Synthesis and antioxidant properties of Lycium barbarum polysaccharides capped selenium nanoparticles using tea extract

Abstract

Selenium nanoparticles (SeNPs) have attracted increasing interest over the last decades because of their activities on redox balance in human body. However, the SeNPs tend to aggregate into large clusters, resulting in lower bioactivity, bioavailability and biocompatibility. Surface-capping agents on SeNPs play crucial roles in its stabilization and biological activity. Here, a green synthesis method for the preparation of Lycium barbarum polysaccharides capped SeNPs using green tea extracts as reductants under mild conditions, at room temperature, is reported. The structure, size, morphology and thermal behaviour were analyzed by various characterization techniques. The functionalized nanoparticles demonstrated high antioxidant activity, including DPPH and ABTS free radical scavenging. Moreover, the SeNPs significantly protected the H₂O₂-induced PC-12 cell death. Taken together, these results evidence the possible application of these SeNPs as antioxidants food supplement or ingredient and neuroprotective agent.

Graphical Abstract

Keywords:

• Selenium nanoparticles
• Lycium barbarum polysaccharides
• green tea
• antioxidants

Introduction

Selenium (Se), a semimetallic chemical element, has been extensively used in medicinal and pharmaceutics as an important micronutrient for maintenance [1–5]. Se is an indispensable micronutrient for human health and constituent part of selenoenzymes, which prevent cellular damage from free radicals [5,6]. In the human organism, free radicals are naturally generated as byproducts of oxygen metabolism, and may importantly contribute to the development and progression of various diseases, such as arthropathy, cardiomyopathy, thyroid dysfunction, immune dysfunction and cancer [5,7,8]. Overflowing experimental evidences have state clearly that selenium supplementation is a popular and broadly adopted complementary cancer treatment option. Selenium-rich dietary supplement generated considerable interest in pharmaceutical and food sciences [9].

However, high doses of selenite give rise to great concerns about its toxicity [10,11]. In this regard, nanoparticles of elemental selenium (SeNPs) have been suggested as safer and more effective platforms for the delivery of selenium for biological purposes [12–15]. However, SeNPs are usually prone to agglomeration into large clusters in aqueous media, reducing their bioactivity, biocompatibility and bioavailability [16,17]. Very recently, the combination of the naturally originated biomacromolecules such as protein or polysaccharide as templates or capping agents with SeNPs through strategic functionalization were used in attempts to tackle challenging with desired function from the bio-functionalized systems [18–22]. Therefore, SeNPs functionalized with polysaccharides extracted from plants, using biosynthesis or green chemically synthesized method, are drawing the attention of researchers recently [23–26].

Lycium barbarum polysaccharides (LBP), a major effective compositions of Chinese wolfberry as an excellent tonic, have shown different biological activities, such as antioxidant, anti-aging, anti-tumor, immunoregulation, hepatoprotection and so on [27,28]. Green tea is one of the most popular beverages worldwide, and several epidemiological and clinical studies have definitely confirmed an energetic relationship between green tea consumption and the prevention of free radicals-associated diseases [29–31].

In the present paper, considering the bioactivities of Chinese wolfberry and green tea, a combined procedure is proposed to the preparation of SeNPs using sodium selenite as precursor, polysaccharides from Lycium barbarum as stabilizer and green tea extracts as reductants under mild conditions, at room temperature, which probably constructed a precise hybrid structure presenting unprecedented properties for myriad biomedical applications. The obtained particles were analyzed by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analyzer and zeta potential analyzer to understand the morphology of SeNPs. Moreover, the antioxidant activity by green tea extract and LBP synthesized SeNPs as evidenced by DPPH and ABTS free radicals scavenging assays. Finally, the neuroprotective role of LBP-GT-SeNPs against oxidative stress-induced cell death was investigated by MTT assay.

Materials and methods

Chemicals

Sodium selenite (Na₂SeO₃), potassium persulfate (K₂S₂O₈), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were purchased from YuanYe Biotechnology Co., Ltd.(Shanghai, China). Green tea and dried fruits of Chinese wolfberry were available from the local market. The ultrapure water used in this study was produced from a Millipore^® filtration system (Millipore, Milford, MA).

Extracts of green tea

The extract of green tea leaves was obtained by using 5 g of tea leaves in 250 ml beaker with 100 ml of boiling water for 10 min before decanting it. The solution was filtered first through a 0.45 µm Millipore membrane and then through a 0.2 µm Millipore membrane for further use.

Preparation of Lycium barbarum polysaccharides (LBP)

Lycium barbarum was extracted from wolfberry. Briefly, wolfberry was dried at 65 °C and ground into powder. The lipids removed using chloroform:methanol solvent (2:1, v/v) into boiling for 30 min. After the filtration, air-dried residues were soaked in 80% ethanol at 78 °C to remove oligosaccharides. The residues were dissolved in 75 °C hot water and filtered, extracted four times, and then and then concentrated and concentrated. The concentrated extract precipitated using 95%, 100% ethanol and acetone, respectively. After centrifugation and washing, the extract was dried in vacuum. The desired LBP obtained were stored in a dry place for further use.

Preparation of Lycium barbarum polysaccharides green tea-selenium nanoparticles (LBP-GT-SeNps)

SeNPs were prepared similarly as previous studies. Briefly, 0.5 ml aqueous solution of 25 mM Na₂SeO₃ was added into a 10 ml beaker, and different concentrations of LBP with agitation. Then 1 ml freshly prepared green tea extract solution was added by dripping slowly into the mixture, and regulated the final volume to 5 ml with ultrapure water. The final concentrations of Se, LBP, and Green tea were 25 μM, 0.1–2 mg/ml and 1% (w/w), respectively. Samples were dialyzed against ultrapure water in a dialysis bag (MWCO= 3500) overnight for investigation to the properties of SeNPs.

Characterization and measurements

The morphology and characterization of SeNPs were investigated using various methods. The size and shape of LBP-GT-SeNPs were observed using transmission electron microscopy (Hitachi HT7700 TEM, Japan). For the observations, a drop of diluted LBP-GT-SeNPs aqueous solution was placed onto a carbon film on copper grids. The morphology of LBP-GT-SeNPs was observed using TEM (Hitachi HT7700 TEM, Japan). For TEM observations, one drop of properly diluted sample (after washing) was placed on a copper grid and then air dried before examination. The TEM image was acquired on HT7700 microscope at 120 kV equipped with an energy-dispersive X-ray spectrometer. FTIR spectra were recorded on a Nicolet 7600 FTIR spectrometer. Samples were prepared by scraping thin films from the LBP-GT-SeNPs substrates and mixing the powder with KBr in the atmosphere using pure KBr as background, and the spectra were scanned at wavenumber range 4000–400 cm⁻¹. The thermal properties of LBP-GT-SeNPs were ascertained using DSC-60 instruments (DSC-60, Shimadzu, Japan). The experiments were used an empty sample as the reference. Powder X-ray diffraction data were collected on a Bruck D8 Advance diffractometer (Bruker AXS, Germany) with Bragg–Brentano (θ, 2θ) geometry using Cu Kα radiation. The scan was performed at 25 °C from 10–80° in a scanning speed was 0.1 s/step. Hydrodynamic diameters and zeta potentials were determined with a dynamic light scattering equipment (Nano ZS90, Malvern Instruments, UK). The measurements were performed at room temperature with a 15° scattering angle.

DPPH radical scavenging ability

The method described previously was used to assess DPPH radical scavenging activity [32]. Samples were dissolved in methanol and the results were given as EC50, which is the amount of extract required to scavenge the initial DPPH radical by 50% and the ordinate the average percent of scavenging capacity from three replicates.

ABTS radical cation decolonization assay

For ABTS assay, the procedure followed the method described by Ren et al. with some modifications [33]. Briefly, the ABTS stock solutions included 7.0 mM ABTS and 2.5 mM potassium persulfate. Next, 60 μL of this solution was diluted in 2.4 ml ethanol to obtain an optical density of 0.7 ± 0.02 units at 734 nm using the UV–Vis spectrophotometer. Sample solutions were prepared by diluting 500 μL of SeNPs dispersion with deionized water (1:9, v:v). The mixture was left to react at room temperature in the dark for 10 min, and then the reduction of the ABTS free radical was measured. The scavenging activity was calculated from the equation

Cells culture and MTT

PC12 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 5% fetal bovine serum, 10% heat-inactivated horse serum, 100 units/ml of penicillin and 100 Ag/ml of streptomycin at 37 °C in a humidified atmosphere with 5% CO₂. The culture medium was changed every two days. PC12 cells were seeded at 1 × 10⁵ cells/well in 96-well plates and their viability was measured using the MTT assay. After before the addition of hydrogen peroxide (H₂O₂, final concentration 500 nM), the cells were incubated for 24 h, then LBP (2 mg/ml), Green tea extracts (1%), Na₂SeO₃ and SeNPs were added to the wells. For 24 h of treatment, 200 μL of a 5 mg/ml MTT stock solution, in PBS, were added to 1 ml medium and the incubation continued at 37 °C for 1 h. Data were expressed as the percentage of MTT reduction relative to the absorbance of control cells.

Results and discussion

Synthesis of LBP-GT-SeNPs

SeNPs were feasibly prepared by a “green” method using water as solvents and natural products as reducing and coating agents. Green tea extract was used as the Na₂SeO₃-reducing agent at room temperature and the LBP acted as the coating agent to prevent SeNPs agglomeration in dispersion, thus enhancing its stability and bioactivity under physiological conditions. Green tea extract mainly consists of flavanols, commonly known as catechins, such as (–)epicatechin-3-O-gallate (ECG) and (–)epigallocatechin-3-O-gallate (EGCG). These phenolic antioxidants deoxidize SeO₃²⁻ to Se⁰, which then forms the selenium core. In addition, the formed selenium core was then stabilized by a corona of LBP, which interacts with water by hydrogen bonds, forming a solvation shell that avoids agglomeration of nanoparticles (Scheme 1).

Scheme 1. Synthetic scheme for the LBP-GT-SeNps and its general mechanism.

The synthetic approach of the selenium nanoparticles involves simple mixing of an aqueous solution of Na₂SeO₃ and the LBP with the stock solution of 1% tea solution. After different concentrations of LBP added to the Na₂SeO₃, 1% green tea solution was added with stirring and turned slowly orange, indicating the formation of SeNPs. Production of SeNPs in this phytochemically mediated process was completed at room temperature within 12 h accompanied by a change in the colour of the solution, going gradually from orange to brown. The brown colour shown by the LBP-GT-SeNps was due to the formation of amorphous selenium in the form of spherical colloids [34].

Characterization of LBP-GT-SeNPs

Particle size is a significant feature in determining performance of nanoparticles in biological applications because it influences the biological properties, such as cellular uptake, circulating half-life and biodistribution [17]. As a result, regulating the size of nanomaterials is an exponential factor. Variances in the reactant ratios can significantly affect the particle size of SeNPs. In the present study, different concentrations of LBP were investigated. Diameters of LBP-GT-SeNPs were determined by DLS to be 690.7, 325.4, 310.2, 270.2, 258.7 nm when prepared with LBP at concentration of 0, 0.1, 0.5, 1, 2 mg/ml respectively (shown in Table 1). The average sizes of LBP-GT-SeNPs were observed to decrease with increasing LBP concentration. This resulted in an intensive ability to act as a surface modifier, thereby able to control the formation of monodispersed and homogeneous spherical of the SeNPs. Based on these results, 2 mg/ml LBP was used for further experiments (Table 1).

Table 1. Size of LBT-GT-SeNPs at different LBP concentrations using 1% green tea.

LBP concentration (mg/ml)	Diameter/nm	PDI
0	693.7 ± 160.1	0.805
0.1	325.4 ± 11.6	0.492
0.5	310.2 ± 5.9	0.338
1	270.2 ± 7.1	0.285
2	258.7 ± 10.6	0.241

Stability of nanomaterials is the most important factors influencing their medicinal application. Therefore, it is necessary to investigate the particle size, size distribution and size stability of LBP-GT-SeNPs. The surface charge, which may be investigated by zeta potential data, importantly affects the colloid stability, and a relatively low zeta potential could be predictive of a tendency of nanoparticles to agglomerate. As given in Figure 1(C), the zeta potential value for SeNPs was −24.1 mV, suggesting that selenium nanoparticles have a tendency to repel each other, rendering the colloid stable. Furthermore, according to results from time course of study on particle size presented desirable size and stability, and there were no significant differences within a month (shown in Figure 1).

Figure 1. Colour change of LBP (2 mg/ml), Green Tea (1%), LBP + Green Tea, LBP-GT-SeNPs at 0 and 30 days (A). Size distribution of LBP-GT-SeNps at 0 and 30 days (B). Time course of size distribution of LBP-GT-SeNps (2 mg/ml LBP and 1% Green Tea (C). Zeta potential distribution of LBP-GT-SeNps at 0 and 30 days (D).

The morphology and structure of SeNPs

To investigate the morphological and structural features, the synthesized LBP-GT-SeNPs were analyzed by TEM without contrasting. The TEM image confirmed that the LBP-GT-SeNPs were in the nanoscale range and comprised spherical and triangular shapes. The size distribution of the SeNPs was obtained in the range of 83–160 nm with an average size of 125 nm. It was found that the sizes observed by DLS were larger than those determined by TEM images, as expected. This is probably due to the solvation shell bound by the LBP coat onto the surface of the selenium core, which contributes to particle diameter only in DLS measurements. Furthermore, the small area of the dark dots was detected with energy dispersive X-ray spectrometry (EDX) (Figure 2(B)). The EDX spectrum demonstrated that the presence of strong signals from the Se atoms, together with signal of C and O atom from LBP confirming the presence of Se in LBP. The Cu and C elements in the EDX spectrum originated from the TEM copper grid and carbon film. Further characterization of the synthesized LBP-GT-SeNps for their structural information and crystallinity nature was conducted by the XRD spectroscopy. As shown in Figure 2(C), a typical XRD pattern of the selenium nanoparticles obtained revealed that the polymorph of as-prepared Nano-Se was amorphous Se (α-Se) [35].

Figure 2. TEM image (A), EDX analysis (B) and (C) XRD spectra of LBP-GT-SeNPs.

FTIR spectra analysis

To further confirm the interaction between LBP and selenium nanoparticles, Fourier transform infrared (FTIR) spectroscopy analysis was performed. According to the spectrum of LBP, a typical major broad stretching peak was at 3392 cm⁻¹ for the hydroxyl group, and a weak band at 2926 cm⁻¹ showed C–H stretching vibration. The band at 1630 cm⁻¹ was due to the bound water. The band at 1024 cm ⁻¹ was ascribed to configuration of the sugar units in the polysaccharide (Figure 3). The FTIR spectrum of LBP-GT-SeNPs resembled that of LBP which confirmed the presence of LBP on the surface of SeNPs. Moreover, the stretching vibration peak of hydroxyl was blue-shifted from 3392 cm⁻¹ to 3377 cm⁻¹, which suggested that selenium interacted with a hydroxyl group from LBP through hydrogen bonding and that is the reason why the LBP-GT-SeNPs were spherical in shape and stable.

Figure 3. FT-IR (A) and TG/DSC (B) analysis of LBP-GT-SeNPs.

TG/DSC analysis

The thermal behaviour of LBP-GT–SeNPs was further characterized by the TG/DSC analysis at a range of 20–600 °C. From the TG curve, the first thermal event can be seen that the mass of the sample loss of about 13.7% from 20 to 150 °C, which is due to the evaporation of water physically adsorbed and absorbed in the inner polymeric network. And then the mass decreases sharply upon heating over 200 °C mainly associated with the decomposition of the LBP capping on SeNPs. The exothermic effect might result from the thermal degradation of LBP in the DSC curve.

Antioxidant properties of LBP-GT-SeNPs

Nanoparticle antioxidants are an emerging strategy for antioxidant therapies to prevent and treat diseases involving oxidative stress. Nano-particles show stronger interactions with biomolecules providing more effective against free radical induced damage than small molecule antioxidants. This work describes a facile strategy for the preparation of selenium nanoparticles. Research shows SeNps, a novel Se species, possess high antioxidant. The antioxidant activity of LBP-GT-SeNps was evaluated by ABTS and DPPH free radical scavenging methods.

One of the main characteristics of antioxidants is the ability to scavenge free radicals. DPPH assay is routinely used for the assessment of free radical scavenging potential and considered as a gold standard assays. The scavenging mechanism of DPPH• is based on the reduction of DPPH in the presence of a hydrogen donating antioxidant due to the formation of the nonracial form. As shown in Figure 4(A), SeNPs showed strong DPPH-scavenging activity in a concentration-dependent manner at 5–25 μM. The 1% Green tea LBP-GT-SeNPs showed high antioxidant activity with low EC50 (22 μM SeNPs able to scavenge 50% of DPPH). And the DPPH scavenging ability of LBP-GT-SeNPs (25 μM) was higher than that of Na₂SeO₃ and LBP, comparing to the activity of green tea extracts and the value can reach up to 52.5% (Figure 4(B)).

Figure 4. Free radical scavenging effect of LBP-GT-SeNPs in different concentration (A). DPPH (B) and ABTS (C) radical scavenging of LBP-GT-SeNPs (25 µM), (D) Green Tea (1%), Na₂SeO₃ (25 µM), and LBP (2 mg/ml). Protective effect against H₂O₂ (0.5 µM)-induced PC-12 cell death using LBP-GT-SeNPs (25 µM), Green Tea (1%), Na₂SeO₃ (25 µM), and LBP (2 mg/ml) by MTT assay.

And the total antioxidant activities of LBP-GT-SeNPs were compared by using ABTS free radical scavenging assay. Unlike the reactions with DPPH radicals, which involve H-atom transfer, the reactions with ABTS•⁺ radicals involve an electron-transfer process. As shown in Figure 4(C), Na₂SeO₃ and LBP showed low antioxidant activity. In contrast, LBP-GT-SeNP at concentrations ranging from 5 to 25 μM significantly inhibited the ABTS free radicals in a dose-dependent manner. These results evidence the antioxidant activity of LBP-GT-SeNPs, which may be at least partially due to the presence of phenolic antioxidants in the green tea extracts. In contrast to hydrophobic systems of DPPH assay, the water-soluble LBP-GT-SeNPs were significantly active toward ABTS•⁺ which is applicable to both hydrophilic and lipophilic antioxidant systems.

The primary antioxidant assays based on chemical reaction do not necessarily reflect the behaviour of antioxidants in biological systems. Therefore, the neuroprotective role of LBP-GT-SeNPs against oxidative stress-induced cell death was investigated by MTT assay. A control experiment showed that ignorable cytotoxicity was observed for SeNPs suggesting the great biocompatibility of SeNPs. Moreover, LBP-GT-SeNPs protected PC12 against H₂O₂ induced toxicity. The viability of cells incubated with H₂O₂ only was reduced to 50%, while cells pre-treated with LBP-GT-SeNPs and then treated with H₂O₂ exhibited 90% of viability. This protective effect was comparatively lower in the presence of LBP, green tea extracts and Na₂SeO₃ alone, demonstrating that SeNPs were able to protect cells from H₂O₂-induced injury. These results confirmed that the LBP-GT-SeNPs possessed positive antioxidant activity in cells, which may be due to the free radical scavenging potential and, occasionally, to pharmacological properties of SeNPs.

Conclusions

A suitable and facile method for the preparation SeNPs under ambient conditions using constituents from Lycium barbarum berries and green tea as a stabilizer and reducing agent was developed. The obtained particles were analyzed by various characterization techniques including transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analyzer and zeta potential analyzer to understand the morphology of SeNPs. The functionalized nanoparticles demonstrated high DPPH scavenging ability and ABTS scavenging ability. Moreover, investigations on nanoparticle in biological system had been displayed protective effect against H₂O₂-induced PC-12 cell death. These results suggest that the SeNPs have potential for utilization as food materials in green synthesis of selenium nanoparticles as well as in applications in biomedical, cosmetic and pharmaceutical products, especially as antioxidant supplement or ingredient and neuroprotective agent.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The authors are grateful for the generous financial support from National Natural Science Foundation of China (No.81573717); The Natural Science Foundation of Shandong Province (ZR2015HL064); National Key Technology R&D Program of the Ministry of Science and Technology (No. 2013GA740103); College Students’ Technology Innovation Project of Weifang Medical University (KX2016047).