Optimized Extraction and Antioxidant Activities of Polysaccharides from Two Entomogenous Fungi

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 antiinflammation, renal dysfunction, asthma, and cancer [1114]. 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][21][22][23][24][25][26].High antioxidant activities provide health benefits in preventing damages due to free radicals produced by biological degeneration [27][28][29][30][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.
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 (%)=A 0 -A P /A 0 × 100, where A 0 was the absorbance of the control and A P 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 FeSO 4 , 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% FeCl 3 .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 (%)=[(A b -A s )/A b ] × 100, where A b is the absorbance of the blank and A s 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 FeCl 3 .H 2 O (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 (t 0 ).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 (t 30 ).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 Fe 2+ 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 FeCl 2 (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 L 9 (3 4 ) method was used.

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

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].
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.NaH 2 PO 4 + MgSO 4 supported maximum EPS (374.86 ± 3.36 mg/L) and IPS production (279.80 ± 1.22 mg/L) in I. sinclairii whereas NaH 2 PO 4 + K 2 HPO 4 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).
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).

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 EC 50 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   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).

Factors
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 EC 50 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 EC 50 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 EC 50 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).

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,

Figure 5 :
Figure 5: Iron chelating activity of EPS and IPS.

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

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

Table 6 :
EC 50 value of EPS and IPS.