INTRODUCTION:

Free radicals and degenerative diseases are associated with aging include cancer, cardiovascular disease, immune–system decline, cerebral dysfunction and cataracts^1,2because oxidative damage to DNA, proteins and other macromolecules accumulates with age and has been postulated to be a major type of endogenous damage leading to aging^3,4. Superoxide, hydrogen peroxide and hydroxyl radicals, which are mutagens produced by radiation, are also by–products of normal metabolism^5,6. Our own immune system at times is unable to combat these reactive oxygen species (ROS). Hence, the need for antioxidants creeps in.

As carcinogenic properties have been reported for some synthetic antioxidants, recent research on the potential applications of natural antioxidants from plants and mushrooms have received much attention. It has been also established that they are less toxic⁷. They may act directly as antioxidant or prevent underlying oxidative stress related pathological conditions such as cancer⁸, heart ailments⁹, diabetes¹⁰, inflammation¹¹, gastric ulcer¹², hepatic damage^13,14, microbial pathogens^15–17etc.

Termitomyces medius R. Heim commonly known as “choto bali chatu” by the tribal people of lateritic West Bengal, have been traditionally used as food for their flavour, texture and delicacy. It is also believed to be improved vitality and as protectant of body defence for the maintenance of good health. As far as our literature survey says the antioxidant activity of polyphenol rich fraction of this mushroom has not yet been published. Hence the present investigation was performed. We have examined the antioxidant activity of polyphenol rich fraction of T. medius employing various in vitro assay models such as chelating of ferrous ion, reducing power ability, superoxide radical scavenging, total antioxidant capacity, DPPH radical scavenging and inhibition of lipid peroxidation tests along with a phytochemical screening for determining the usefulness of this mushroom as a food stuff as well as a medicine.

MATERIALS AND METHODS:

Basidiomata sampling and morphological studies:

Basidiocarps of T. medius were collected from West Midnapore district of West Bengal, India during the month of July, 2012. The morphological and ecological features of the collected specimens were noted in the field. Microscopic features were obtained from dried material by mounting free–hand sections of basidiocarps in 5% potassium hydroxide (KOH), Melzer’s reagent, Congo red or lactophenol–cotton blue. Microscopic features were examined with a Carl Zeiss AX10 Imager A1 phase contrast microscope. Spore statistics include: Q, the quotient of spore length by spore width in any one spore, indicated as a range of variation in n spores measured; Q_av, the mean of Q–values in a single specimen; n, total number of spores measured; s, the number of specimens. The specimen is identified with the help of standard literature^18,19and finally the voucher specimen has been deposited in the Calcutta University Herbarium (CUH) with the accession number AM080 (CUH).

DNA extraction, Polymerase Chain Reaction and sequencing:

Genomic DNA was extracted from dried (50°C) basidiocarps (10–50 mg) using the ‘Fungal gDNA Mini Kit’ (Xcelris Genomics, Ahmedabad, India). Internal Transcribed Spacer–1 (ITS–1) region was amplified using ITS1 (forward) and ITS2 (reverse) primer pair. For the primer pair ITS1/ITS2 a hot start of 2 min at 94°C was followed by 30 cycles consisting of 30 s at 94°C, 1 min at 56°C, 1 min at 72°C and a final elongation step of 5 min at 72°C. PCR products were checked on 2% agarose gel stained with ethidium bromide. PCR products were purified using QIAquick® Gel Extraction Kit (QIAGEN, Germany) and sequencing was done using Sanger methods.

The obtained sequence was used for molecular identification of the Termitomyces species. The sequence generated for this study was submitted to GenBank (www.ncbi.nlm.nih.gov).

Preparation of extract:

Polyphenol rich fraction was extracted according to the method of Khatua et al. (2013)²⁰ with slight modification. Dried and powdered basidiocarps of T. medius were extracted with ethanol at 25ºC for 2 days. It was then filtered using Whatman No. 1 ﬁlter paper. After filtration, the residue was then re–extracted with ethanol. The filtrate was air dried. It was boiled with distilled water for 8 hours with continuous stirring. After filtration, 4 volume of ethanol was added to the supernatant, it was then kept at 4ºC, undisturbed for one whole night. After centrifugation the supernatant was concentrated under reduced pressure in a rotary evaporator. Now, this concentrated polyphenol rich extract of T. medius (TmedPre) was stored at 4ºC.

Standards and reagents:

All chemicals used were of analytical grade and freshly prepared before use. L–methionine, nitro–blue tetrazolium (NBT), riboflavin, 2–Deoxy–D–ribose, ferric chloride, hydrogen peroxide (H₂O₂), trichloro acetic acid (TCA), thiobarbituric acid (TBA), ferrous chloride, ferrozine, potassium ferricyanide, 2, 2–Diphenyl–1–picrylhydrazyl (DPPH), ammonium molybdate, ferrous sulphate, sodium bicarbonate, Foin–ciocalteu reagent, aluminium nitrate, acetone, n–hexane, acetic acid, sodium acetate, hydrogen peroxide, sodium phosphate, standards such as L–ascorbic acid, ethylenediamine tetra acetic acid (EDTA), butylated hydroxylanisole (BHA), gallic acid, quercetin were purchased from Sigma Chemicals Co. (St. Louis, MO, USA).

Antioxidant Assays:

Total antioxidant capacity assay:

The assay is based on the reduction of Mo (VI) to Mo (V) by the extract; as was done by Prieto et al. (1999)²¹. When acidic pH is maintained then subsequent formation of a green phosphate/Mo (V) complex is seen. The tubes containing extract (1 mg/ml) and reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) were incubated at 95˚C for 90 min. Then the mixture was cooled to room temperature. Absorbance was recorded spectrophotometrically for each solution at 695 nm against blank. The antioxidant capacity was expressed as ascorbic acid equivalent (AAE).

Chelating effect on ferrous ions:

Chelating capacity of TmedPre on ferrous ions was estimated by the method described by Dinis et al. (1994)²². Reaction mixture (4 ml) was made up with different concentration of TmedPre (100 – 500 μg/ml) mixed with 3.7 ml of water and 0.1 ml of 2 mM ferrous chloride. The reaction was initiated by the addition of 0.2 ml of 5 mM ferrozine. It was incubated for 10 min at room temperature. Absorbance was determined at 562 nm against blank. EDTA was used as positive control. The percentage of inhibition is given by this formula:

% inhibition = {(A₀–A₁)/ A₀} × 100.

Where A₀ was the absorbance of the control and A₁ was the absorbance in the presence of TmedPre.

Reducing power:

Reducing power of TmedPre was determined following the method of Oyaizu (1986)²³. Variable concentrations (1.0 –2.0 mg/ml) of TmedPre were added to 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml 1% potassium ferricyanide. The reaction mixture was incubated for 20 min. After that 2.5 ml of 10% trichloroacetic acid was added to the mixture. It was then spun for 10 min at 12000 rpm. 2.5 ml of the supernatant was mixed with 2.5 ml distilled water and 0.5 ml. of 0.1% ferric chloride. Absorbance was measured at 700 nm. An increase in absorbance of the reaction mixture signified increase in reducing power of the sample.

Superoxide radical scavenging assay:

The scavenging potential of TmedPre for superoxide radical was recorded as described by Martinez et al. (2001)²⁴with some modification in the riboflavin–light–NBT system. Each 3 ml reaction mixture sequentially contained 50 mM sodium phosphate buffer (pH 7.8), 13 mM methionine, different concentrations (0.5–1.5 mg/ml) of TmedPre, 100 μM EDTA, 75 μM NBT and 2 μM riboflavin. Reaction was started by illuminating sample with light for 10 min. Increase in absorbance was measured at 560 nm. Identical set of tubes with the similar reaction mixtures were kept in dark and served as blank. BHA was used as a positive control. The inhibition percentage was calculated by the following equation:

Scavenging effect (%) = {(A₀–A₁)/ A₀} × 100

A₀ and A₁ were the absorbance of the control and absorbance in presence of sample respectively.

DPPH (2, 2 –diphenyl–1–picrylhydrazyl) radical–scavenging activity:

As per the method of Shimada et al. (1992) 25 0.004% methanolic solution of DPPH was prepared. Then various concentrations (0.2–0.6 mg/ml) of TmedPre were added to it. The mixture was shaken vigorously and left to stand for 30 min in the dark. Gradual fading of a purple colour against various concentrations were measured at 517 nm against a blank. EC₅₀ value is the effective concentration of extract that scavenged DPPH radicals by 50%. Ascorbic acid was used as positive control. The degree of scavenging was calculated by the following equation:

Scavenging effect (%) = {(A₀–A₁)/ A0} × 100

Where A₀ was the absorbance of the control and A₁ was the absorbance if sample is present.

Hydroxyl Radical Scavenging Activity:

The method used by Halliwell et al. (1987)²⁶was followed in this study. The reaction mixture (1ml) consisted of KH₂PO₄– KOH buffer (20 mM, pH 7.4), 2–deoxy–D–ribose (2.8 mM), variable concentration (10 – 30 μg/ml) of TmedPre, FeCl₃ (100 mM), EDTA (104 μM), ascorbate (100 μM) and H₂O₂ (1 mM). It was incubated at 37˚C for 1 h. 2ml thiobarbituric acid (TBA) and trichloroacetic acid (TCA) solution (0.375 w/v TBA, 15% TCA and 0.25 N HCl) was added and incubated at boiling water bath for 15 min. After cooling, absorbance was monitored at 535 nm. EC₅₀ value in µg/ml expressed the effective concentration at which the scavenging free radical activity is 50%. BHT was used as positive control.

Lipid peroxidation:

In accordance with the method of Ohkowa et al. (1979)²⁷ yolk portion of an egg is required to make 10% egg homogenate by using 0.1 (M) Na–phosphate buffer. Then TmedPre, freshly prepared FeSO₄ and water was added and left for 30 min at a temperature of 37°C. Then after acetic acid, TBA and TCA were added to the reaction mixture and incubated for 1 hour at 95°C. The intensity of the red coloured formagen formed was reduced by the addition of butanol. After a spin of 3000 rpm for 10 min, OD was measured at 532 nm against the supernatant obtained. Inhibition of lipid peroxidation (%) by the extract was calculated according to

[(1– E/C * 100]

where C is the absorbance value of the fully oxidised control and E is (Abs532_+TBA –Abs532_{_TBA}).

Bioactive compounds:

The content of total phenolic compounds in TmedPre was estimated using Folin–ciocalteu reagent. Gallic acid was used as standard²⁸for this experiment and expressed as μg of gallic acid equivalents per g of dry tissue. Aluminium nitrate and potassium acetate were required to determine total flavonoid content²⁹ and Quercetin (5–20 μg) was used as standard, in this case to express results as μg of quercetin equivalents per g of dry tissue. Quantification of Ascorbic acid was done by titration against 2, 6–dichlorophenol indophenol dye using oxalic acid³⁰. β–carotene and lycopene were estimated by measuring absorbance at 453, 505 and 663 nm³¹.

Detection of Phenols and flavonoids by HPLC:

Eleven standards of Sigma Aldrich (MO, USA), gallic acid, chlorogenic acid, vanillic acid, p–coumaric acid, ferulic acid, myricetin, salicylic acid, quercetin, cinnamic acid, pyrogallol and kaempferol were used. For quantitative analysis, a calibration curve (10 – 50 μg /ml) for each phenolic standard was constructed. Compounds present in the concerned samples were identified on the basis of the retention times and absorption spectra of standard materials. Components were quantified based on their peak areas in comparison to those of standard curves.

Dried TmedPre was dissolved with 1 ml of 50% methanol and diluted to concentration of 1.0 mg /ml. The suspension was filtered through 0.2 μm filter paper. The mobile phase consisted of eluent A (acetonitrile) and eluent B (aqueous phosphoric acid solution, 0.1% v/v). TmedPre was run across Agilent Eclipse Plus C18 column (100 mm × 4.6 mm, 3.5 μm) with a flow rate of 0.8 ml /min at 25°C for the separation. 20 μl of TmedPre was loaded on the HPLC system (Agilent, USA). The absorbance of standard and TmedPre were measured at 280 nm.

Statistical Analysis:

Results were subjected to statistical analysis using Student’s t test. Values are mean ± SD of 3 replications.

RESULTS AND DISCUSSION:

Taxonomy:

Termitomyces medius R. Heim & Grassé:

Pileus 12˗25 mm broad, convex, with a very sharp umbo, buffy brown, surface somewhat viscid, glabrous, margin striate; context cream, thin. Lamellae somewhat sinuate to subfree, cream coloured, close, edges even to wavy. Stipe 3˗7 cm long, up to 2 mm broad at apex, somewhat thicker towards base, central, well developed, cylindrical, with a well developed pseudorhiza, hollow. Odor and taste mild. Basidiospores 6.8˗7.16 × 3.58˗3.9(˗4.29) µm [Q=1.58˗1.81, Q_av=1.73, n=30 spores, s=1 specimen], ellipsoid, inamyloid, hyaline. Basidium (20˗)21˗22(˗25) × 7.16 µm, clavate, oil granules present when observed under KOH , tetrasterigmatic; sterigmata 3.58 × 0.7˗14 µm. Pleurocystidia present, 28.36˗32.2 × 7.16 µm, clavate, hyaline. Cheilocystidia club shaped to subfusiform, 41.4˗51.1 × 14.3˗18 µm, hyaline. Lamelle–edge fertile. Lamellar trama composed of parallel, septate, thin to thick wall, 3.58˗4 µm, broad, hyaline hyphae. Pileipellis composed of long, 36 × 3.6 µm, hyaline, septate hyphae. Stipitipellis composed of 53˗85 (˗161) × 3.58˗14.3 µm, parallel, septate, hyphae. Clamp–connection absent.

Ecology:

Gregarious to caespitose near the termite nests under the ground.

The amplified fragment of Termitomyces medius with the combination of primer set ITS1 (forward) and ITS2 (reverse) resulted in 293 bp long stretches among which the ITS1 region comprised 243 bp long sequence. The sequence generated for this study was submitted to GenBank with accession number KJ768983.

Antioxidant assays:

Total antioxidant capacity assay:

Total antioxidant capacity is measured by the formation of green phosphomolybdenum complex. The TmedPre results in the reduction of Mo (VI) to Mo (V) and form a green phosphate/Mo (V) complex. The colour intensity is determined with the maximal absorption at 695 nm. Ascorbic acid is used as standard. In the assay conducted it was found that 1mg of extract is as functional as approximately 0.12 ± 0.031 mg of ascorbic acid, expressed as 120 µg AAE. In comparison Russula albonigra was reported to have 30 µg AAE³².

Chelating effect on ferrous ions:

Ferrous ions have the ability to enhance free radical formation, as other transition metals. It has been recognized that transition metals are involved in both initiation and propagation of lipid peroxidation³³. Undoubtedly, the compounds which interfere with the catalytic activity of metal ions can also affect the preoxidative process. Ferrous ions are most powerful prooxidants among various species of metal ions³⁴. Chelating activity here was determined by the ferrozine assay. Ferrozin has the ability to quantitatively form complexes with Fe²⁺. If other chelating agents are present then this complex formation is disrupted with the decrease in the intensity of the coloured formazen³⁵. Lower EC₅₀ value indicates higher antioxidant activity. That of TmedPre was calculated from the graph in Fig. 1a, and it was found to be 0.54 ± 0.02 mg/ml (Table 1). EC₅₀ values of different polyphenolic extracts of mushrooms were found to be in the descending order of Russula laurocerasi > TmedPre > Russula albonigra^20,32.

Reducing power:

Reducing power of any compound can be an identifying character so that it can be considered as an antioxidant. Such a compound is TmedPre, as the assay revealed the fact that it could reduce Fe³⁺ to Fe²⁺. This change can be monitored at 700nm, by measuring the intensity of the Perl’s Prussian Blue colour. The reducing power of TmedPre was compared to that of BHA, a synthetic antioxidant. As seen from Fig. 1b, EC₅₀ value of TmedPre was 1.55 ± 0.15 mg/ml (Table 1). Polyphenol rich fraction of R. laurocerasi had EC₅₀ at a much higher concentration of 4.483 mg/ml²⁰. On the other hand R. albonigra was reported to have EC₅₀at 1.12 mg/ml³².

Superoxide radical scavenging assay:

Superoxide radical is known to be very harmful to cellular components and plays a major role in the formation of other reactive oxygen species such as hydroxyl radical, hydrogen peroxide and singlet oxygen in living system. Regarding data presented in Fig. 1c, the scavenging activity of TmedPre increased with increasing concentration of the polyphenol rich extract. The EC₅₀ value was found to be 0.425 ± 0.005 mg/ml for TmedPre (Table 1). On the other hand the polyphenol rich extract of R. laurocerasi and R. albonigra showed EC₅₀value at 1.56 ± 0.04 mg/ml and 0.74 mg/ml respectively^20,32. These results suggested that TmedPre exhibited scavenging effect on superoxide anion radical generation that could help prevent or ameliorate oxidative damage.

DPPH (2, 2–diphenyl–1–picrylhydrazyl) radical–scavenging activity:

DPPH has the capacity to accept an electron or hydrogen to earn stability. Antioxidants, on the other hand are capable of donating electron or hydrogen atom³⁶. A solution containing DPPH and methanol gives violet colour. But when electrons are donated to DPPH, then solution gets faded. Free radical scavenging is known as one of the mechanisms by which antioxidants inhibit lipid oxidation. This particular test is a standard assay in antioxidant activity studies and offers a rapid technique for screening the radical–scavenging ability of a specific extract³⁴. A lower absorbance at 517nm indicates a higher radical–scavenging activity of the extract³⁷. From Fig. 1d EC₅₀ value of TmedPre with regards to DPPH radical scavenging activity was seen to be 0.60 ± 0.01 mg/ml (Table 1), which was much lower than the EC₅₀ value (4.3 ± 0.3 mg/ml) of ascorbic acid, a potent scavenger. Though polyphenol rich fraction of R. albonigra had the ability to inhibit 50% DPPH radicals at 0.47 mg/ml³² but that of R. laurocerasi was reported to have EC₅₀ at 1.09 ± 0.03 mg/ml which was higher than that of TmedPre.

Hydroxyl Radical Scavenging Activity:

Hydroxyl radicals are the major active oxygen species capable of modifying almost every molecule in the living cells. This radical has the capacity to cause strand damages in DNA leading to carcinogenesis, mutagenesis, and cytotoxicity. Furthermore, this radical is also capable of stealing hydrogen atoms from unsaturated fatty acids leading to quick initiation of lipid peroxidation process^38,39.

Detection of Phenols and flavonoids by HPLC:

HPLC has been done to predict phenolic composition of a fraction extracted after thermal processing. As shown in Fig. 2a and 2b, eleven phenolic substances were analysed and two of them were detected in TmedPre. Our findings revealed that among the 11 standards that were run dominant phenolic compound in TmedPre was pyrogallol (11.21 µg/mg of dry weight of mushroom).

CONCLUSION:

At the end of the study we may conclude that the polyphenol rich extract of T. medius contains many bioactive compounds such as ascorbic acid, β–carotene, lycopene, flavonoids and phenols. It can also be said as a potent antioxidant for the results in various in vitro assays including ferrous iron chelating, ferric iron reducing, superoxide radical scavenging, DPPH free radical scavenging, hydroxyl radical scavenging, lipid peroxidation and total antioxidant activity. Hence, it can be suggested that this mushroom can be used as curatives to promote health for human and wealth for pharmaceutical industries.

ACKNOWLEDGEMENT:

The author PM is grateful and acknowledges Innovation in Science Pursuit for Inspired Research of Department of Science and Technology for financial support.

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Received on 04.04.2019 Modified on 28.04.2019

Research J. Pharm. and Tech 2019; 12(9):4287-4294.

DOI: 10.5958/0974-360X.2019.00737.6

Chelating	Reducing	SOD	DPPH	OH	Lipid peroxidation
0.54±0.02 mg/ml	1.55±0.15 mg/ml	0.425 ± 0.005 mg/ml	0.60±0.01 mg/ml	0.0195±0.0025 mg/ml	1.65±0.0 mg/ml

Ascorbic acid (µg/mg)	β–carotene (µg/mg)	Lycopene (µg/mg)	Total flavonoids (µg/mg)	Total phenol (µg/mg)
0.385 ± 0	0.0523 ± 0.01	0.020 ± 0.0141	1.6 ± 0.10	2.333 ± 0.15