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Acetic acid (HAc, 99.5%), KH2PO4 (99.5%), NaH2PO4 (99.9%), Na3PO4, H3PO4, NaOH, chloroform (≥ 99%) and FeCl3•6H2O (99%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ethylene glycol (> 99%), sulfuric acid (96%), 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP, 99%), 4-formylphenylboronic acid (FPBA, 98%), methanol (99.5%), 1,6-hexamethylenediamine (99%) and anhydrous sodium acetate (99.5%) were purchased from Titan Scientific Co., Ltd. (Shanghai, China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH, 98.5%), thiazoyl blue tetrazolium bromide (MTT, > 99%), HPLC-grade acetonitrile (ACN), trifluoroacetic acid (TFA, 99%), anhydrous ethanol and phenol were obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). Pullulan polysaccharide (PPS, 99%), tea polysaccharide (TPS, 50%), soybean polysaccharide (SPS, 70%), Lycium barbarum polysaccharide (LBPS, ≥ 50%), trypan blue (60%) and deoxyguanosine (99%) were from Yuanye Bio-Technology (Shanghai, China). NaBH3CN (95%), guanosine (99%), adenosine (99%), deoxyadenosine (99%) and cytidine (99%) were from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Human breast cancer cell line (MCF-7), human lung carcinoma cell line (A549), Roswell Park Memorial Institute 1640 medium (RPMI-1640, containing 2.0 mg/mL D-glucose, 0.3 mg/mL glutamine, 2.0 mg/mL NaHCO3, 80 U/mL penicillin, and 0.08 mg/mL streptomycin), Dulbecco’s Modified Eagle Medium (DMEM, containing 4.5 mg/mL D-glucose, 0.3 mg/mL glutamine, 0.11 mg/mL sodium pyruvate, and penicillin streptomycin), parenzyme cell digestion solution (containing 0.25% trypsin and 0.02% EDTA), and phosphate buffer solution for cell culture (PBS) were purchased from Keygen Biotech (Nanjing, China). Fetal bovine serum (FBS) was purchased from Gibco (Life Technologies, Australia). Cell culture bottles (25 cm2 in growth area) and glass bottom cell culture dishes (Φ 30 mm) used for cell culture and microimaging were obtained from NEST Biotechnology (Wuxi, China). Real-world beverage plants, including Lycium barbarum, tea leaves (green tea) and soybeans, were purchased from a local supermarket. Water used in this study was produced by a Milli-Q system (Millipore, Milford, MA, USA). Except as otherwise noted, the purity grades of all chemicals without specific stipulations were analytically pure and phosphate buffer (PB) solutions used in this work was especially referred to the 0.1 M, pH 8.5 phosphate buffer.
Synthesis of MNPs@B(OH)2
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MNPs@B(OH)2 were prepared referring to our previous reports[4,5] with slight modifications, in which the preparation of amino group-capped MNPs (bare MNPs) and then their post-modification with FPBA were contained. To synthesize bare MNPs, 480 mL ethylene glycol, 16.0 g FeCl3 and 32.0 g anhydrous sodium acetate were mixed, then the mixture was slowly heated to 55 °C and kept vigorously stirring until the solution became transparent, followed by adding with 104.0 g 1,6-hexamethylenediamine and mixing evenly. After that, the resulting solution was transferred into a polytetra-fluoroethylene autoclave reactor and kept under reaction for 10 h at 198 °C. Finally, the prepared bare MNPs were magnetically separated and washed three times with ethanol and water, then the obtained MNPs were dried in a vacuum oven at 55 °C and stored in an air tight container.
For the boronic acid functionalization, 2.0 g the above prepared MNPs were dispersed into 100 mL absolute ethanol containing 5.0 mg/mL FPBA and 1.0 mg/mL NaBH3CN. After ultrasound for 30 min, the mixed solution was mechanically agitated at 40 °C for 12 h. When the reaction finished, the MNPs@B(OH)2 were magnetically isolated and respectively rinsed three times with ethanol and water, and then harvested after drying at 55 °C and stored under air tight conditions for upcoming assays.
Binding selectivity and adsorption dynamics of BA-MSPE
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Deoxyguanosine, deoxyadenosine, cytidine, guanosine and adenosine were served as model compounds to investigate the binding selectivity of MNPs@B(OH)2. Briefly, 8 mL aliquots of PB containing 1.0 mg/mL model analytes were supplemented with 35.0 mg MNPs@B(OH)2 or bare MNPs, after fully dispersing by ultrasonic, the resulting mixture solutions were incubated at room temperature for 0.5 h. Then the analyte-extracted MNPs@B(OH)2 or bare MNPs were magnetically separated and redispersed into 1.0 mL desorption solution composed by 0.1 M acetic acid. After desorption for 2 h, the materials were removed by external magnet while the supernatants were collected individually, and their UV-vis absorption spectra were tested on a UV-1800PC spectrophotometer (Mapada Instruments, Shanghai, China). The equilibrium binding capacity of MNPs@B(OH)2 and bare MNPs was figured out with standard curve method, which measured by UV-vis spectrometry and plotted by licensed OriginPro 2016 software. The wavelengths used for plotting standard curves of adenosine, guanosine, cytidine, deoxyadenosine and deoxyguanosine were, respectively, set at 258, 252, 278, 258 and 258 nm.
For the investigation of adsorption dynamics, guanosine, a cis-diol containing compound which can form boronate affinity effect with MNPs@B(OH)2, was selected as a target analyte. The steps were as follows: 35.0 mg MNPs@B(OH)2 was fully dispersed into 5 mL PB containing 0.1 mg/mL guanosine by vortex and ultrasonic. After incubation at room temperature for the appropriate time (0, 5, 10, 15, 20, 30 and 50 min), 200 μL aliquots of this mixture solution were taken out and the supernatants were individually collected after discarding the materials by external magnetic field. Finally, the absorbance of supernatants was measured on a spectrophotometer. The binding dynamics were plotted according to the relationship between the absorbance of supernatants and extraction time.
Influence of pH on BA-MSPE
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To probe the effect of pH on the extraction performance of MNPs@B(OH)2, the main operations were the same as above except that the consumption of MNPs@B(OH)2 was 10 mg and the solvents used for the preparation of guanosine solutions were replaced by PB with pH ranging from 2.5 to 12.5 with intervals of one pH unit. The absorbance of desorption solutions was tested and the extraction amounts of MNPs@B(OH)2 at binding equilibrium were calculated using the standard curve method. This assay was repeated in triplicate and the data were averaged for plotting. The relationship between equilibrium extraction amounts and system pH was applied to assess the impact of pH on BA-MSPE.
Adsorption isotherms and the comparison of extraction performance between bare MNPs and MNPs@B(OH)2
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Since the direct spectrometric measurement of polysaccharides was difficult due to their poor UV-vis light absorption properties, color development by phenol-sulfuric acid method was utilized for spectral tests of polysaccharides. With the help of phenol-sulfuric acid chromogenic reaction, standard curves of polysaccharides were tested by UV-vis spectrometry as follows: polysaccharide stock solutions with certain concentration gradients (0.75, 0.5, 0.25, 0.1, 0.05 and 0.005 mg/mL for TPS; 1.0, 0.75, 0.5, 0.25, 0.1, 0.05 and 0.01 mg/mL for PPS; 0.5, 0.4, 0.3, 0.2, 0.1 and 0.075 mg/mL for LBPS; 1.0, 0.75, 0.5, 0.25, 0.1, 0.05 and 0.01 mg/mL for SPS) were prepared at first using 0.1 M HAc aqueous solutions as solvents, then phenol solution (5%, wt%), sulfuric acid and the prepared polysaccharide solutions were mixed in volume ratio of 1:5:1, followed by incubating at 55 °C for 10 min. Thereafter, the digital photos of all solutions were recorded using a smartphone (Vivo X80, Guangdong, China) and their UV-vis spectra were determined on a UV-vis spectrophotometer. Standard curves were plotted by absorbance at 488 nm against polysaccharide concentrations, and the extraction amounts of polysaccharides by MNPs and MNPs@B(OH)2 were inferred from linear regression equations for quantitative comparison. All data were obtained by three tests in parallel for quantifications.
Regarding adsorption isotherms, the details were as follows: A series of TPS stock solutions with a concentration gradient of 0.05, 0.1, 0.5, 1.0, 2.0, 4.0, 6.0, 10.0, 20.0 and 50.0 mg/mL were prepared using PB as solvents, then to each an aliquot of 40.0 mg MNPs@B(OH)2 was added, followed by incubation for 2 h at room temperature. After removing the solvents by magnetic separation, the resulting materials were washed three times with PB, and then supplemented with 1.0 mL aliquots of desorption solution composed of 0.1 M HAc aqueous solutions. After desorption for 2 h, the supernatants were collected via magnetic isolation and their absorbance was measured after color development by phenol-sulfuric acid method. The binding isotherms were obtained through the extraction amounts deduced by the absorbance at 488 nm plotting against TPS concentrations. The Hill equation given below was applied to fit data and estimate the binding properties of MNPs@B(OH)2.
$ y={Q}_{max}\cdot {x}_{n}/({x}_{n}+{K}_{d}^{n}) $ Herein, n was the Hill slope while Qmax and Kd were, respectively, the maximum binding capacity and dissociation constant.
As for the dosage-dependent extraction experiments, the main operations were the same as above except that the dosages of MNPs@B(OH)2 were set as 5, 10, 25, 50, 75, 100 and 150 mg using TPS and LBPS as model analytes both with a concentration of 1.0 mg/mL (dissolved in PB).
Extraction of polysaccharides from real-world beverage plants
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Three beverage plants, including tea leaves (green tea), soybeans, and Lycium barbarum, were devoted as real samples to extract polysaccharides. Three steps, i.e., the pretreatments of raw materials, the preparation of leaching liquors of crude polysaccharides, and subsequent BA-MSPE, were contained in this experiment.
For the pretreatments of raw materials, the above-mentioned three beverage plants were washed with water several times to remove impurities, followed by drying to a constant weight at 55 °C in an electric oven. Then the powders of raw materials were obtained by quickly crushing in a high speed pulverizer.
To prepare the leaching liquors of crude polysaccharides, the above obtained raw material powders were immersed into PB with a material-to-liquid ratio of 1:50 (1.0 g powder per 50 mL PB). After incubation for 2 h at 60 °C, the leaching liquors and solid matters were centrifugally separated (5000 rpm for 10 min) and individually collected, then the solids were immersed into PB again with the same feed ratio and incubation for another 2 h at 80 °C. After further centrifugal separation, the supernatants were collected and gathered with the filtrates of the previous separation. The total leaching liquors were centrifuged again (10,000 rpm for 5 min) and stored at 4 °C after discarding the precipitation.
BA-MSPE were carried out as follows: 4.0 mL aliquots of freshly prepared leaching liquors were respectively added with 100 mg MNPs or MNPs@B(OH)2, followed by incubating for 0.5 h at room temperature. After magnetic separation, the supernatants were removed and 0.1 M HAc aqueous solutions was supplemented into the polysaccharides-extracted materials with 1.0 mL for each. After desorption for 2 h, the desorption solutions were magnetically collected. After color development with phenol-sulfuric acid method, the UV-vis spectra were measured and their colors were simultaneously recorded. The desorption operations were repeated several times until the color of chromogenic solution was indistinguishable by naked eye. The extraction amounts were figured out and weighted according to standard curve method using the absorbance at 488 nm.
Relative purities of polysaccharide extractives analyzed by UV-vis spectrophotometry
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In order to survey the relative purities of polysaccharides extracted by MNPs@B(OH)2, the extractives from tea leaves and Lycium barbarum were freeze dried and corresponding standard polysaccharides were devoted as controls in this assay. The main steps for the BA-MSPE of TPS and LBPS were the same as above except that the dosages of leaching liquors and materials were enlarged. In detail, the volume of leaching liquors was set as 400 mL and the usage of MNPs@B(OH)2 was 2.0 g. Concentrations of extracted and standard polysaccharide samples for absorbance tests were set at 1.0 and 0.5 mg/mL, respectively. The relative purities of polysaccharides extracts were calculated by dividing the amount of polysaccharides obtained from extracts into that of standard polysaccharides.
HPLC analysis
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Freeze-dried extracts of TPS and LBPS are denoted as model polysaccharides herein. Due to their poor light-absorbing properties, PMP-based pre-column derivatizations[13,14] of polysaccharides were essential for HPLC analysis. In detail, 100 μL methanol solution containing 0.5 M PMP was added with equal volume of 0.5 mg/mL polysaccharide solutions, followed by incubation at 70 °C for 2 h. When the labelling reaction was finished, 1.0 mL chloroform was added into the solution, then vortex for 1 min to extract the unreacted PMP reagents. After centrifuging for 1 min at 1000 rpm, the remaining PMP in the bottom layer was removed. Such a PMP separation operation was repeated three times and the resulting PMP-tagged polysaccharides were loaded for HPLC analysis. The standard polysaccharides labelled in the same way were used as benchmarks for qualitative identification and the determinations of HPLC standard curves.
TFA-based hydrolysis was performed to identify the monosaccharide composites of polysaccharides by HPLC, and the details were as follows: polysaccharides were separately dissolved into 2.0 M TFA solution with a final concentration of 10.0 mg/mL, and then reacted in air tight conditions for 6 h at 105 °C. When the reaction completed, the supernatants were centrifugally collected and their pH was adjusted to neutral using 1.0 M NaOH solution. Subsequently, the PMP labelling of hydrolysates and standard monosaccharides was implemented as above. The resulting solutions were finally stored at 4 °C for use.
HPLC conditions were selected referring to an existing method[15]: A Adamas C18-Classic column (SepaChrom S.R.L., 5 μm, 250 mm × 4.6 mm) was equipped for chromatographic separations; The mobile phase was composed by acetonitrile (A) and monopotassium phosphate aqueous solution with pH 6.8 (B) in an isocratic mode with 20% A; Injection volume was set as 20 μL; A pre-equilibration period of 30~60 min was applied between two consecutive separations; The flow rate and column temperature were, respectively, set as 1.0 mL/min and 40 °C, and the wavelength of 248 nm was selected for detection. All buffer and sample solutions were filtrated using a 0.45 μm filter membrane before running.
HPLC standard curves were plotted in terms of the function between peak areas and the concentrations of standard polysaccharides derived by PMP. Briefly, PMP-marked TPS and LBPS stock solutions with concentration gradients of 1.0, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 and 0.01 mg/mL, and 0.3, 0.2, 0.1, 0.05, 0.01 and 0.005 mg/mL were prepared and their chromatographic retentions were surveyed with the aforementioned conditions. Each sample was tested in triplicate and the weighted peak area was used for plotting standard curves, then the concentrations of polysaccharides in extracts were deduced by linear regression equations.
Reusability tests of MNPs@B(OH)2
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The recyclability of MNPs@B(OH)2 was investigated by consecutive extraction and desorption 10 times. In detail, 80.0 mg MNPs@B(OH)2 was dispersed into 20 mL PB containing 3.0 mg/mL TPS, then incubation for 1 h at room temperature. After that, the materials were magnetically collected and washed with 5 mL PB for 5 min, followed by desorption for 0.5 h with 1 mL HAc aqueous solution (0.1 M). Finally, the supernatants were separately collected after magnetic isolation. After color development by phenyl-sulfuric acid method, their UV-vis spectra were tested and digital photos were recorded. Such an extraction-elution process was repeated for 10 cycles. A pre-equilibrium period of 1 h by PB (10 mL) was adopted for MNPs@B(OH)2 between two extractions.
Antioxidant capacity assessed by DPPH radical scavenging assay
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Two aliquots of 1.0 mL ethanol solution containing 0.06 mg/mL DPPH were, respectively, mixed with 0.3 mL TPS and LBPS solution (dissolved in ethanol, and both with a concentration of 0.5 mg/mL). The DPPH solution combined with an equal volume of ethanol was used as the control group. After incubation in the dark for appropriate periods (0, 1, 3, 5, 10, 20, 30, 45 and 60 min), the digital photos were recorded using a smartphone and the UV-vis spectra were measured on a spectrophotometer. The radical scavenging activities of TPS and LBPS were evaluated by the following expression:
$ Radical\;scavenging\;acitivity\;\left({\text{%}}\right)\;=\;\left(1-\frac{{A}_{T}}{{A}_{C}}\right)\;\times\; 100 $ In which, AT and AC were, respectively, on behalf of the absorbance of polysaccharides-treated group and control group (without additives).
Cell culture and antitumor activity evaluation
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MCF-7 cell was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified chamber containing 5% CO2. A549 and DU145 cells were cultured in DMEM medium with 10% fetal bovine serum at 37 °C in a humidified chamber containing 5% CO2. All the cell experiments were implemented when the confluence reached ~80%. MTT assays and trypan blue staining experiments were adopted to estimate cell viability and apoptosis, respectively.
Details for MTT assay: The cells cultured in 96-well microplate were washed with 1 × PBS three times, followed by supplementing with relevant culture medium containing polysaccharide extracts with different concentrations ranging from 0 (control), 0.25, 1.0, 1.5, 2.5 to 5.0 mg/mL (200 μL per well). After culturing for another 24 h, the culture medium was removed and the remaining cells in microplate were cultured with PBS containing 5.0 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 50 μL per well), and then cultured for 4 h. The remaining MTT reagent was discarded and 100 μL DMSO was supplemented into each well, and then slowly shaken for 10 min to dissolve the formazan in cells. Finally, the absorbance of the DMSO solutions was determined at 492 nm on a Synergy H1M microplate reader (BioTek, Winooski, VT, USA). The formula given below was utilized to calculate the cell viability (%):
$ Cell\;viability=\frac{{{A}_{T}-A}_{b}}{{{A}_{C}-A}_{b}}\times 100{\text{%}} $ Herein, AT, AC and Ab represented the absorbance value of treatment group, control group (cells without additives) and 96-well microplate substrate, respectively.
Trypan blue staining trials were performed as follows: The preculture with polysaccharide extract-added medium was the same as above, but only the cells cultured by the medium containing 0 and 5.0 mg/mL polysaccharide extractions were selected for staining and comparison. The pretreated cells were subsequently stained by 0.4% (wt%) trypan blue solution for 3 min, then microscopic images of the resulting cells were recorded on an inverted fluorescence microscope (Sunny Optical XD-FRL, 10 × objectives).
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Boronic acid-functionalized magnetic nanoparticles (MNPs@B(OH)2) have been prepared and used as sorbents for boronate affinity-mediated MSPE of polysaccharides in this work. Boronate affinity effect and its work parameters were investigated, the mechanism for the binding of polysaccharides by MNPs@B(OH)2 was discussed, and the extraction conditions were also optimized. Three polysaccharides, including TPS, LBPS and SPS, were successfully extracted from relevant real-world beverage plants, and the main active ingredients in extracts were identified by several instrumental analysis techniques, such as UV-vis/FT-IR spectrometry and HPLC. In the end, the extracted TPS and LBPS were experimentally proven to be of fine antioxidant and antitumor bioactivities in terms of DPPH radical scavenging experiments, trypan blue staining, as well as MTT assays. Since the operations of BA-MSPE were straightforward and do not necessitate the use of organic solvents or other intricate impurities elimination steps during BA-MSPE, coupled with the fine recyclability of MNPs@B(OH)2, this approach may have more potential for the simple separation and purification of cis-diol containing compounds in the fields of food and agricultural product processing.
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About this article
Cite this article
Ding Y, Li H, Liu T, Liu Y, Yan M, et al. 2023. Boronate affinity-mediated magnetic solid phase extraction and bioactivities of polysaccharides from beverage plants. Beverage Plant Research 3:14 doi: 10.48130/BPR-2023-0014
Boronate affinity-mediated magnetic solid phase extraction and bioactivities of polysaccharides from beverage plants
- Received: 19 April 2023
- Revised: 07 May 2023
- Accepted: 11 May 2023
- Published online: 14 June 2023
Abstract: Polysaccharides are of great significance in food production, but their isolation highly relies on multi-staged liquid-liquid extraction. In this study, a boronate affinity-mediated magnetic solid phase extraction (BA-MSPE) method was initiated for the effortless and efficient extraction of polysaccharides using boronic acid-grafted magnetic nanospheres (MNPs@B(OH)2) as extractants. MNPs@B(OH)2 showed fine class selectivity toward cis-diol containing compounds at weak alkaline condition (pH 7.5~8.5) and higher binding capacity than that of MNPs without boronic acid functionalization. Fast binding dynamics with a binding equilibrium within 10 min, stronger affinity toward polysaccharides (Kd as low as 10−3~10−6 M level) than that of small molecular cis-diol compounds (Kd in the range of 10−1~10−4 M level), and good recyclability (the binding capacity decreased less than 13% after ten times consecutive extraction) could also be observed for MNPs@B(OH)2. Finally, the BA-MSPE of polysaccharides was performed with three beverage plants as real samples, including tea leaves, soybeans, and Lycium barbarum. Antioxidant activity of polysaccharide extractives was verified by DPPH radical scavenging assays, giving a radical scavenging rate of 31.4% and 18.8% for crude extractives of TPS (tea polysaccharide) and LBPS (Lycium barbarum polysaccharide), respectively. Microscopic imaging combining with MTT and trypan blue staining trials uncovered that the extractives were of dosage-dependent antitumor bioactivities, giving the cell mortality rates over 91.8% and 77.2% for MCF-7 and A549 cells in the presence of 5.0 mg/mL TPS, and 56.6% and 40.0% with the equal dosage of LBPS, respectively. As the BA-MSPE strategy is simple and eco-friendly, there will be more potential for the application of cis-diol compound purification.
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Key words:
- Boronate affinity /
- Solid phase extraction /
- Polysaccharide /
- Bioactivity /
- Beverage plant