm was related to the absorption signal of C2–C5 in monosaccharides, while the resonance signal region of C6 was located at δC 60–65.1 ppm . Meanwhile, the δC 109.3 ppm and δC 107.4 ppm signals were attributed to different C-1 bonds of Rha, and the δC100 ppm signal was attributed to different C-1 bonds of Gal .
Apparent morphology analysis
The surface morphology of polysaccharides can be effectively observed by FE-SEM (Fig. 3). It was found that the three components had different surface structures. Among them, EUP1 resembled a honeycomb with loose and porous surface morphology, while EUP2 presented a rod-like shape with rough and branching surface, and EUP3 was mainly a flake structure with smooth surface. The differences in surface morphology of three components indicated that the strengths of aggregation and bonding were different (Fig. 3). 3.2 Analysis of physicochemical property of EUP1, EUP2 and EUP3.
Zeta potential and particle size analysis
Zeta potential analysis showed that EUP1 potential was − 11.47 mV, while EUP2 potential and EUP3 potential were − 14.5 mV and − 16.63 mV, respectively. Therefore, the three elution components of E. ulmoides polysaccharides were acidic polysaccharides, and the absolute values of ζ potential of three components increased with the increase of eluent concentration, which was consistent with the elution mechanism of ion chromatography . The average particle size of EUP1 (964.97 nm) was the largest, followed by EUP2 (816.97 nm) and EUP3 (284.93 nm). The average particle size of the three components decreased with the increase of eluent concentration, which may be because the larger the absolute value of ζ potential, the stronger the electrostatic repulsion between particles and the formation of a more dispersed system between particles . The greater the absolute value of ζ potential, the smaller the mean particle size (Fig. 4A).
Thermal stability analysis
Thermal stability is an important feature of various biomolecules for biological applications. DSC curves showed that the endothermic reactions of EUP1, EUP2 and EUP3 occurred within 100–150 ℃, among which the crystallization temperatures of EUP2 and EUP3 were about 120 ℃, and that of EUP1 was 142 ℃ (Fig. 4B). The melting of polysaccharides occurred with an exothermic reaction between 270 and 300 °C. Therefore, the thermodynamic curve showed that these three components have certain thermal stability in the temperature range below 270 ℃. Additionally, the reaction enthalpy of EUP1 was 103.6 J/g, which was much higher than that of EUP2 (66.78 J/g) and EUP3 (52.94 J/g). EUP1 had the highest thermal stability, while EUP2 and EUP3 had similar thermal stability. The difference in thermal stability of three components may be caused by the different monosaccharide composition and molecular weight .
Analysis of antioxidant activity of EUP1, EUP2 and EUP3
Free radicals are naturally generated in human metabolism and can easily attack protein, DNA, lipids and other biological macromolecules to induce oxidative stress. Studies have shown that they are related to tumors, cancers, cardiovascular diseases and other diseases . ESR method is the most direct method to detect free radicals. It is based on the principle that unpaired electrons absorb electromagnetic radiation in the DC magnetic field and transition from a low energy level to a high energy level, which can avoid the influence of sample color .
DPPH radical scavenging activity analysis
The DPPH free radical is a relatively stable free radical at room temperature and has been widely used to evaluate the antioxidant capacity of polysaccharides. In the range of 0.04–0.24 mg/mL, all three components had strong DPPH free radical scavenging ability, and the free radical scavenging ability increased with the increase of concentration, showing a significant dose relationship. When the concentration was 0.24 mg/mL, the DPPH radical scavenging ability of EUP1, EUP2 and EUP3 reached 58.6%, 68.4% and 74.5%, respectively (Fig. 5A). Meanwhile, the IC50 values of EUP1, EUP2 and EUP3 scavenging DPPH free radical were 0.13, 0.07 and 0.06, respectively (Fig. 5B). EUP2 and EUP3 had no significant difference in the DPPH scavenging activity, but both of them were superior to EUP1 (p < 0.05).
OH radical scavenging activity analysis
In the range of 0.04–0.24 mg/mL, the scavenging ability of the three components to OH radicals increased with the increase in concentration, and the IC50 of EUP1, EUP2 and EUP3 were 0.15 mg/mL, 0.11 mg/mL and 0.12 mg/mL, respectively (Fig. 5C and D). Similar to the DPPH radical scavenging activity, there was no significant difference in OH scavenging ability between EUP2 and EUP3, but both of them were superior to EUP1 (p < 0.05).
The differences in the antioxidant capacity of EUP1, EUP2 and EUP3 may be caused by different molecular weight, monosaccharide composition, substituent position and branching degree. EUP1 had the highest molecular weight, but the scavenging capacity of DPPH and OH radical was lower than that of EUP2 and EUP3 with smaller molecular weight. This result indicated that polysaccharides with smaller molecular weight had higher antioxidant capacity within a certain range, which was consistent with the results of Long et al. . In addition, the study by Lo et al.  showed that the free radical scavenging ability of polysaccharides depended on their monosaccharide composition, and Rha was the most important factor related to the free radical scavenging ability of polysaccharides. In this research, the mole ratio of Rha in EUP1 was 3.66 mol%, which was much lower than that in EUP2 (7.92 mol%) and EUP3 (18.67 mol%).
In this research, crude polysaccharides from E. ulmoides leaves were extracted by microwave assisted hot water with an extraction rate of 6.5 ± 0.3%, and three components EUP1, EUP2 and EUP3 were purified and obtained through anion exchange column. The structure, physicochemical property and antioxidant activity of EUP1, EUP2 and EUP3 were studied by SEC–MALLS, GC–MS, FT-IR, XRD, NMR, FE-SEM, DSC and ESR methods. The results showed that the three components all had typical polysaccharides absorption peaks, which may be polysaccharides with β configuration of pyranose structure, and amorphous structure of acid polysaccharides with good thermal stability below 270 ℃. However, the molecular weight, monosaccharide composition and apparent morphology of the three components were different, leading to the superior scavenging ability of DPPH and OH radicals of EUP2 and EUP3. The results will provide a reference for developing E. ulmoides leaves as well as a basis for developing a new antioxidant.
All data generated or analyzed during this study are included in this published article.
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This work was supported by the National key research and development project (2017YFD060130205).
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Liu, M., Wang, Y., Wang, R. et al. Structural characterization, physicochemical property, and antioxidant activity of polysaccharide components from Eucommia ulmoides leaves. Chem. Biol. Technol. Agric. 10, 122 (2023). https://doi.org/10.1186/s40538-023-00495-4