Isolation, Structural Characterization and Antioxidant ...

American Journal of Biology and Life Sciences 2015; 3(5): 168-175 Published online September 7, 2015 (http://www.openscienceonline.com/journal/ajbls)

Isolation, Structural Characterization and Antioxidant Activities of Polysaccharide from Ganoderma lucidum (Higher Basidiomycetes)

Kannan Mohan1, *, Muthusamy Padmanaban1, Venkatachalam Uthayakumar2

1PG and Research Department of Zoology, Sri Vasavi College, Erode, India 2Departmentof Zoology, School of Life Sciences, Bharathiar University, Coimbatore, India

Email address

[email protected] (K. Mohan)

To cite this article Kannan Mohan, Muthusamy Padmanaban, Venkatachalam Uthayakumar. Isolation, Structural Characterization and Antioxidant activities of

Polysaccharide from Ganoderma lucidum (Higher Basidiomycetes). American Journal of Biology and Life Sciences.

Vol. 3, No. 5, 2015, pp. 168-175.

Abstract

To study the isolation, structural characterization and antioxidant activities of polysaccharide from the medicinal mushroom G.lucidum. In this study structural characterization of Ganoderma lucidum polysaccharides was conducted by Fourier Transform Infrared spectroscopy (FT-IR) and Scanning Electron Microscopy (SEM) analyses. Antioxidant activities of Ganoderma lucidum

polysaccharides was measured by 1,1–diphenyl–2–icryl-hydrazyl (DPPH-), Hydroxyl radical scavenging assay (HO-), superoxide anion (O-) radical scavenging assay and Ferrous ion (Fe2+) chelating assay. Scavenging effect of G. lucidum

polysaccharides and ascorbic acid on the DPPH radical scavenging, Hydroxyl radical scavenging, superoxide anion radical scavenging and Ferrous ion (Fe2+) chelating radical scavenging-dependently increased and was 30.45% and 85.40%, 35.70% and 90.50%, 49.68% and 90.10%, 32.69% and 80.26% at the dose of 4 mg/ml respectively. The FT-IR spectra revealed the general characteristic absorbance peaks of the GLP. The SEM image demonstrated surface features of the GLP. This study suggests that the Ganoderma lucidum polysaccharides could potentially be used as natural antioxidants.

Keywords

Ganoderma lucidum Polysaccharide, Isolation, Structural Characterization, Antioxidant Activities

1. Introduction

Oxidation is an essential biological process to many living organisms for the production of energy. However, the uncontrolled production of oxygen-derived free radicals is hostile and damaging the cells. It can also cause a chain reaction resulting to the multiplication of new free radicals. The damage cause includes tissue loosening, genetic damage and the promotion of disease and aging. In order to reduce oxidation damage to the human, many synthetic antioxidants are widely used at present. However, recent researches suggested that synthetic antioxidants were restricted due to their potential hazards to health, such as liver damage and carcinogenesis [1]. Thus, it is essential to develop and utilize effective natural antioxidants to protect the human body from free radicals and reduce risk to many diseases such as heart diseases, cancer, arthritis and the aging process [2].

Mushrooms have become attractive as a functional food and

as a source for the development of drugs and nutraceuticals [3]. Higher basidiomycete’s mushrooms have been used in folk medicine throughout the world since ancient times [4]. Ganoderma lucidum, a medicinal fungus belonging to the Polyporaceae family, is used extensively in traditional Chinese medicine. Modern studies have revealed that Ganoderma lucidum contain a variety of bioactive ingredients, including triterpenoids, polysaccharides, sterols, fatty acids, nucleosides and alkaloids [5], and possess multiple pharmacological activities, such as antitumor [6], Immunomodulation [7,8], anti-inflammatory [9], antiviral [10], antioxidant [11], anti-aging [12] and anti-diabetic [13] effects. Due to its ability to cure many different diseases it received names like “Elixir of life”, “Food of Gods”, “Mushroom of Universe” [14, 15]. Triterpenoids and polysaccharides are two main categories of the bioactive

American Journal of Biology and Life Sciences 2015; 3(5): 168-175 169

components from G.lucidum and it has been found previously that polysaccharides exert their effect mainly through an immunomodulatory mechanism [16, 17].

Modern pharmaceutical research shows that G. lucidum

polysaccharide (GLP) has several physiological and health effects including strong antioxidant activities [18]. In addition its therapeutic effects, the Ethanolic extracts from G. lucidum

and G. tsugae also possess antioxidant abilities [19]. Various components of G. lucidum, in particular polysaccharides and triterpenoids, show high antioxidant activity in vitro [20-25]. Polysaccharides were also reported to protect the immune cells from oxidative damage [26]. Methanol extracts of G.

lucidum were reported to prevent kidney damage (induced by the anti cancer drug cisplatin) through restoration of the renal antioxidant defense system [27]. In vitro antioxidant an activity of G. lucidum polysaccharides was carried out by Jia and others using streptomycin induced diabetic rats. The results indicated G. lucidum polysaccharides could significantly and dose-dependently increase non enzymic/enzymic antioxidants and reduce lipid peroxidation [28]. Although antioxidant activities of the G. lucidum

polysaccharides (GLP) were investigated, there was a dearth of information about the physiological properties and structure of GLP. Therefore, isolation of GLP was necessary and could to better understand structural characteristics, antioxidant activity and the relationship between chemical characteristic and activity of GLP.

The objective of the present study was evaluating isolation, structural characterization and antioxidant activities (1, 1-diphenyl-2-picryl-hydrazyl (DPPH) radical-scavenging activity, Superoxide anion radical-scavenging activity, Hydroxyl radical-scavenging activity and ferrous ion chelating activity) of polysaccharide from the medicinal mushroom G. lucidum.

2. Material and Methods

2.1. Chemicals

1, 1-diphenyl-2-picryl-hydrazyl (DPPH) was purchased from sigma-Aldrich (USA). Ascorbic acid was purchased from E.MERCK (India). Stock solutions of DPPH were prepared in methanol and methanol buffered with acetic acid buffer (0.1M, pH5.5) respectively. Buffered methanol was prepared by mixing 40 ml of 0.1 M acetic acid buffer (pH5.5) with 60 ml methanol. The solvent and other chemicals were of analytical grade. The reaction tubes, in triplicate were wrapped in aluminum foil and kept at 30 °C for 30 min in dark.

2.2. Collection and Identification of

Ganoderma lucidum

Matured Ganoderma lucidum fruiting bodies were collected from July 2013 to December 2013 on Maruthamalai hills region, Bharathiar University, Coimbatore, Tamilnadu, India and authenticated by Botanical Survey of India, Coimbatore.

2.3. Isolation of Ganoderma lucidum Polysaccharides (GLPs)

Sporocarps were cut into small pieces dried at 40-50o C for 48 hand powdered. Polysaccharides were isolated by the method of [29] with slight modification. The crushed powder (100g) was removed the impurities for 24 h with 80% ethanol at room temperature. The extract was filtered and centrifuged at 7500 rpm for 30 min at room temperature. The supernatant was concentrated in a rotary evaporator under reduced pressure at 50 °C and removed free protein layer by the use of method of sevage. At last the extract was subjected to the precipitation with four fold volumes of ethanol. The crude polysaccharide was collected by centrifugation, washed with ethanol twice, and then freeze dried. The crude polysaccharides was dissolved in water and reprecipitated with equal volume of cetyl trimethal ammonium hydroxide and kept for overnight. The supernatant obtained was precipitated with chilled ethanol. After centrifugation the precipitate obtained was run thought DEAE cellulose column and eluted with deionized water. The precipitate thus obtained was lyophilized to get light brown polysaccharides (3.1g).

2.4. Fourier Transform - Infrared

Spectroscopy (FT-IR)

For FTIR analysis of GLP, 1.0 g sample were ground to get her with 20 mg potassium bromide (KBr) powder and passed into pellet. FTIR spectrum was recorded with in wave number of 4000-500 cm-1 with resolution of 4 cm-1, on a Perkin Elmer-Spectrum RX1 instrument.

2.5. Scanning Electron Microscopic (SEM)

Analysis of GLP

Scanning Electron Microscopic (SEM) analysis was done in Karunya University, Coimbatore, Tamilnadu, India. After the preparation of the Polysaccharides, the suspension of the particles in water was used for SEM analysis by fabricating a drop of suspension on to a clean electric Stubs and allowing water to completely evaporate and allowed to dry by putting it under a mercury lamp for 5 min. SEM observations were carried out on a Hitachi S-4500 SEM machine.

2.6. Antioxidant Activities of Ganoderma lucidum Polysaccharides

2.6.1. 1-Diphenyl-2-Picryl-Hydrazyl (DPPH)

Radical - Scavenging Activity

The free radical scavenging activity of the G. lucidum

polysaccharide was measured by 1-diphenyl-2-picryl-hydrazyl (DPPH) test according to the method of [30], with some modifications. 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) is a stable free radical is the radical which has an unpaired valence electron at one atom of Nitrogen Bridge [31]. Scavenging of DPPH radical is the basis of the popular DPPH antioxidant assay [32]. 100 ml of a DPPH radical’s solution in ethanol (60ml) was mixed with 100 µL of sample (0.10, 0.20, 0.40, 0.8, 1.4, 3.2, and 4.0

170 Kannan Mohan et al.: Isolation, Structural Characterization and Antioxidant Activities of Polysaccharide from

Ganoderma lucidum (Higher Basidiomycetes)

mg/mL) solution in ethanol (0.5-2 µg/ml). A control, containing 100 ml of DPPH solution and 60ml of ethanol was prepared. The mixture was incubated at room temperature for 30 min and then the absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity, which was analyzed from the graph plotted of inhibition percentage against compound concentration. Ascorbic acid was used as positive controls. The experiment was carried out in triplicate and averaged. The capability to scavenge the DPPH radical was calculated using the following equation,

Scavenging effect of DPPH (%) = [(A0-A1) / A0] X 100

Where A0 was the absorbance of the control and A1 was the absorbance of the mixture containing extracts IC50 of reference antioxidant compounds.

2.6.2. Superoxide Anion Radical - Scavenging

Activity

Superoxide anion radical-scavenging activity was measured by a non-enzymatic method [33] modified slightly [34]. 0.025 ml of sample solutions with various concentrations (0.10, 0.20, 0.40, 0.8, 1.4, 3.2, and 4.0 mg/mL) was treated with 0.1ml of 25 mM phosphate buffer (pH7.2), 2mM NADH (0.025 ml) and 0.5 mM NBT (0.025 ml), and absorbance at 560 nm was measured as a blank value. After a 10 min incubation at ambient temperature with 0.025 ml of 0.03 mM PMS, the absorbance was a gain measured. Ascorbic acid was used as the positive control.

2.6.3. Hydroxyl Radical-Scavenging Activity

Hydroxyl radical-scavenging Activities of polysaccharide from G. lucidum were determined according to previous method with slightly modification [35]. 0.5 ml of sample solutions with various concentrations (0.10, 0.20, 0.40, 0.8, 1.4, 3.2, and 4.0 mg/mL) were mixed with 1.5 ml of 2.0 mmol/LFeSO4, then 1.5 ml of 6.0 mmol/LH2O2 and 1.5 ml of 6.0 mmol/L sodium salicylate were added. The mixture was incubated at 37 °C water bath for 30 min. The absorbance of the mixture was measured at 510 nm after cooling to the room temperature. Ascorbic acid was used as the positive control.

2.6.4. Ferrous Ion Chelating Activity

Ferrous ion chelating activity was estimated by the decrease in the maximal absorbance of the iron-ferrozine complex [36]. Briefly, 1 ml of sample solutions with various concentrations (0.10, 0.20, 0.40, 0.8, 1.4, 3.2, and 4.0 mg/mL) were mixed with 100 µl of 2.0 mmol/L FeCl2.4H2O solution and 3.7 ml distilled water. The reaction was initiated by the addition of 200 µl of 5.0 mmol/L ferrozine and after the mixture had reached equilibrium (20 min), the absorbance of the reaction mixture was measured at 562 nm using a spectrophotometer. Ascorbic acid was used as the positive control.

2.7. Statistical Analysis

All experiments were done in triplicate, and mean values are presented. The results were expressed as mean±SD values.

Statistical analyses were performed using SPSS version 16.0. Means were statistically analyzed using analysis of variance (ANOVA). P values < 0.05 were regarded as being statistically significant.

3. Results

3.1. Fourier Transform-Infrared Spectroscopy

(FT-IR)

FTIR spectroscopy is a powerful technique for the identification of characteristic organic group in the polysaccharide. A typical FT-IR spectrum of GLP is presented in (Figure 1). Various absorption bands within the 4000-400 cm range were recorded in the FT-IR spectra of polysaccharide, prepared from Ganoderma lucidum. The FT-IR shows GLPs representative bands at 3398.89, 2924.88, 2853.70, 1647.18, 1554.25, 1456.89, 1383.82, 1316.17, 1243.79, 1154.42, 1043.46 and 566.86 cm-1. The absorbance bands at 3398 cm-1 represented the stretching vibration of O-H in the constituent sugar residues. The strong band at around 1647 cm-1 and the small band at around 2853 cm-1 were associated with stretching vibration of C-H in the sugar ring. The 1500- 800 cm-1 region usually allows the identification of major chemical groups in polysaccharides. The position and intensity of the bands are specific for every polysaccharide [37]. The broader band of 1156 cm-1 was representative of C-O-C and –OH in pyran structure. The small absorption bands at about 565 cm-1 in the spectrum could be associated with, β - glycosidic linkages between the sugar units. As a result, it can be GLP belongs to a β- type polysaccharide with pyran group.

3.2. Scanning Electron Microscopic (SEM)

Analysis of GLP

The SEM images of the G. lucidum polysaccharides are shown in (Figure 2). Fig. A shows that GLP have a flat surface. Fig. B shows that GLPs had a rough surface with characteristic large wrinkle, and that its surface was very distinct pore openings 2–5 µm in diameter. In summary, the G.

lucidum polysaccharides extracted by the same kind of methods were qualitatively identified by comparing their micrographs with those of the standards.

3.3. Antioxidant Activities of Ganoderma lucidum Polysaccharides

3.3.1. Scavenging Ability on DPPH

DPPH is a free radical compound that has been widely used to determine the free radical- scavenging ability of various samples [38]. The color of the DPPH radical solution becomes lighter and its absorbance goes down in the presence of an antioxidant compound [39]. Figure 3 illustrates that the scavenging effect of G. lucidum polysaccharides and ascorbic acid on the DPPH radical concentration- dependently increased and were 30.45% and 85.40% at the dose of 4mg/ml respectively.


3.3.2. Superoxide Anion Radical Scavenging

Activity

The superoxide radical (O2-) is a highly toxic species could

be generated by numerous biological and photochemical reactions. In addition directly attack important biological molecules, O2

- may also decompose to form single oxygen and hydroxyl radicals, which may increase local oxidative stress and initiate cellular damage or lipid peroxidation and pathological incidents such as arthritis and Alzheimer’s

diseases [40]. In the present study, we investigated the scavenging capacity of G. lucidum polysaccharides against the superoxide anion free radicals. As illustrated in Figure 4, the superoxide anion radical scavenging effects of the G.

lucidum polysaccharides increased with increasing concentrations. At a concentration of 4mg/ml, the scavenging activities were 35.70% and 90.50% for the GLP and ascorbic acid respectively. The GLP extract had the highest scavenging activity, which was higher than that of Ascorbic acid.

Fig. 1. Fourier Transform-Infrared Spectroscopy (FT-IR) analysis of G. lucidum polysaccharides.

Fig. 2. Scanning electron microscope analysis of G. lucidum polysaccharide extract (A) GLPs (2500x) (B) GLPs (10000x).

SAMPLE NAME : FT-3493 FT-IR SPECTRUM Date: 1/30/2014

4000.0 3000 2000 1500 1000 400.0

35.036

38

40

42

44

46

48

50

52

54

56

58

60

62

64

65.8

cm-1

%T

3398.89

2924.88

2853.70

1647.18

1554.25

1456.89

1383.82

1316.171243.79

1154.42

1043.46

566.86



Fig. 3. DPPH radical scavenging activity of G. lucidum polysaccharides, Results are expressed as means ± SD (n=3).

Fig. 4. Superoxide anion radical scavenging activity of G. lucidum polysaccharides, Results are expressed as means ± SD (n=3).

Fig. 5. Hydroxyl radical scavenging activity of G. lucidum polysaccharides, Results are expressed as means ± SD (n=3).


Fig. 6. Ferrous ion chelating activity of G. lucidum polysaccharides, Results are expressed as means ± SD (n=3).

3.3.3. Hydroxyl Radical - Scavenging Activity

The hydroxyl radical is one of representative reactive oxygen species generated in the body. Hydroxyl radical can easily cross cell membranes, and can readily react with biomolecules including carbohydrates, proteins, lipids and DNA in cells and cause tissue damage or cell death. Thus, removing of hydroxyl radical is important for the protection of living systems [41]. The results of hydroxyl radical scavenging activities of the GLP and ascorbic acid were given in Fig 5 However, the scavenging activities of the GLP were higher than that of ascorbic acid (P<0.05). At a concentration of 4mg/ml, the scavenging activities were 49.68% and 90.10% for the GLP and ascorbic acid respectively.

3.3.4. Ferrous Ion Chelating Activity

Metal chelating capacity is important since it reduces the concentration of transition metals that may act as catalysts to generate the first few radicals and initiate the radical mediated oxidative chain reaction in biological systems. In the present study, the G. lucidum polysaccharides were compared to ascorbic acid for their Fe2+ chelating capacity. The antioxidant capacity of GLP were shown in Fig and compared with ascorbic acid as a positive control. The antioxidant capacities of GLP were higher than that ascorbic acid (P<0.05). At the concentration of 4mg/ml, the scavenging activities were 32.69 % and 80.26% for the GLP ascorbic acid respectively (Figure 6). The lower absorbance indicates a higher chelating power.

4. Discussion

FT-IR spectra were obtained to investigate the molecular properties of mushroom polysaccharides, and to evaluate correlations between their structural characteristics and bioactivity. As shown in Fig. 1, all mushroom extract spectra showed typical carbohydrate patterns, with strong and broad absorption near 3398 cm-1 which indicates the presence of OH groups. The weak peaks at 2922cm-1 are interpreted to be due

to CH2 stretching and bending vibrations [42]. Carbonyl groups (C=O) show two bands: an asymmetrical stretching band around 1650 cm-1 and a weak symmetric stretching band near1400 cm-1 [43]. The structural characteristics of the polysaccharides, such as molecular weight, monosaccharide composition, configuration and type of glycosidic linkage, were found to modulate and affect their antioxidant activities [44].

The DPPH radical scavenging assay is a widely accepted model to assess free radical-scavenging activity [45]. The ability of antioxidants to scavenge DPPH is attributed to their hydrogen donating activity [46]. The IC50, meaning the concentration of antioxidant needed to decrease [by 50%] the initial substrate concentration, is a parameter widely used to measure the antiradical efficiency. The lower IC50 values show the higher antioxidant activity [47]. G. lucidum polysaccharides exhibited a relatively high level of radical scavenging activity. The reason for this could be that the Ganoderma lucidum polysaccharides were rich in antioxidant components, such as proteins, amino acids, peptides, phytosterols, ascorbic acid and microelements, which contributed to their antioxidant properties. Lin et al, found that methanolic extracts from other medicinal mushrooms were extremely effective in inhibiting the lipid peroxidation [6.41% for Ganoderma lucidum, 2.62% for Ganoderma lucidum and 2.30% for Ganoderma tsugae at 0.6 mg/mL [48]. The result suggested that the G. lucidum polysaccharide is a good scavenger for DPPH radicals.

It has been suggested that the overall radical scavenging ability was related to the number of hydroxyl or amino groups in a polysaccharides molecule such as Chitosan [49]. It has been reported that the polysaccharide extracts of G. lucidum

and Grifola umbellata possess superoxide radical scavenging activity [50]. The activities of antioxidant have been attributed to various mechanisms, such as prevention of chain initiation, binding transition metal ion catalysts, decomposition of peroxides, reductive capacity and radical scavenging [51]. Since ferrous ion are the most effective pro-oxidants the food system [52], the high ferrous ion chelating abilities of



polysaccharides from G.lucidum could added to food quality. That result demonstrated that the GLP showed the strong antioxidant activities.

5. Conclusion

This study demonstrates the isolation, structural characterization and Antioxidant activities of G. lucidum

polysaccharides. Our results suggest that mushroom Ganoderma lucidum polysaccharide is a very good source of naturally-derived antioxidant and potential functional food ingredient. It could also employ to combat several diseases caused by pathogenic microorganisms.

Acknowledgements

We extend our sincere thanks to University Grant Commission for funding this project work (Grant No: MRP-5540/15SERO/UGC).

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