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ISSN: 1948-593X
Journal of Bioanalysis & Biomedicine
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Optimized Extraction and Antioxidant Activities of Polysaccharides from Two Entomogenous Fungi

Sapan Kumar Sharma*

Centre for Mushroom Research and Training, Department of Plant Pathology, CSK, Himachal Pradesh Agriculture University, India

*Corresponding Author:
Sapan Kumar Sharma
Centre for Mushroom
Research and Training, Department of Plant Pathology
CSK, Himachal Pradesh Agriculture University
Palampur-176062, India
Tel: +91 9857-481-650
E-mail: [email protected]

Received Date: April 14, 2014; Accepted Date: November 10, 2015; Published Date: November 13, 2015

Citation: Sharma SK (2015) Optimized Extraction and Antioxidant Activities of Polysaccharides from Two Entomogenous Fungi. J Bioanal Biomed 7: 180-187. doi: 10.4172/1948-593X.1000141

Copyright: © 2015 Sharma SK. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Submerged cultures of two entomogenous fungi Isaria sinclairii and I. tenuipes have been optimized for polysaccharides production and their biological activities have been evaluated under present studies with using one- factor-at-a-time method. Combination of various factors influenced the higher polysaccharides production in both of the fungi. Polysaccharides production was influenced by different carbon, nitrogen and mineral sources. In vitro evaluation of EPS and IPS of both species showed significant antioxidant activities and high inhibition rate of peroxidation of polyunsaturated fatty acids.

Keywords

Isaria sinclairii; I. tenuipes; EPS; IPS; Antioxidants

Introduction

Genus Isaria belongs to phylum Ascomycota and class Sordariomycetes with worldwide distribution [1-3]. Many Ascomycetes species have been used as the source of disease combating natural products with tremendous biological, pharmacological and immunomodulatory activities [4-10]. Cordyceps militaris, C. sinensis, C. ophioglossoides etc. have been found to contain abundant kind of active compounds and extensively used in East Asia for treatment of anti- inflammation, renal dysfunction, asthma, and cancer [11-14]. Biological active compounds extracted from many species of entomopathogenic genera have shown far reaching liver protective effects, anti-oxidative activity, enhancing the T-cell and macrophages activity, vascular endothelial growth factor levels in the lungs and liver by exopolysaccharide fraction, reduction in cholesterol and triglyceride [15-19].

Polysaccharides extracted from the mycelium of these fungi constitute the main bioactive agents and exhibit multiple pharmacological activities including antitumor, anti- inflammatory, immunopotentiation, hypoglycemic and hypocholesterolemic effects, protection of neuronal cells against the free radical- induced cellular toxicity, steroidogenesis and antioxidant activities [20-26]. High antioxidant activities provide health benefits in preventing damages due to free radicals produced by biological degeneration [27-31]. Polysaccharides protect neuronal cells against the free radical-induced cellular toxicity and stimulate steroidogenesis. Polysaccharides possess great potential and considered as important tool for studying the development of nutraceutical products. However, under submerged culture conditions, the productivity of polysaccharides has been observed to vary with environmental conditions (medium composition, carbon source, nitrogen source, pH, etc.). Commercial cultivation through submerged culture is becoming quite useful nowadays because of higher mycelial yield with fewer chances of contamination [32,33]. In view of this, present studies have been conducted to optimize the EPS and IPS production by one-factor-at-a-time method and orthogonal matrix design, and to evaluate the antioxidant activities of EPS and IPS obtained under submerged culture conditions.

Materials and Method

Culturing and optimization studies

Isaria sinclairii and I. tenuipes were collected on Hymenopteran and Coleopteran insect respectively from Mcleodganj, Dharamshala (Himachal Pradesh, India, 2200 m above mean sea level) and were deposited at Herbarium, Department of Botany, Punjabi University, Patiala (I. sinclairii PUN 7074; I. tenuipes PUN 7073). Isolation was done on Potato Dextrose Agar (PDA) slants by tissue culture technique (Figure 1). The slants were incubated at 25°C for 10 days. Cultures were maintained by repeated sub-culturing after 30 days. Submerged cultivation was done in a standard medium (sucrose 30.0 g/L, yeast powder 5.0 g/L, peptone 5.0 g/L, MgSO4·7H2O 1.0 g/L and KH2PO4 0.5 g/L) [34,35]. Effect of medium capacity (50, 100, 150, 200 and 250 mL), rotation speed (50, 100, 125, 150, 175 and 200 rpm) of culture medium, incubation time (2-10 days), pH (3.0-8.0), temperature (20, 22, 25, 27, 30, 33°C), carbon sources (glucose, galactose, sucrose, mannitol, maltose and fructose), nitrogen sources (peptone, yeast extract, NaNO2 (NH4)2SO4 and L-Arginine HCL) and C/N (1:5, 1:10, 1:20, 1:30 and 1:40) ratio was studied by orthogonal experiments using one-factorat- a-time method were tested for maximum EPS and IPS production.

bioanalysis-biomedicine-wild-fruit-bodies

Figure 1: Wild fruit bodies of Isaria sinclairii and I. tenuipes.

Extraction of EPS and IPS

EPS were extracted by the method described by Fang and Zhong [36] with minor modifications. Briefly, mycelial biomass in the medium was centrifuged at 10,000 × g for 12 min. The supernatant obtained was mixed with three volumes of pure ethanol and left for 24 hours at 4°C. The resulting precipitate was then separated by centrifugation at 8000 × g for 12 min. The precipitate (EPS) was washed with ultrapure water and subsequently lyophilized for quantitative assessment and analysis.

For IPS, mycelial biomass was subjected to extraction with boiling water for an hour and the mixture was filtered through Whatman no. 1 filter paper. The filtrate was allowed to precipitate using pure ethanol and left overnight at 4°C. The polysaccharides thus precipitated were separated by centrifugation at 8000 × g for 10 min. The precipitate (IPS) were washed with ultrapure water and subsequently lyophilized for quantitative analysis [37].

Polysaccharides composition

Monosaccharide composition of polysaccharides was determined by high performance liquid chromatography coupled to an evaporative light scattering detector [38]. Polysaccharide fraction (0.1 g) was extracted with 2.5 ml of 70% aqueous methanol followed by 1.5 ml of 70% aqueous methanol and then 1 ml of 70% aqueous methanol. This extract was centrifuged at 4000 rpm at 4°C for 10 min. Supernatant was collected and volume made up to 5 ml with 70% methanol. The extract was passed through Millipore filter (0.45 μm) prior to injection on the HPLC.

Antioxidant assays

DPPH radical scavenging activity: The DPPH scavenging activity was measured following standard method [39]. For this, DPPH (200 μm) solution at different concentrations (2-10 mg/mL) was added to 0.05 mL of the samples dissolved in ethanol. An equal amount of ethanol was added to the control. Ascorbic acid was used as the control. The absorbance was read after 20 min. at 517 nm and the inhibition was calculated using the formula:

DPPH scavenging effect (%)=A0-AP/A0 × 100, where A0 was the absorbance of the control and AP was the absorbance in the presence of the sample.

ABTS radical scavenging assay: ABTS radical scavenging activity was measured by the method described by Li et al. [40]. For this, 10 μL of the sample was added to 4 mL of the diluted ABTS•+ solution (prepared by adding 7 mM of the ABTS stock solution to 2.45 mM potassium persulfate, kept in the dark, at room temperature, for 12-16 h before use). The solution was then diluted with 5 mM phosphatebuffered saline (pH 7.4) to an absorbance at 730 nm. The absorbance was measured at 30 min. Ascorbic acid was used as control. The ABTS radical-scavenging activity was calculated as = (Acontrol-Asample/ Acontrol) × 100.

Reducing power: Reducing power was estimated by standard method given by Papuc et al. [41]. Briefly, samples (200 μL) were mixed with sodium phosphate buffer (pH 6.6), 1 mM FeSO4, and 1% potassium ferricyanide and incubated for 20 min at 50°C after that trichloroacetic acid was added and the mixtures were centrifuged. Supernatant (2.5 mL) was mixed with an equal volume of water and 0.5 mL 0.1% FeCl3. The absorbance was measured at 700 nm.

Ferrous ion chelating assay: For this, 1 mL of the sample (2-10 mg/mL) was mixed with 3.7 mL of ultrapure water, following which the mixture was reacted with ferrous chloride (2 mmol/L, 0.1 mL) and ferrozine (5 mmol/L, 0.2 mL) for 20 min. and the absorbance was read at 562 nm. EDTA was used as positive control. The chelating activity on the ferrous ion was calculated using the formula: chelating activity (%)=[(Ab-As)/Ab] × 100, where Ab is the absorbance of the blank and As is the absorbance in the presence of the extract [42].

Scavenging ability on superoxide anion radicals: The scavenging activity of superoxide anion radicals was measured with standard method with minor modifications [43]. A tube containing polysaccharide sample (0-2.0 mg/mL, 1 mL) and Tris-HCl buffer (50.0 mM, pH 8.2, 3 mL) was incubated in a water bath at 25°C for 20 min and after this pyrogallic acid (5.0 mM, 0.4 mL) was added. HCl solution (8.0 M, 0.1 mL) was added to terminate the reaction after 4 min. The absorbance of the mixture was measured at 320 nm.

The scavenging ability of superoxide anion radicals was calculated using the following formula: scavenging ability (%)=(1–A sample/A control) × 100, where A control is the absorbance of control without the polysaccharide sample, and A sample is the absorbance in the presence of the polysaccharide sample.

Ferric reducing antioxidant power (FRAP) assay: The Ferric reducing antioxidant power was determined by standard method [44,45]. For this, FRAP reagent was prepared by mixing TPTZ (tripyridyltriazine) (2.5 mL, 10 mM in 40 mM HCl), 25 mL of 300 mM acetate buffer, and 2.5 mL of FeCl3.H2O (20 mM). Freshly prepared FRAP reagent (1.8 mL) was taken in a test tube and incubated at 30°C in water bath for 10 minutes. Then, absorbance was taken at 0 min (t0). Immediately, 100 μl of sample extract or standard and 100 μl of distilled water was added to the test tube, mixed and incubated at 30°C for 30 minutes. Then, the absorbance was taken at 593 nm (t30). Ferrous sulphate was used as standard. The antioxidant potential of the sample extract was determined against a standard curve of ferrous sulphate and the FRAP value was expressed as μM Fe2+ equivalents per gram of extract and calculated using the following equation:

FRAP value=Absorbance (sample+FRAP reagent) –Absorbance (FRAP reagent)

Inhibition rate of peroxidation of polyunsaturated fatty acid from lipoprotein: Inhibition rate of polyunsaturated fatty acid (PUFA) peroxidation was determined following a standard method described by Zhang et al. [46]. Briefly, yolk suspension was prepared with addition of fresh egg yolk to 0.1 M pH 7.4 phosphate buffers. Yolk suspension (0.2 mL) was mixed with 0.1 mL different concentration of polysaccharides (1-10 mg/mL). After that, 25 mM FeCl2 (0.2 mL) and phosphate buffer (1.5 mL, 0.1 M, pH 7.4) were added to the mixture and incubated at 37°C for 15 min with continuous shaking. After this, TCA 20% (0.5 mL) was added to the mixture and centrifuged at 5,000 × g for 10 min. Supernatant (2 mL) was added with 1 mL 0.8% thiobarbituric acid, and the solution was incubated at 75°C for 10 min. The absorbance was determined at room temperature at 532 nm. The inhibition rate was calculated using the following formula: Inhibition rate %= [controlsample] / control.

Experimental design

One-factor-at-a-time: In each experiment, one factor was varied, while all other factors were holding constant. Different carbon sources, nitrogen, mineral sources and different conditions were initially studied by single factor experiments in both of the species.

Orthogonal matrix method: To investigate the relationships between variables of medium components and optimize their concentrations for EPS and IPS production, the orthogonal matrix experimental design L9 (34) method was used.

Chemicals

All the chemicals were purchased from Sigma Aldrich and all other unlabelled chemicals and reagents were of analytical grade.

Results and Discussion

Optimization of submerged culture conditions for polysaccharides production

To find the suitable medium capacities for maximum EPS and IPS production, Isaria sinclairii and I. tenuipes were grown in the media with different capacities. The maximum EPS (544.40 ± 1.76 mg/L) and IPS (340.12 ± 3.67 mg/L) production was observed in 150 mL of the medium, while least values of EPS (229.11 ± 1.40 mg/L) and IPS (202.10 ± 2.42 mg/L) production were observed in 50 mL of the medium. Similarly, submerged culture of I. tenuipes resulted maximum EPS (359.62 ± 1.81 mg/L) and IPS (164.20 ± 2.01 mg/L) production in the 150 mL medium and least values of EPS (220.11 ± 2.12 mg/L) and IPS (127.13 ± 1.49 mg/mL) were observed in 50 mL of the medium (Table 1). However, no significant difference (p<0.05%) was observed in IPS production in the medium capacities from 150-200 mL in I. tenuipes. Variation in rotation speed in submerged cultures of Isaria sinclairii and I. tenuipes showed significant effect on EPS and IPS production. Submerged culture of I. sinclairii resulted maximum EPS (521.28 ± 1.18 mg/L) and IPS (321.16 ± 2.72 mg/L) production with rotation speed 150 rpm. Similar was observed in I. tenuipes with EPS (278.34 ± 1.10 mg/L) and IPS (174.21 ± 2.62) yield. Medium capacities showed significant effect on EPS and IPS production. Previous studies also revealed t he effect of medium capacities on EPS and IPS production and has been studied in other entomogenous fungi viz., I. farinosa, Cordyceps ophioglossoides and other ascomycetes [34,47].

Sources I. sinclairii I. tenuipes
Medium capacity/ml EPS IPS EPS IPS
50 229.11 ± 1.40b 202.10 ± 2.42b 220.11 ± 2.12b 127.13 ± 1.49a
100 292.18 ± 3.30b 244.14 ± 4.34b 258.12 ± 3.47b 158.13 ± 1.40a
150 544.40 ± 1.76e 340.12 ± 3.67c 359.62 ± 1.81b 164.20 ± 2.01a
200 453.19 ± 2.10d 254.18 ± 4.12b 342.14 ± 345c 161.62 ± 2.12a
250 393.20 ± 1.40c 243.22 ± 4.47b 242.23 ± 2.49a 149.13 ± 3.41a
Rotation speed (rpm)        
50 253.19 ± 2.15b 203.11 ± 2.26b 212.19 ± 2.32a 112.10 ± 1.51a
100 214.10 ± 4.74b 214.13 ± 3.22b 242.11 ± 2.13a 144.26 ± 4.06a
125 241.31 ± 3.26b 241.13 ± 4.22b 250.56 ± 2.21a 159.20 ± 3.22a
150 521.28 ± 1.18e 321.16 ± 2.72c 278.34 ± 1.10b 174.21 ± 2.62b
175 414.36 ± 3.19d 309.33 ± 1.40c 267.10 ± 3.12b 166.12 ± 2.16b
200 402.11 ± 4.14d 285.21 ± 5.20b 255.10 ± 3.15a 152.13 ± 3.04a
Incubation time/d        
2 195.16 ± 2.17a 90.00 ± 1.10a 169.17 ± 2.19a 63.11 ± 1.42a
3 256.35 ± 3.19b 163.12 ± 2.20a 208.11 ± 3.11b 108.21 ± 1.29a
4 328.45 ± 2.23c 182.12 ± 1.32a 216.13 ± 4.15b 119.10 ± 2.10a
5 377.19 ± 1.11c 187.19 ± 1.11a 253.64 ± 3.17b 127.42 ± 1.71a
6 443.18 ± 3.14d 194.04 ± 1.27a 352.34 ± 2.20c 166.15 ± 1.21a
7 437.12 ± 2.91d 167.42 ± 1.11a 256.51 ± 3.50b 154.12 ± 2.52a
8 372.48 ± 3.71c 142.25 ± 1.12a 248.91 ± 5.15b 147.10 ± 1.16a
9 359.64 ± 4.30c 140.14 ± 1.37a 234.17 ± 2.16b 131.20 ± 1.83a
10 214.39 ± 1.92b 104.32 ± 2.55a 226.51 ± 2.15b 120.12 ± 1.22a
pH        
3.0 261.10 ± 2.10c 131.12 ± 1.17c 205.15 ± 2.44a 102.65 ± 1.19a
4.0 298.14 ± 3.00c 148.22 ± 1.20c 244.22 ± 2.11a 109.06 ± 1.71a
5.0 365.35 ± 4.21c 175.31 ± 2.25c 264.34 ± 4.12a 135.91 ± 1.94a
6.0 385.48 ± 3.20c 185.10 ± 1.27c 242.54 ± 2.31b 110.21 ± 1.52b
7.0 343.61 ± 3.12c 153.19 ± 2.32c 212.21 ± 4.35a 109.39 ± 1.62a
8.0 262.37 ± 2.17b 102.19 ± 1.72b 182.14 ± 2.76a 80.11 ± 2.85a
Temperature        
20 253.39 ± 2.28b 153.30 ± 1.29a 220.12 ± 2.10b 125.11 ± 1.12a
22 329.11 ± 2.18c 215.16 ± 1.22b 265.25 ± 2.33b 137.10 ± 1.76a
25 392.19 ± 2.20c 222.11 ± 1.21b 238.27 ± 2.10a 120.41 ± 3.12a
27 325.27 ± 3.20c 215.18 ± 2.27b 221.10 ± 4.40a 119.19 ± 2.42a
30 203.52 ± 3.12b 237.13 ± 2.12b 212.20 ± 2.80a 116.21 ± 1.81a
33 118.15 ± 3.10a 108.16 ± 2.11a 63.19 ± 1.32a 028.19 ± 1.32a

Table 1: Effect of different factors on IPS production (mg/L) in submerged culture of Isaria sinclairii and I. tenuipes.

Similar observations were made in Cordyceps ophioglossoides, in which maximum values for IPS production were obtained in the flasks containing 150 mL of medium and 150 rpm rotation speed [34]. Incubation time and pH range showed significant effect on EPS and IPS production in both I. sinclairii and I. tenuipes under submerged culture conditions. I. sinclairii culture incubated for 7 days showed high EPS (437.12 ± 2.91 mg/L) and IPS (167.42 ± 1.11 mg/L) production. Under submerged conditions I. sinclairii resulted maximum EPS (385.48 ± 3.20 mg/L) and IPS (185.10 ± 1.27 mg/L) production was observed at pH 6.0 (Table 1). However, submerged culture of I. tenuipes showed maximum EPS (352.34 ± 2.20 mg/L) and IPS (166.15 ± 1.21 mg/L) production at incubation time of 6 days. Maximum EPS and IPS production was observed at pH 5.0 for this species (Table 1). Temperature 25°C and 22°C were observed ideal for EPS and IPS production in I. sincalirii and I. tenuipes respectively (Table 1). Present findings are in conformity with the results obtained in medicinal insect hosting fungi in which incubation period of 5-6 days and slightly acidic pH 5.0-6.0 promoted maximum IPS production [34,48]. The best temperature for polysaccharide production in Cordyceps sinensis as observed as 20°C and 25°C for C. ophioglossoides [49].

Eight different carbon sources were studied to find the suitable medium source for the production of EPS and IPS in I. sinclairii and I. tenuipes. Although, all the tested carbon sources yielded EPS and IPS in both of entomogenous species, but maximum EPS and IPS production took place in the medium supplemented with glucose as carbon source. The results are same as obtained for many entomogenous species of genus Cordyceps Fr. species, as glucose was found to be the most favourable carbon source for polysaccharide production [50,51]. To find the best nitrogen source, six different nitrogen sources were selected. Amongst them, peptone yielded maximum EPS (379.10 ± 2.35 mg/L) and IPS (244.18 ± 1.30 mg/L) in I. sinclairii. I. tenuipes yielded maximum EPS (274.15 ± 3.45 mg/L) and IPS (214.10 ± 1.41 mg/L) in the medium supplemented with yeast extract as nitrogen source. Previous studies have shown that supplementation of medium with nitrogen sources supported the production of polysaccharides under submerged culture conditions. Present results are in conformity with previous reports on Cordyceps and its anamorphic species [49]. Five different mineral sources were studied for EPS and IPS production. NaH2PO4 + MgSO4 supported maximum EPS (374.86 ± 3.36 mg/L) and IPS production (279.80 ± 1.22 mg/L) in I. sinclairii whereas NaH2PO4 + K2HPO4 resulted maximum EPS (312.67 ± 1.21 mg/L) and IPS (256.40 ± 2.37 mg/L) in I. tenuipes. C/N ratio 10:1 promoted maximum EPS and IPS production in both species (Table 2). Similar results have been obtained for other entomogenous species like Cordyceps ophiogllosoides, as C/N ratio 10:1 provided maximum IPS (653.79 ± 5.24 mg/L) production [27]. Orthogonal experiments for the different factors on the yield of EPS and IPS showed a significant effect in both of the species. Results revealed the effect on EPS and IPS production in the order as: temperature>incubation time>pH>rotary speed>medium capacity (Tables 3 and 4).

Factors   I. sinclarii I. tenuipes
  EPS (mg/L) IPS (mg/L) EPS IPS (mg/L)
Carbon sources        
Sucrose 365.22 ± 1.14c 234.23 ± 3.10b 296.42 ± 1.48b 246.42 ± 2.37b
Mannitol 301.17 ± 3.15c 221.16 ± 2.18b 246.18 ± 5.17b 188.42 ± 2.12a
Galactose 287.10 ± 2.21b 207.12 ± 3.29b 213.10 ± 3.39b 173.60 ± 1.34a
Starch 211.20 ± 1.75b 201.22 ± 2.57b 235.32 ± 1.10b 188.37 ± 2.11a
Glucose 337.14 ± 1.37c 267.19 ± 2.30b 318.12 ± 2.32b 208.15 ± 3.17b
Maltose 242.11 ± 1.62b 202.10 ± 3.74b 157.52 ± 1.18a 157.32 ± 2.19a
Fructose 252.11 ± 2.19b 212.11 ± 3.80b 214.47 ± 3.33a 119.34 ± 2.92a
Lactose 172.46 ± 2.94a 76.12 ± 3.17a 174.10 ± 3.27a 78.10 ± 3.27a
Nitrogen source        
Yeast Extract 322.89 ± 2.11c 225.72 ± 1.71b 274.15 ± 3.45b 214.10 ± 1.41b
Peptone 379.10 ± 2.35c 244.18 ± 1.30b 263.32 ± 2.63b 201.19 ± 1.60b
NaNO2 273.32 ± 2.76b 204.90 ± 3.15b 197.23 ± 2.43b 149.11 ± 2.70a
(NH4)2SO4 224.33 ± 5.62b 194.11 ± 1.17a 194.52 ± 5.63a 124.12 ± 1.60a
L – Arginine HCL 209.10 ± 3.15b 164.17 ± 2.42a 187.24 ± 4.45a 107.19 ± 2.46a
DL – Ascorbic Acid 263.70 ± 2.16b 203.11 ± 1.19b 193.15 ± 2.26a 103.12 ± 2.21a
Mineral Sources        
NaH2PO4+CaCl2 317.14 ± 6.11c 212.19 ± 2.21b 199.10 ± 6.17a 169.61 ± 2.47a
NaH2PO4+K2HPO4 341.14 ± 2.45c 211.25 ± 1.15b 312.67 ± 1.21c 256.40 ± 2.37b
NaH2PO4+MgSO4 374.86 ± 3.36c 279.80 ± 1.22b 278.18 ± 3.63b 248.22 ± 2.62b
KH2PO4+CaCl2 334.89 ± 2.68c 233.71 ± 2.22b 245.37 ± 2.17b 157.46 ± 1.46a
KH2PO4+MgSO4 327.18 ± 3.17c 217.11 ± 1.19b 237.53 ± 4.23b 137.30 ± 1.87a
C/N ratio        
40:1 117.16 ± 2.10a 76.12 ± 1.11a 82.29 ± 2.57a 42.29 ± 3.12a
30:1 294.19 ± 4.61b 196.81 ± 4.61a 142.20 ± 1.33a 142.20 ± 3.10a
20:1 313.11 ± 3.29c 213.67 ± 2.21b 153.72 ± 4.46a 153.72 ± 1.23a
10:1 388.19 ± 4.12c 248.10 ± 4.12b 281.64 ± 4.48b 135.12 ± 2.13a
5:1 334.84 ± 1.50c 231.12 ± 2.28b 219.15 ± 3.50b 114.14 ± 1.34a
1:1 293.72 ± 1.24b 103.13 ± 1.14a 192.29 ± 3.16a 57.16 ± 1.35a

Table 2: Effect of different carbon, nitrogen, mineral sources and C/N ratio on EPS and IPS yield in submerged culture of I. sinclairii and I. tenuipes.

Experimental group Temperature (ºC) pH Rotary speed/r·min−1 Culture time/d EPS (mg/L) IPS (mg/L)
1 1 1 1 1 312.13 ±3.10 262.13 ±2.62
2 1 2 2 2 370.21 ±9.10 282.12 ±11.15
3 1 3 3 3 380.20 ±11.21 228.21 ±12.16
4 2 1 2 3 578.23 ±20.15 294.10 ±24.19
5 2 2 3 1 562.60 ±12.17 392.10 ±19.23
6 2 3 1 2 642.65 ±7.22 411.16 ±10.52
7 3 1 3 2 242.12 ±40.14 188.15 ±12.31
8 3 2 1 3 322.10 ±10.71 241.11 ±12.70
9 3 3 2 1 220.10 ±22.11 166.16 ±12.10
K1 9.84 7.12 13.21 12.14    
K2 19.12 12.14 13.42 14.21    
K3 14.60 8.28 14.12 14.21    
R 12.20 3.27 0.82 3.22    
K1* 440.60 325.46 444.11 318.42    
K2* 575.69 512.21 519.10 426.11    
K3* 298.13 332.20 358.20 425.17    
R* 387.17 79.26 32.21 78.18    

Table 3: Results obtained for orthogonal design I. sinclarii.

Experimental group Temperature (ºC) pH Rotary speed/r·min−1 Culture time/d EPS (mg/L) IPS (mg/L)
1 1 1 1 1 221.12 ±5.21 236.81 ±2.62
2 1 2 2 2 322.11 ±9.22 334.12 ±11.12
3 1 3 3 3 311.12 ±12.71 324.21 ±11.14
4 2 1 2 3 398.10 ±10.23 458.11 ±32.14
5 2 2 3 1 391.61 ±16.13 398.61 ±12.11
6 2 3 1 2 442.61 ±8.17 342.21 ±4.15
7 3 1 3 2 178.10 ±12.22 163.22 ±12.13
8 3 2 1 3 262.12 ±11.70 212.15 ±8.15
9 3 3 2 1 176.25 ±18.12 151.71 ±11.23
K1 9.43 6.91 12.13 11.29    
K2 12.16 10.15 10.13 13.52    
K3 8.64 7.22 12.12 12.38    
R 11.25 2.94 0.97 3.15    
K*1 325.31 310.18 314.12 322.41    
K*2 439.41 384.21 421.21 212.11    
K*3 242.13 241.20 311.13 362.11    
R* 322.10 68.10 32.12 72.11    

Table 4: Results obtained for orthogonal design I. tenuipes.

Monosaccharide composition analysis of polysaccharides obtained from I. sinclairii and I. tenuipes showed glucose as chief component. However, xylose, rhamnose, mannose, galactose and fructose were also detected in small percentages (Table 5).

Monosaccharides I. sinclairii I. tenuipes
Xylose 15.17±2.10 12.26±5.19
Glucose 54.10±6.13 44.67±3.75
Rhamnose 32.11±2.53 21.18±4.57
Mannose 11.89±3.75 25.80±2.70
Galactose 0.10±0.0 0.13±0.01
Fructose 0.06±0.0 0.01±0.00
Lactose nd nd
Maltose nd nd

Table 5: Monosaccharide composition of polysaccha rides (%).

Antioxidant activities of EPS and IPS

The DPPH scavenging activity of EPS and IPS extracted from the mycelium of I. sinclairii and I. tenuipes showed positive and direct correlation with the concentration of the sample (Figure 2). The EPS showed higher DPPH scavenging activity than IPS. EPS and IPS extracted from both of species showed high DPPH scavenging activities. The results are also supported by high EC50 values of both EPS and IPS (Table 6). DPPH radical scavenging activities of both these species were observed same as obtained for other medicinally important entomogenous species like Cordyceps militaris and C. sinensis [52,53]. The inhibition percentage of the ABTS radical by EPS and IPS of both showed a direct positive relation with the concentration of the sample. The ABTS radical scavenging activity of IPS was found to be higher as compared to EPS in both of species (Figure 3). At a concentration of 10.0 mg/mL, the percentage inhibition of EPS and IPS was found to be maximum and showing their ability to quench the free radicals in the system. The results indicated that the EPS and IPS of both entomogenous species possessed significant scavenging power for the ABTS radicals. However EPS showed higher ABTS radical scavenging activity than IPS (Figure 3).

bioanalysis-biomedicine-scavenging-activity

Figure 2: DPPH scavenging activity of EPS and IPS.

bioanalysis-biomedicine-radical-scavenging-activity

Figure 3: ABTS radical scavenging activity of EPS and IPS.

EC50 I. sinclairii (mg/mL) I. tenuipes (mg/mL)
  EPS IPS EPS IPS
DPPH radical scavenging activity 7.52 ± 0.10 3.17 ± 0.20 6.12 ± 0.10 2.71 ± 0.14
ABTS radical scavenging activity 6.29 ±0.13 4.25 ± 0.19 5.43 ±0.13 3.11 ± 0.19
Reducing power 7.17 ± 0.22 3.11 ±0.17 6.36 ± 0.22 2.43±0.27
Iron chelating activity 2.04 ± 0.31 1.24±0.16 1.84 ± 0.31 0.85±0.31
FRAP 1.76 ± 0.05 1.51± 0.04 1.51 ± 0.04 1.51± 0.04
Scavenging activity superoxide ion 2.14 ± 0.20 1.74±0.10 1.88 ± 0.30 0.75±0.11

Table 6: EC50value of EPS and IPS.

The results obtained for reducing power abilities of EPS and IPS in submerged culture showed that both types of polysaccharides possess significant reducing capacities. The reducing powers of EPS and IPS increased as the sample concentration increased (Figure 4). The reducing power of EPS was found to be higher than reducing power of IPS in both of the species. However at highest concentration 10 mg/ mL, the reducing power of EPSs of both species was found to be same (1.11 ± 0.01 mg/mL). Reducing power of EPSs was found to be higher as compared to IPSs in both of the species (Figure 4). The results are further supported by EC50 values. Present results showed that EPS and IPS of both of these species contained reductones which react with certain precursors of peroxides to prevent peroxide formation. The iron chelating ability of the EPS and IPS was found to be related with the concentration of sample. At initial concentrations 2 mg/mL, there was not much difference observed between the iron chelating ability of both EPS and IPS (49-54%). However, at higher sample concentrations EPS showed higher iron chelating ability than IPS in both of the species (Figure 5). EPSs showed maximum iron chelating activity in both of the species at concentration 10 mg/mL. The results further supported by EC50 values (Table 6). EPS and IPS of both of the species showed high scavenging ability on superoxide anion radicals. At high concentration the scavenging ability was found to be maximum (Figure 6). I. sinclairii showed higher values for scavenging radicals than I. tenuipes however at concentration 8 mg/mL, EPSs of both species possessed same activity (Figure 6). EPS and IPS of both species showed high FRAP activity. The results are supported by EC50 values (Table 6). Experiments were performed to study the inhibition rate of polyunsaturated fatty acids. Present investigations showed that EPS as well as IPS of both species showed high inhibition rate of polyunsaturated fatty acids. At the highest polysaccharide concentration the inhibition rate was found to be high (Figure 7).

bioanalysis-biomedicine-reducing-power

Figure 4: Reducing power of EPS and IPS.

bioanalysis-biomedicine-iron-chelating-activity

Figure 5: Iron chelating activity of EPS and IPS.

bioanalysis-biomedicine-scavenging-superoxide-anion

Figure 6: Scavenging ability on superoxide anion radicals.

bioanalysis-biomedicine-inhibition-peroxidation-polyunsaturated

Figure 7: Inhibition rate of peroxidation of polyunsaturated fatty acid from lipoprotein.

Conclusion

Submerged culture of I. sinclairii and I. tenuipes required several factors for the production of EPS and IPS. Factors such as effect of temperature, rotation speed, pH, incubation time, carbon, nitrogen, mineral sources, and carbon to nitrogen ratio showed significant effects on the production of EPS and IPS. Composition analysis of polysaccharides obtained from I. sinclairii and I. tenuipes showed glucose as chief component. As observed in the present studies EPS and IPS of both species exhibited significant DPPH radical scavenging activities, ABTS radical scavenging activities, reducing power, Iron chelating activities, scavenging activities of superoxide anion radicals, ferric reducing antioxidant power, as well as inhibition rate of peroxidation of polyunsaturated fatty acid from lipoprotein. Due to high antioxidant potential of EPS and IPS, present studies will be helpful for its large scale industrial fermentations and commercial use like other useful ascomycetes species namely Cordyecps sinensis, C. militaris and C. ophioglossoides.

Acknowledgements

Author wish to thank Science and Engineering Board, Department of Science and Technology, New Delhi for research grant under Young Scientist Scheme (SB/ FT/LS-04/2013) to carry out present studies.

References


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