Antioxidant and organic acid evaluation of Geaster saccatum mushroom by chemical and electrochemical assay at carbon nanotube paste electrode

2.1 Chemicals and reagents

The standards used namely, oxalic acid, ascorbic acid, Succinic acid, malic acid, ketoglutaric, gallic acid, quinic acid, fumaric acid were purchased from Sigma (St. Louis, MO, USA). All the solvents (ethanol, methanol) used were of analytical grade and were purchased from either Anala R or Himedia Laboratories Pvt. Ltd. Mumbai.

2.2 Sample preparation and extraction

Geaster saccatum fruiting bodies were collected from some parts of central India (Jabalpur, Sagar) during the rainy season in 2019 and dried in an oven at 40 °C for 4 h. The dried fruiting bodies were crushed to powder by using electronic blender. Twenty grams (20 g) of powder was taken in 200 ml of 95% methanol in soxhlet extraction unit for extraction at 30C⁰ for 12–18 h. The extraction was filtered through Whatman no.4 filter paper. The methanolic extract was then evaporated in vacuum rotary evaporater at 40 °C to dryness. The extract was re-dissolved in acid water (0.1% formic acid). The aqueous solution was then passed through an Isolute C₁₈ column at a ratio of 3:7(methanol and acid water). The organic acids (polar compounds) eluted with aqueous solution while non polar compounds hold back, respectively. Under reduced pressure the aqueous mushroom extract was evaporated to dryness and re-dissolved in 1 ml of H₂SO₄ (0.02 N), out of which 30 µL was thoroughly analyzed by HPLC at UV detector. Meanwhile, a standard mixture was prepared in H₂SO₄ (0.02 N) out of which the same volume was analyzed. The organic acids were separated at 0.1 ml/min flow rate at 110 min with 0.02 N H₂SO₄ as mobile phase. The quantitative analysis of organic acids were done by comparing the absorbance chromatograms of standards by using their peak areas and retention time values.

2.3 DPPH radical scavenging assay

The DPPH assay was done as per authentic protocol with some modifications. Different concentrations of mushroom methanolic extract (0.1–1 ml) were thoroughly mixed with 1 ml of DPPH (0.2 mM) solution. The mixture was thoroughly whir pooled vigorously and then left stagnant for 50 min at 20 °C in dark. The absorbance of the resulting solution was measured at 517 nm against the blank using a UV/Vis spectrophotometer (Elico SL164 Double beam UV–VIS spectrophotometer). Similarly, the absorbance of standards were also measured for comparison. On reduction by antioxidant compound, the DPPH is decolorized, which was quantitatively recorded on spectrophotometer, as absorbance changes. The IC₅₀ value (mg/ml) was recorded as interject by using linear regression analysis.

2.4 ABTS radical scavenging assay

The antioxidant assay of mushroom was determined by using ABTS•+ (2,2′ –azinobis, 3-ethylbenzothiazoline-6-sulphonic acid) with small modifications in pH (7.8). The radicals were produced by reacting ABTS with potassium per sulfate (K₂S₂O₈). 3 mmol/L of ABTS was prepared by dissolving it in 60 ml phosphate buffered saline as a stock solution, from which 60 ml were taken and kept in dark at normal room temperature for overnight. The various concentrations of mushroom extract solutions (0.5 ml) were mixed with 1.5 ml ABTS•+ solution, and absorbance was measured at 734 nm against blank (PBS). The sample analysis were carried in triplicates and their average values were taken as a statistical tool. The absorbance of mushroom extract concentration where 50% radicals were scavenged was taken its IC₅₀ value. The average radical-scavenging assay of samples were calculated as: $$\mathit{RS}\%=\left(\left[A_{\mathit{control}}--A_{\mathit{extract}}/A_{\mathit{control}}\right]\right)\times 100$$ where A _control; absorbance of the control (ABTS•⁺ alone) and A_test is absorbance of the test sample (ABTS•⁺ with mushroom extract).

2.5 Electrochemistry

Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were performed on a Ω Metrohm 757 VA Computrace (Ion analyzer, version 3.1) using a compact standard triple electrode cell. A single walled carbon nanotube paste electrode (SWCNPE) was used as the working electrode and a platinum (Pt) wire as the counter electrode. All potentials are refer to an Ag/AgCl 3 M KCl reference electrode (Methrom). Prior to use, the working electrode was polished in an aqueous suspension of 0.3 lm alumina (Beuhler) on a Master-Tex (Beuhler) polishing pad, then rinsed with water. Subsequently, in a chemical treatment, the electrode was soncated in 6 M HCl for 1 min, and then in methanol, as a cleaning procedure before any electrochemical measurements. The nitrogen gas was purged for 5 min. A systronics digital µpH meter model-361 was used for pH measurements.

3.1 Antioxidant activity

3.1.3

3.1.3 Organic acid analysis

The organic acid evaluation of Geaster saccatum mushroom by HPLC-UV chromatogram revealed the presence of eight organic acids (Fig. 1), reported first time in the studied mushroom. However, some of these compounds were also been reported from some other mushrooms as well (Valentao et al., 2005). The quantified data of organic acids in the investigated mushroom ranges from 536 mg/kg to 664 mg/kg. Among all the identified organic acids, gallic acid was found in the highest concentration (193.34%), followed by Ketoglutaric acid (181.16%) and oxalic acid (64.54%). The other organic acid were found in absolutely good proportion namely, ascorbic acid (57.22%), succinic acid (43.74%) and mallic acid (40.66%),respectively. Quinic acid (4.04%) and fumaric acid (8.86%) were found in lower concentrations among all the quantified organic compounds (Table 2).

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Fig. 1

Chromatogram (HPLC-UV) of organic acids of Geaster saccatum, Detection at 214 nm: (a) oxalic acid, (b) ascorbic acid, (c) succinic acid, (d) malic acid, (e) ketoglutaric acid,(f) gallic acid, (g)Not identified, (h) Quinic acid, (i) fumaric acid.

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Table 2 Organic acid quantification in three different concentrations of five samples of Geaster saccatum (mg/kg dry wt).

Organic acids	RT	S1 (c1,c2,c3)	S2 (c1,c2,c3)	S3 (c1,c2,c3)	S4 (c1,c2,c3)	S5 (c1,c2,c3)	G.M
Oxallic acid	2.37	33.4 ± 04	64.7 ± 0.2	69.0 ± 1.1	76.2 ± 0.2	79.4 ± 1.2	64.54
Ascorbic acid	2.99	45.4 ± 1.4	49.3 ± 1.2	57.7 ± 0.6	64.5 ± 1.6	69.2 ± 1.3	57.22
Succinic acid	3.12	37.9 ± 1.3	40.4 ± 0.4	43.9 ± 0.3	45.8 ± 1.4	50.7 ± 0.2	43.74
Malic acid	3.46	31.6 ± 1.3	37.4 ± 0.4	40.2 ± 0.3	45.3 ± 1.6	48.8 ± 0.2	40.66
Ketoglutaric acid	3.75	168.2 ± 1.2	174.6 ± 0.2	179.4 ± 1.3	187.2 ± 0.3	196.4 ± 1.5	181.16
Gallic acid	3.90	185.2 ± 1.4	187.3 ± 0.8	193.6 ± 1.5	197.4 ± 0.5	203.2 ± 0.6	193.34
NI	4.67	nq	nq	nq	nq	nq
Quinic acid	6.1	2.5 ± 0.7	3.2 ± 1.3	4.0 ± 0.4	4.8 ± 1.2	5.7 ± 0.6	4.04
Fumaric acid	7.1	7.1 ± 0.5	8.2 ± 0.4	8.9 ± 1.1	9.7 ± 0.7	10.4 ± 1.3	8.86
Summation (Σ)		536.3 ± 8.1	565.1 ± 4.9	596.7 ± 6.6	630.9 ± 7.5	663.8 ± 6.9

Values are: Mean ± SD (standard deviation) of five concentrations, RT = Retention time, S = Sample, c¹,c²,c³ = three different concentrations of each sample, GM = Grand Mean, NI = Not identified, nq = Not quantified.

The organic acid profile of our studied mushroom was found in higher concentration than most of the earlier reports (Valentao et al., 2005). The HPLC-UV confirms only a few organic acids in L. giganteus mushroom with highest concentration of citric acid as main organic acid (Ribeiro et al., 2007), which was absent among the eight organic acids of our studied mushroom. Barros et al. (2009) used UFLC-PDA of some mushroom species and found five organic acids namely, oxalic acid, quinic acid, malic acid, citric acid and fumaric acid, with mallic acid at higher concentration (6 g/100 g), respectively, but comparatively at lower concentration than our studied mushroom. The presence of oxalic acid in our studied mushroom was also been found in other mushrooms as well including Amanita spissa, F. hepatica and B. nigrescens and most of the plants including tea as main antioxidant compounds (Ribeiro et al., 2008) but was in slightly lower proportion comparatively. Patriacia et al. (2005), reported nine organic acids in S. collinitus and X. chrysenteron mushrooms, supported our results with only the absence of shikimic acid in our mushroom. The organic acid analysis of this investigated mushroom revealed a virgin report in its nature so far, indicated its chemo taxonomic importance, a key to food composition and identification.

4.1 Cyclic voltammetry (CV)

The CV of Geaster saccatum was used to assess the constant chemical reactions following electrochemical oxidation reaction. Both standards shown normal irreversible oxidation phenomenon, a diagnostic feature of antioxidant compounds (Cosio et al., 2006). The CV of gallic acid produced a single anodic peak at Epa = 0.29 ± 0.01 V (Fig. 2a), while CV of ascorbic acid produced double anodic peaks, at Epa = 0.41 ± 0.01 V and at Epa = 0.69 ± 0.01 V (Fig. 2b), respectively. The mushroom extract also produced, a diagnostic irreversible anodic peak at 0.69 ± 0.01 V, The E^1/2 (half-wave potential) value of mushroom in CV is 0.78 V (Fig. 2c), confirmed that mushroom extract undergone oxidation at relatively higher positive potential in comparison to standards used. The voltammogram showed a linear relation from 2 mg/ml to15mg/ml of mushroom concentration to positive potential in calibration plot (Fig. 2d), with R² = 0.969, confirmed the phenomenon of diffusion restrain. The positive potential as a peak current shown by the mushroom ensured its concentration dependence, electron transfer kinetics and diffusion coefficient of its electroactive compounds which was agreeable with earlier studies (Arulpriya et al., 2010). This CV character of our studied test mushroom accepted the fact that mushrooms having lower oxidation potential do carry higher oxidative current densities possessing excellent antioxidant property and in turn higher antibiotic capacity, and was found comparable with prior studies (Cosio et al., 2010). The results also indicated that both gallic and ascorbic acids went oxidation reaction slightly at low positive potentials than mushroom extract, forming an electrochemical responses, clearly shown the absence of used standards in test mushroom. The electron flow as a current assessment of the mushroom extract ahead and after pulse potential developed unprecedented character because of adsorption on working electrode and were proportionally same at main potential intervals, this electrochemical feature of the mushroom extract was found similar to other reports (Dandamudi and Nageswara, 2011). The mushroom showed extremely low oxidation potential, produced higher oxidative current density, hence possessed excellent antioxidant activity, a character found in other reports as well (Barros et al., 2008).

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Fig. 2

CV, in 0.1 M methanol/acetate buffer at scan rate of 100 mV/s (pH 4) solutions of: (a) 0.5 mM ascorbic acid, (b) 0.5 mM gallic acid, (c) 5 mg/ml of mushroom extract, (d) 2–15 mg/ml of mushroom extract.

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4.2 Differential pulse voltammetry (DPV)

The DPV of mushroom was featured by higher resolution, absolute for peak separation, promotes the characterization of electro-oxidation process to measure the differential current (di). The DPV of gallic acid at 0.2 mg/ml produced two anodic peaks at 0.41 V and 0.71 V (Fig. 3a), while ascorbic acid produced one anodic peaks at 0.29 V (Fig. 3b). DPV of mushroom was done at 2–5 mg/ml, produced a well defined peak current at 0.56 ± 0.01 V (Fig. 3c). At 2 mg/ml, peak current was low, while increasing to 5 mg/ml, the peak current also increased, simultaneously. The DPV of gallic acid produced peak currents at different concentrations such as, 0.0125 mg/ml, 0.025 mg/ml, 0.05 mg/ml, 0.1 mg/ml, 0.15 mg/ml and 0.2 mg/ml each and concentration of ascorbic acid were 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml and 5 mg/ml, respectively (Fig. 4). At each increasing concentration of standards, a linear relation existed between the concentration and its peak current. Similar character was seen in mushroom extract also where the peak current increased along the different concentrations namely, 2 mg/ml, 4 mg/ml, 6 mg/ml, 8 mg/ml, 10 mg/ml and 12 mg/ml (Fig. 5). The slope of peak current density (j) of mushroom was 0.798 (Table 3), while the slope of peak current density of ascorbic and gallic acid were 203.5 and 237.6, respectively. The slope values of mushroom and standards were different because it depends upon concentration, kinetics of electron transfer and diffusion coefficient of electro-active species (Brett and Brett, 1993). The relation between current density (j) and mushroom concentration was different from standards, because neither of standards were present in mushroom extract. However, on slope comparison of two standards (Table 3), which depicted their different diffusion coefficients, but while comparing with mushroom extract, the difference was due to electro-active mass amount, and was found comparable with other reports (Cosio et al., 2006). Therefore, DPV of mushroom extract confirmed the presence of antioxidant compounds which undergone either oxidation or reduction and produced differential current (DI) which increased at each increasing concentration of mushroom. The electrochemical method used in the present study was an accurate and authentic drive to assess the antioxidant activity of the studied mushroom due to its precision and cheap disbursement (Blasco et al., 2005). The main compounds present in this mushroom were organic acids, acted as reducing agents, undergone oxidation in electrolytic set up cell at inert electrodes. In view of this fact, enormous cited authors developed a relationship between electrochemical behavior of the organic acids and their resultant antioxidant activity, where low oxidation potential corresponds to high antioxidant rate (Cosio et al., 2006).

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Fig. 3

Differential pulse voltagram of 0.2 mg/ml of (a) gallic acid (b) ascorbic acid (c) 2–5 mg/ml of mushroom extract in methanol/acetate buffer 0.1 M (pH = 4) solution at 100mv/s scan rate in methanol/ acetate buffer 0.1 M (pH = 4) solution at 100mv/s scan rate.

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Fig. 4

Calibration plot of concentration of gallic acid and ascorbic acid versus their oxidation currents in DPV (1-gallic acid,2-ascarbic acid) at 0.0125 mg/ml, 0.025 mg/ml, 0.05 mg/ml, 0.1 mg/ml, 0.15 mg/ml and 0.2 mg/ml concentration each.

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Fig. 5

(a) DPV of 2 mg/ml, 4mg/ml, 6mg/ml, 8mg/ml, 10 mg/ml and 12 mg/ml mushroom extract in methanol/acetate buffer 0.1 M (pH = 4). (b) Variation of the peak current in DPV of mushroom extract at different concentrations.

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4.3 Total anti-oxidant power (TAP)

The TAP of Geaster saccatum mushroom is a measure of its quenching free radical creation in terms of chemical measurement, but in terms of electrochemical measurement, it is the capacity of any substance to undergo redox in an electrochemical reaction and its transfer ability of ions to move from the electrolytic solution to the surface of working electrode. The TAP of this mushroom was found 4.6 ± 0.35 mg g⁻¹ in terms of ascorbic acid and 4.9 ± 0.25 mg g⁻¹ in terms of gallic acid, respectively (Table 3). While, TAP of ascorbic acid was measured 1005 mg g⁻¹ in terms of ascorbic acid, whereas, 710 mg g⁻¹ in terms of gallic acid, respectively. The antioxidant power of gallic acid was measured 1120 mg g⁻¹ in terms of ascorbic acid, whereas, 1005 mg g⁻¹ in terms of gallic acid, respectively. The slope variation of ascorbic and gallic acids were because of difference in their diffusion coefficients. Whereas, On comparison, slopes of mushroom with slopes of standards, the difference was due to electro active mass of mushroom rather than the diffusion coefficients, which in turn resulted in single peak potential characterization of CV. The higher TAP of this mushroom produced more current density (j), and was found highly agreeable with other reports as well (Blasco et al., 2005).