Exploring a novel edible mushroom Ramaria subalpina : Chemical characterization and Antioxidant activity

Background: During macrofungal inventorisation from an unexplored subalpine forest of Sikkim Himalayas, a new edible mushroom, Ramaria subalpina was found and documented in consultation with the forest dwellers. Objective: The aim of the present study was to understand the antioxidative potentiality and bioactive constituents of a novel taxon, Ramaria subalpina, that is highly prized by the ethnic people of subalpine Sikkim Himalayas, India, for its flavor, texture and gastronomic delicacy since time immemorial. Methods: Chemical composition and antioxidant properties of methanolic extract of dried basidiocarps were assessed using HPLC and several in vitro assay systems. Results: Methanolic extract had phenolics in highest amount, among which pyrrogallol was identified. It also showed potent antioxidant activity. Conclusion: The present study suggests that Ramaria subalpina has strong medicinal prospects. This novel mushroom can safely be added to the world’s edible mushrooms list.


INTRODUCTION
Rural communities of India, utilize certain local wild mushrooms for food and medicine, but such indigenous knowledge is either not reported or poorly documented and unsystematically recorded.Claims of their efficacy to be considered as food or medicine need to be scientifically verified prior to their acceptance.It is therefore necessary to know and correctly identify these mushroom species, used by the locals as food or medicine.][3] Choice of food with their myriad usages and cuisine are the signature of cultural, regional and national identity.They are key elements for the dietary patterns in different countries and are consequently important in accurately estimating population dietary intakes.However, throughout Europe, traditional foods are threatened with extinction due to altered lifestyles, and nutritional information of these foods is missing from most current food composition databases. 4,5n recent days, consumers are looking for new foods with various organoleptic characteristics for their daily lunch basket.Traditional foods such as mushrooms might fulfill this demand, as they are quite rich in carbohydrate, fiber and protein with all essential amino acids and have a low fat content. 6In a general approxi-mation, the fruit body of a mushroom contain about 56.8% carbohydrate, 25.0% protein, 5.7% fat and 12.5% ash on a dry weight basis. 7In addition, edible mushrooms are also rich in various polyphenolic compounds that are recognized as an excellent antioxidant due to their ability to scavenge free radicals by single-electron transfer.The enhancement of antioxidant systems for the prevention of cellular oxidative damage via consumption of antioxidant rich foods is of great interest. 8n search of wild edible mushrooms (WEM) that are commonly prized by the local forest dwellers across the country, we came across quite a number of mushroom taxa that has been previously identified and reported therein. 3,9,10Ramaria subalpina K. Das & K. Acharya [commonly called as 'thokrechyau' (Nepali; 'thokre' = in bunch and 'chyau' = mushroom)], a newly described taxa, 10 traditionally consumed as an edible mushroom (Figure 1A) by the ethnic people of subalpine Sikkim Himalaya for its flavor, texture and gastronomic delicacy since time immemorial was explored to understand its antioxidative potentiality and bioactive constituents.

Preparation of methanolic extracts
Basidiocarps of the collected specimen were cleaned to remove residual compost and dried at a maximum temperature of 45 ± 3°C.Dried basidiocarps were ground to obtain fine particles.One gm of the powder was extracted by stirring with 50 ml of methanol for overnight and subsequently separated by Whatman filter paper.The residue was then re-extracted with 30 ml of methanol and the combined methanolic extracts were evaporated at 40°C (Rotavapor R3 Buchi, Switzerland) to dryness.The methanolic fraction was stored at -20°C in a dark bottle until analysis, for no more than 1 month.A stock solution of 20 mg/ml of the fraction was prepared.Successive dilutions were made to evaluate chemical characterization and antioxidant activity. 11

Determination of total phenolic compound
Total phenolics were measured using Folin-Ciocalteu reagent. 121 ml of methanolic extract was mixed with 1 ml Folin-Ciocalteu reagent and incubated for 3 minutes at room temperature.After incubation, 1 ml of 35% saturated Na 2 CO 3 solution was added in the reaction mixture and volume was adjusted to 10 ml with distilled water.The reaction mixture was incubated in dark for 90 min, after which the absorbance was read at 725 nm by a spectrophotometer.Gallic acid was used as standard.Total phenol content of the sample was expressed as µg of gallic acid equivalent per mg of extract.

Determination of total flavonoid content
Flavonoid concentration was determined following the method as described by Adebayo et al. 13 1 ml methanolic extract was diluted with 4.3 ml of 80% aqueous methanol and subsequently, 0.1 ml of 10% aluminum nitrate and 0.1 ml of 1M aqueous potassium acetate were added to it.After 40 min of incubation at room temperature, the absorbance was determined spectrophotometrically at 415 nm.Total flavonoid concentration was calculated using quercetin as standard.

Determination of ascorbic acid content
Ascorbic acid was determined by titration as described by Rekha et al. 15 With little modification.Standard ascorbic acid (0.1 mg/ml) was taken in a conical flask and made up to 10 ml by 0.6% oxalic acid.It was titrated with a dye 2, 6-dichlorophenol indophenol (21 mg sodium bicarbonate and 26 mg of dye in 100 ml water).The amount of dye consumed (V1 ml) is equivalent to the amount of ascorbic acid.The methanolic extract (w µg/ml) was similarly titrated with the dye (V2 ml).The amount of ascorbic acid was calculated using the formula-Ascorbic acid (µg /mg) = ({(10 µg/V1 ml) × V2 ml} × w µg) × 1000

Total antioxidant activity
The total antioxidant capacity was determined as described by Prieto et al. 16 With little modification.In brief, 0.3 ml of methanolic extract with varying concentration (0.1-1 mg/ml) was added to 3 ml of the reaction mixture (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate).The tubes were capped and incubated in a thermal block at 95°C for 90 min.After cooling to room temperature, the absorbance of the aqueous solution of each was measured at 695 nm against a blank.Ascorbic acid was used as standard and the total antioxidant capacity is expressed as equivalents of ascorbic acid.

DPPH radical scavenging activity
Radical scavenging activity was determined using a DPPH assay as described by Shimada et al. 17 Various concentrations of the methanolic extract (0.25, 0.5 and 1 mg/ml) were added to 2 ml of 0.004% methanol solution of DPPH (w/v).After 30 min incubation at room temperature in dark, the absorbance was read against a methanol blank at 517 nm.EC 50 value is the effective concentration at which DPPH radicals were scavenged by 50%.Ascorbic acid was used for standard.The degree of scavenging 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.

Ferrous ion chelating ability
Chelating ability was determined according to the method of Dinis et al. 18 Different concentrations of methanolic extract (0.25, 0.5 and 1 mg/ml) were mixed with 0.1 ml of 2 mM ferrous chloride.The reaction was initiated by addition of 0.2 ml of 5 mM ferrozine.After 10 min at room temperature, the absorbance of the mixture was determined at 562 nm against a blank.EDTA was used as positive control.EC 50 value is the effective concentration at which ferrous ions were chelated by 50%.The percentage of inhibition of ferrozine-Fe 2+ complex formation is given by this formula:  Table 1: Total phenolic compound, flavonoid, ß-carotene, lycopene and ascorbic acid concentrations of the methanolic extract of Ramaria subalpina.
A0 and A1 were the absorbance of the control and absorbance in presence of sample respectively.

Determination of reducing power
The reducing power of the methanolic extract was determined according to the method of Oyaizu 19 .Different concentrations of the fraction (0.2, 0.4 and 0.6 mg/ml) were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and 2.5 ml of 1% potassium ferricyanide.The mixture was incubated at 50ºC for 20 min and then 2.5 ml of TCA (10%) was added to the mixture.2.5 ml of the solution was mixed with distilled water (2.5 ml) and FeCl 3 (0.5 ml, 0.1%).The reaction mixture was incubated for 15 min and absorbance was measured at 700 nm.A higher absorbance indicates a higher reductive capability.Ascorbic acid was used as standard.EC 50 value indicates the effective concentration at which the absorbance was 0.5 for reducing power.

Chemical composition
Table 1 shows total phenolic compound, flavonoid, ß-carotene, lycopene and ascorbic acid concentrations of the methanolic extract of Ramaria subalpina.Total phenolic compound are the major bioactive component found in extract; followed by flavonoid, ascorbic acid, ß-carotene, and very small concentration of lycopene.The results revealed that R. subalpina had exhibited quite higher amount of total phenolic compound and total flavonoid content as compared to previously reported methanolic extract of edible Ramaria flava (Schaeff.)Quél.which showed 10.51 ± 0.47 µg/mg of total phenolic compound and 0.51 ± 0.01 µg/mg of total flavonoid. 20PLC helps to predict phenolic composition of the extract.Figure 2A depicts a typical HPLC chromatogram of eleven phenolic compounds and Figure 2B represents HPLC chromatogram of the extract at 10 mg/ml concentration.The results showed a qualitative profile of extract where 1 peak had been identified.The component in the fraction was found to be pyrrogalol that being present as much as 6.287 ± 2.473 µg/mg of extract.In addition, two unrecognized phenolic substances (λ max in inset) had been detected in the fraction at 10.697 min and 11.692 min possessing area of 3966.797 and 587.216 respectively.

Antioxidant activity of the mushroom fraction
All living organisms are equipped with stress-response systems that regulate the processes of somatic maintenance and repair. 21Administration of dietary supplements help to combat oxidative stress and related ailments.Result showed that, 1 mg of Ramaria subalpina was equivalent to 230 µg of ascorbic acid.Total antioxidant activity of R. subalpina suggests the electron donating capacity, which could act as a radical chain terminator by transforming reactive free radicals into more stable non-reactive products.DPPH radical scavenging activity can be used to evaluate the anti oxidant activity of an extract in a short span of time at room temperature by converting it to 1,1-diphenyl-2-(2,4,6-trinitrophenyl) hydrazine.The amount of yellowing indicates the scavenging potentials of an antioxidant compound. 22,23The utilization of stable DPPH radical has the benefit for being unaffected by side reactions, such as enzyme inhibition and metal chelation. 24Thus a lower absorbance at 517 nm indicates a higher radical scavenging activity of the extract.Result indicated that R. subalpina exhibited significant radical scavenging activity with EC 50 value of 0.3±0.05mg/ml (Figure .1B).As compared to the previously reported R. flava (94.78 ± 0.06% at 12 mg/ml) and R. formosa, 20,25 the methanolic extract of R. subalpina showed grater electron donating capacity.Ferrous ions are considered to be effective pro-oxidants in food system that increases the risk of free radical damage and cancer.So, moderate ferrous ion chelating abilities would be beneficial for the human life. 26,27owndhararajan et al. 28 Reported that chelating agents are helpful as secondary antioxidants, as they decreases the redox potential and thereby stabilises the oxidised forms of metal ions.Figure .1C reveals that the R. subalpina exhibited a marked capacity for iron binding ability of 50% at a concentration of 0.46 ± 0.03 mg/ml.Reducing power of any bioactive compound is directly related to the electron donating capacity and can reduce the oxidized intermediates of lipid peroxidation processes in such a way so that they can act as primary and secondary antioxidants. 29,30Presence of reducers causes the conversion of Fe 3+ /ferricyanide complex to the ferrous form.Figure 1D revealed that R. subalpina showed reducing power of 0.5 at concentration of 0.44 ± 0.02 mg/ml.

CONCLUSION
Exploration of the medicinal properties of dried basidiocarps of Ramaria subalpina were subjected for preparation of heat stable phenol rich extract using methanol as solvent system.The fraction showed its potentiality in chelating ferrous ion, DPPH radical scavenging, reducing power and total antioxidant.Total phenols were also found to be the key bioactive component of the extract; followed by flavonoid, ascorbic acid, ß-carotene, and lycopene.Molecular phenolic profiling of the extract through HPLC-UV indicated probable existence of pyrrogalol in a higher amount.All these results led to consider the potentiality of the studied mushroom to have a good source of bioactive molecules such as phenolic components for dietary supplements and functional food.

FIGURE 1 :
FIGURE 1: Ramaria subalpina (A) Fresh basidiocarps in the field (B) DPPH radical scavenging activity (C) Ferous ion chelating ability (D) Reducing power.Results are the mean ± SD of three separate experiments, each in triplicate.