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SHORT COMMUNICATION - Characterization and Antioxidant Activity of an Exopolysaccharide Produced by Ginkgo polypores.

Byline: Xuewei Jia, Yu Jiang, Zhifei Chen and Chunping Xu

Summary: In this work, an exopolysaccharide (EPS) produced by submerged culture of Ginkgo polypores was purified and its structure and antioxidant activity were investigated. Lactose and yeast extract were chased as optimal carbon and nitrogen sources respectively for EPS production with cultivation cycle of 8 days. EPS fraction was purified from the culture filtrates by gel filtration chromatography on Sepharose CL-6B. Preliminary chemical structure of EPS fraction was determined by FT-IR and GC-MS. Furthermore, the chain conformational was characterized by SEC-MALLS-RI and Ubbelohde capillary viscometer. The antioxidant activity was studied by DPPH and OH methods. Chemically, EPS was found to be consisting of a-glucopyranose and D-mannopyranose. The chain conformation study indicated EPS a kind of water-soluble polysaccharide with the moderate molecular weight (18.4 x 104) and highly stretched chain conformation.

The antioxidant activity tests suggested that the EPS have the great potential application as a natural antioxidant material in food and biomedical area. Thermal stability test revealed EPS stability at 220 AdegC. The EPS from G. polypores may be a novel source of natural antioxidants with potential as healthy food and therapeutics.

Keywords: Ginkgo polypores; Exopolysaccharide; Antioxidant; Chain conformation.

Introduction

In the twenty-first century, the usage of polysaccharides in food and medicinal industry has become an international frontier because of its low toxicity and high immunological activity to the human body [1, 2]. Among the various natural resources, polysaccharides, a class of biopolymers naturally-originated from plants or animals, as functional ingredients has been attracting great interest in recent years [3, 4]. Natural polysaccharides exhibited unique properties including good biocompatibility, stability and biodegradability, which were the basic characteristics for polymers used as food additives and medicinal materials. Extracts from fruiting body of fungi have been used as the traditional Chinese medicine (TCM) due to their antitumor and immunostimulating activities [2]. Moreover, much interest has been focused on exopolysaccharide (EPS) produced by medical fungi for their biological activities [5]. Ginkgo polypore is a polypore mushroom of the genus fomitopsis.

It is one of the most widely distributed species in the northern hemisphere. Polypores are the most important agents of wood decay and play a very important role in carbon cycle of forest ecosystem and nutrient requirement. Most of the polypores are edible or at least nontoxic [6], while some have been used in traditional medicines, such as Trametes versicolor and Ganoderma lucidum [7,8], which have been proved to have anti-tumor activity. All of fungal polysaccharides have complex chemical composition and chain conformation, and their bioactivity is related to their molecular properties and water-solubility [9]. Therefore, an understanding of the chemical and physical parameters of the polysaccharides is essential for interpretation of their bioactivities. The present study explores the suitability of a newly isolated fungus (G. polypores) for the production of EPS. The culture conditions of G. polypores were optimized to obtain the neutral EPS with good water solubility.

The chemical structure of the EPS was determined by FT-IR and gas chromatography-mass spectrometry (GC-MS). Furthermore, size exclusion chromatography combined with multiangle laser light scattering (SEC-MALLS) and viscosity measurements were employed to investigate the molecular weight and chain conformation of the EPS. The thermal stability of the EPS was also analyzed to evaluate the potential value.

Experimental

Strain and culture condition

Ginkgo polypores was collected by our laboratory of food production and safety and was used throughout this study. It was originally isolated form a mountainous district in Sichuan Province, then authenticated and preserved at the Henan Province Microbiological Culture Collection Center (HPMCC no. 427876). Stock cultures were maintained on potato dextrose agar (PDA) slants. Flask culture experiments were performed in 250 mL flasks containing 50 mL media after inoculating with 4% (v/v) of the seed culture. G. polypores was initially grown on PDA medium in a petri dish, and then transferred into the seed medium by punching out 5 mm of the agar plate culture with a self-designed cutter [10].

Purification of EPS

Samples collected from various shake flasks was centrifuged at 8000 g for 30 min. Mycelium was transferred to a dish and dried at 70 AdegC overnight to a constant weight. The supernatant was concentrated under reduced pressure, and precipitated with 3 volumes of ethanol at 4 AdegC overnight. The crude polysaccharide was recovered by centrifugation (11,000 g, 15 min). The precipitates was redissolved in distilled water and centrifuged at 11,000 g for 20 min to remove insoluble materials. The concentration of the EPS was tested for total carbohydrate by the phenol-H2SO4 method [11]. The crude EPS was further treated by Sevag method to remove most of proteins [12], then the EPS was dissolved in 0.2 M NaCl, filtered by 0.45 im membrane and loaded onto a Sepharose CL-6B column. The column was eluted with the same buffer and flow rate was settled at 0.8 ml/min. Protein concentration was monitored at 280 nm [13] and the total carbohydrate by the phenol-H2SO4 method (monitored at 490 nm) [11].

Fourier transform infrared (FT-IR) spectroscopy

The FT-IR spectrum of the EPS was recorded on a spectroscopy (Bruker Tensor 27). EPS fraction (1 mg) was prepared by using the KBr disk method to perform IR spectral measurement on a Mattson Instrument from 550 to 4,000 cm -1.

Monosaccharide composition analysis

To determine the monomer units, the purified EPS (5 mg) was hydrolyzed with 3 mL of 2 M trifluoroacetic acid (TFA) at 121 AdegC for 2 h. The hydrolysate was repeatedly co-concentrated with methanol to dryness, then silanized with N,O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA): Trimethylchlorosilane (TMCA) (99:1, 0.1mL) and pyridine (1 mL) at 80 AdegC for 2h. All the samples (including the standard sugars) were finally filtered through 0.22 im membranes and analyzed by gas chromatography (GC). GC analysis was carried out on a Trace GC Ultra-ISQ (Supelco Inc., Bellefonte, PA, USA) fitted with a fused silica capillary column (Na form, 300mmx0.25mm, Supelco Inc., Bellefonte, PA, USA) and a flame ionization detector. Several kinds of standard sugars (maltose, ribose, mannose, glucose, rhamnose, galactose, arabinose, trehalose and xylose) were prepared and subjected to GC analysis separately in the same way.

Molecular weight and chain conformation

The molecular weight and the gyration of radius of the EPS were measured on size exclusion chromatography (SEC) columns (Wyatt Technology, Santa Barbara, CA, USA) by combination with a multi-angle laser light scattering (MALLS) system (DAWN HELLEOS, equipped with a He-Ne laser, e = 663.7 nm,) and a refractive index(RI) detector (Optilab T-rEX, e = 658.0 nm). The EPS sample was dissolved in a phosphate/chloride buffer (ionic strength = 0.1, pH 6.8) containing 0.04 % diaminotetraacetic acid-disodium salt (Na2EDTA) and 0.01 % sodium azide and then filtered through a 0.25 um Millipore filter prior to injection into the SEC/MALLS system. The SEC system consisted of a degasser, a high-performance pump, an injection valve fitted with a 100 ul loop, and two SEC columns connected in series. The flow rate was set at 0.6 ml/min, and the injection volume and concentration were 100 ul and 2 mg/ml, respectively.

The values of dn/dc were determined using a refractive index (RI) detector (Optilab T-rEX, Wyatt Technology Co., Santa Barbara, CA, USA) at 658.0 nm and 25 AdegC. The dn/dc values of samples in the buffer was 0.138 mL/g. Astra software (Version 4.72, Wyatt Technology Co., Santa Barbara, CA, USA) was utilized for data acquisition and analysis. The viscosity of EPS in aqueous solution was measured at 25 AdegC by using an Ubbelohde capillary viscometer. The flow time was always beyond 120 s, then the kinetic energy correction was negligible. Huggins and Kraemer equations were used to estimate intrinsic viscosity [n] by extrapolation to infinite dilution as follows:

nsp/c = [n]+k' [n]2 c

lnnr/c = [n]-a[n]2 c

where, k' and a are constants for a given polymer under given conditions in a given solvent; nsp/c, the reduced specific viscosity; lnnr/c, inherent viscosity.

Antioxidant activity

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of EPS was assayed according to the method described previously by Zou et al [14]. Briefly, various concentrations (0.5, 1.5, 2.0, 2.5, and 3.0 mg/mL) of EPS or blank control (50% ethanol) were mixed with 2 mL of 0.1 g/L DPPH radical solution. The vortex-mixed mixture was left to stand for 1h in the dark,absorbance at 517 nm was measured using a spectrophotometer (UV-17001C, Shanghai, China). The experiment was carried out in triplicate and averaged. The DPPH radical scavenging activity (%) was calculated according to the equation below:

scavenging ability (%) =[ (1 - (Asample - Ablank)/Acontrol ] x 100,

where Acontrol is the absorbance of control without the tested samples, Ablank is the absorbance of the blank control (without DPPH) and Asample is the absorbance in the presence of the tested samples. Ascorbic acid was used as positive control in all antioxidant assays. The scavenging activity of hydrogen peroxide was measured according to the method described by Zhao et al [15]. EPS at various concentrations (0.5 - 3.0 mg/mL) was mixed with a solution containing phenanthroline (7.5 mM, 1 mL), phosphate buffer (0.02 mM, pH 7.4, 1 mL), FeSO4 (3.25 mM, 1 mL) and H2O2 (1.5%, 1 mL) at 37 AdegC for 1h. The absorbance was read at 510 nm using a spectrophotometer (UV-17001C, Shanghai, China). The experiment was carried out in triplicate and averaged. The hydrogen peroxide scavenging activity (%) of EPS was calculated by using the following equations:

scavenging rate (%) = (A sample - A blank)/(A control - A blank) x 100

where A sample, A control, and A blank were defined as absorbance of the sample, control (without EPS), and blank (without H2O2 and EPS).

Thermogravimetric analysis of EPS

Thermogravimetric analysis of the EPS was investigated in a TAQ5000IR TGA apparatus using 10 mg EPS fraction, with the heating rate of 10 AdegC min-1in the temperature range of 25 - 760 AdegC. TGA curve plot TGA signal (converted to percent weight change on the Y-axis) against the reference material temperature (on the X-axis).

Statistical analysis

All the results were expressed as means +- standard deviation (SD) of three independent experiments. The obtained data were subjected to one-way ANOVA and the differences between means were measured at the 5% probability level using Duncan's new multiple range tests.

Results and Discussion

Optimum Culture Cycle and the Effect of Carbon, Nitrogen in Shake Flask Cultures G. polypores was cultivated in the basal medium at 26 AdegC. The cultivation cycle was mapped by using the cultivate times as the abscissa, the EPS and mycelial dry weight as the ordinate. It could be seen in the Fig 1a, along with the extension of cultivated time, EPS and mycelial dry weight are increasing rapidly, the content of EPS reached a maximum of 1.0 g/L at 8 d, then fell slightly. On this basis, the optimal cultivation cycle was selected for 8d. To find the suitable carbon source and nitrogen sources for EPS production by G. polypores, various sources of carbon and nitrogen were provided at a concentration level of 30 g/L for 8 days in the basal medium. As shown in Fig 1b and 1c, where the lactose and yeast extract were chosen as optimal carbon and nitrogen source for EPS production, respectively.

Purification of EPS

As shown in Fig 2, the main single fraction (EPS) was purified on Sepharose CL-6B from the crude EPS produced under the above optimal culture conditions in shake flask. One main fraction was collected (i.e. 30th - 50th tube) and dialysis enrichment for further analysis of structure and activity.

Chemical structure of EPS

The chemical structure of the EPS was analyzed initially through the FT-IR result. As shown in Fig 3. It shows bands around 3322 cm -1, 2928 cm -1,1423 cm-1 and 990-1200 cm -1, common to all polysaccharides, representing stretching vibration of the hydroxyl group, hydroxyl stretching vibration of the -CH2 groups, a shear stretching vibration of CH2 and a stretching vibration of C-O, respectively. A characteristic absorption band appeared at 1640 cm -1 assigned to the asymmetric stretching of -COO-. The anomeric absorption peak at 890 cm-1 is typical signal for a-configuration glucan in the pyranose form. The strong characteristic absorption signal at 870 cm -1 indicated the presence of D-mannopyranose units.

To further determine the monomer units, the silanization of EPS fraction was analyzed by GC-MS. Monosaccharide composition analysis indicated that it was composed of glucose. The detailed monosaccharide composition of EPS was illustrated in Table-1. The result indicated that glucose and mannose were the major monosaccharide, which was in agreement with the result of FT-IR analysis, indicating that the main structure of EPS is a-glucopyranose and D-mannopyranose.

Table-1: Carbohydrate composition of EPS fraction produced from by G. polypores.

Monomer composition###Molar ratios(%)

###Mannose###59.3

###galactose###12.7

###glucose###28.0

Chain conformation

To clarify the chain conformation of the EPS, conformational parameters such as mol weight (Mw), intrinsic viscosity ([c]), mean-square radii of gyration (Rg), hydrodynamic radius (Rh) and the ratio of the geometric-to-hydrodynamic radii (n) for EPS in water were determined by SEC, multi-angle laser light scattering (MALLS) and viscometer at 25 AdegC, and the experimental results are summarized in Table-2. Mw can be derived from SEC data by a variety of approaches. Using a LLS photometer as a detector, Rg can be calculated from Debye, Zimm or Berry plots, depending on the conformation of the molecules in solution. When a viscometer is used, the [n] value for EPS could be obtained and the Rh can be determined by the Einstein-Simha relation[16, 17].

Vh = Mw[n]/(2.5dNA)

Rh = (3Vh/4)1/3

Vh is the hydrodynamic volume, Mw is the mol weight from LLS, [n] NA is the Avogadro's number. The Rh can be calculated from Vh and the ratio of the geometric-to-hydrodynamic radii n can be determined as follows:

n = Rg/Rh

Rg is regarded as the mean square of the distance between the segment and the mass center, whereas Rh arises in the investigation of the dynamic properties of polymers moving in a solution and it is defined as the parameter to characterize the dimension of macromolecules in a solvent taking into account the hydrodynamic interactions. The molecular shape and stiffness of polymers in dilute solution can be described from the value of n. It is well known that, for a uniform and nondraining sphere, n ~ 0.8; for a loosely connected hyperbranched chain or aggregate, n ~ 1.0; for a linear flexible random coil chain, n ~ 1.5; and for an extended rigid chain, n [greater than or equal to] 2.0 [18]. Substituting the data of Mw, and [c] into equations (3) and (4), the n value of EPS was found to be 1.71, indicating a relatively stretch of semi-rigid chain structure. Moreover, the Huggins constant (k') of the EPS in water was estimated according to Huggins and Kraemer equations (1) and (2), and the result was illustrated in Table-1.

The Huggins constant can provide an indication of the hydrodynamic interactions of the polymer with the solvent [19]. The k' usually has value roughly between 0.3 and 0.5 for a polymer in good solvents, and the k' of EPS indicating that it can be molecularly dispersed in water [20]. Therefore, aggregates hardly coexist in the solution.

Table-2: Conformational parameters of EPS produced by G. polypores.

Sample###Mwx104(g/mol)###Rg(nm)###[c](mL/g)###k'###Rh(nm)###n

EPS###18.4###34.1###274###0.39###20.0###1.71

Antioxidant Properties of the EPS

There are various types of free radicals in biological systems, but some of the most concern are derived from oxygen [21-23]. Oxidative stress is the state of imbalance between the level of antioxidant defense system and manifestation of reactive oxygen species (ROS) [24]. High oxidative stress may play an important role in the development of many human diseases [21], including cancer, aging, neurodegenerative disease, atherosclerosis and even AIDS 28[25-28]. So, it have been attracted a large number of health professionals pay attention to antioxidants because they are supposed to relieve oxidative damage caused by ROS [29]. In the large number of natural antioxidants, EPS play an important role in scavenging ROS effects [30]. To investigate the antioxidative activity of the EPS in vitro, hydroxyl, DPPH radical scavenging assays were used. As shown in Fig 4a and 4b. The scavenging effect of hydroxyl and DPPH radical exhibited a dose-dependent manner.

EPS represented obviously better activity for scavenging OH radical than the DPPH radical. The OH scavenging rate of EPS showed a steady increase significantly with the increase in the concentration of EPS, and finally reached 38% at the concentration of 4 mg/mL (Fig 4a). These results showed that the EPS may be used as potential source of antioxidants. The antioxidant activity of polysaccharides could be influenced by various factors including chemical components (functional groups), structure (monosaccharide constituent and configuration of glycosidic bond), molecular mass and chain conformation. According to FT-IR spectra for EPS fraction, the unsaturated groups, such as -CHO and -COOH, could donate electrons to decrease the ROS to a more stable form [31]. In addition to the chemical structure, molecular size and shape will affect the bioactivity [1].

It has been reported previously that only moderate molecular weight (3.0x105 ~ 8.0x105) polysaccharides has good biological activity. More stretch chain conformation will facilitate the active group to contact with ROS.

TGA of EPS

Thermal stability of EPS is very important for its applications in food and biomedical field. TGA is commonly used to determine a sample's change in mass with variation of temperature and is mainly used to investigate the thermal stability of polymers. As shown in Fig 5, the TGA analysis of EPS was carried out dynamically between weight loss and temperature. When the temperature reached to 120 AdegC, weightlessness rate of EPS was 5.0%, indicated that the polysaccharide contained adsorbed water. The degradation temperature of 220 AdegC was determined from the TGA curve. The weight of EPS was stable eventually around 400 AdegC and the final residue was 28%.

Conclusion

In this work, lactose and yeast extract were chosen respectively as optimal carbon and nitrogen sources for EPS production with cultivation cycle of 8 d. One fraction of EPS was obtained by gel filtration chromatography on Sepharose CL-6B. The component of EPS was characterized by FT-IR and GC-MS analysis, and the main structure of EPS consisted of a-glucopyranose and D-mannopyranose. Furthermore, the antioxidant capacity of EPS was determined. The EPS from G. polypores may be a novel source of natural antioxidants with the nutritional and therapeutic potential values. To better understand the bioactivity of EPS, further investigation of their bioactivity and mechanisms in vivo will be carried out in our laboratory.

Conflicts of interest

There are no conflicts of interest

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Author:Jia, Xuewei; Jiang, Yu; Chen, Zhifei; Xu, Chunping
Publication:Journal of the Chemical Society of Pakistan
Article Type:Technical report
Date:Dec 31, 2018
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