Materials and Methods

The East Khasi hills constitute the most populated district among the seven districts of the State of Meghalaya (Fig. 1). The district occupies an area of 2 748 Km² and lies between 25°07" & 25°41" N and 91°21" & 92°09" E (^{Tripathi et al., 2005}). Forests occupy 35.34 % of the land area and have been exploited over the years for natural resources such as timber, medicinal plants, fodder, fuel wood and food resources like mushrooms (Tripathi et al., 2005).

Fig. 1: Mushrooms collection sites (forests and markets) in the East Khasi Hills district of Meghalaya, India. 

Table 1: Markets and forests in the East Khasi hills district of Meghalaya from which the mushrooms were collected during the study period 

Thematic services of Bhuvan (Geoportal of Indian Space Research Organisation, showcasing Indian imaging capabilities in Multisensor, Multi-platform and Multi-temporal domain), available at http://bhuvan-noeda.nrsc. gov.in/theme/thematic/theme.php, was used to obtain the Land Use Land Cover (LULC) data of the areas near which the local markets (Table 1) selling the mushrooms were located. Land Use Land Cover (LULC-50K) datasets with an overall accuracy ranging from 79 % (for agro-horticulture) to 97 % (for water bodies) of the area near the capital city of Shillong in Meghalaya State, India, from two timeframes i.e. 2005-06 and 2011-12 (http:// bhuvan-noeda.nrsc.gov.in/theme/thematic/ theme.php, accessed 04/01/2013) were visualized to study the changes of evergreen and semi-evergreen forest cover. Habitat information of the wild edible mushrooms exhibiting ecto-mycorrhizal symbiosis with trees or saprotrophic relationships with wood as reported in previous literature (^{Ogawa, 1976}; ^{Sabota, 1996}; ^{Joshi et al., 2013}) was correlated with the forest cover patterns available in the LULC datasets. Additional information on the annual rainfall distribution and agro-climatic zone characterization (^{APIB, 2013}http://megapib. nic.in) obtained from the Agriculture Planning and Information Bank (APIB) available at http://megapib.nic.in/, of the East Khasi hills district of Meghalaya along with the traditional mushroom harvesting knowledge of the ethnic tribes (^{Khaund & Joshi, 2013}), were taken into consideration to arrive at the best possible locations for the mushroom specimens in their wild original habitats in the forests (geographical coordinates in Table 1).

Mushroom samples were collected from the forests and the local markets (Fig. 1; Table 1) in the East Khasi hills district of Meghalaya during the study period from April to October, 2011. Sampling of around 4 000 specimens per distinct morphological type, and a total of four rounds of sampling were carried out during the entire course of the study period. The samples were wrapped in aluminium foil and stored in sterile containers prior to their transport to the laboratory for preservation and identification. Care was taken to avoid distortion of the fleshy fungi. The collected specimens were assigned a unique identifier accession number and were stored in the culture collection of the parent department in 4% formaldehyde (^{Tanti et al., 2011}). Alternatively, the specimens were also oven dried using protocols modified from ^{Gezer et al. (2006}) at 80 °C for five days, wrapped in aluminium foil and stored at -20 °C in labelled containers for further analysis. A part of the dried sample was used for the preparation of crude extracts. The morphological characters of the fruiting bodies were noted following the standard manuals for mushroom identification (^{Svrcek & Coxon, 1975}; ^{Purkayastha & Chandra, 1985}) and mycokeys available at www.mushroomexpert.com and www. mycokey.com.

Lentinula edodes sample preparation and estimation of bioactive compounds:One of the most widely harvested and used mushroom specimen is L. edodes (Accession no:PKSR14). In this study, even though samples were taken for different species in the area, this study selected this species as a representative mushroom for further analysis, to evaluate the nutritional and medicinal properties with relevance to its demand, common availability and health benefits to the ethnic tribes. For this, dried mushroom samples were ground into coarse powder (20 mesh). A total of 10 g of the powdered sample were extracted by stirring with 100 mL ethanol at 30 °C at 150 rpm for 24 h and filtered through Whatman No. 4 filter paper. The residue was then, extracted with two additional volumes of 100 mL of ethanol as described above. The combined ethanolic extracts were then rotary evaporated at 40 °C to dryness, redissolved in ethanol for the antioxidant assays to a final concentration of 10mg/ mL and stored at 4 °C for further use (^{Gezer et al., 2006}).

Total phenolic content: The total phenolic compounds (TPC) in the mushroom extract were estimated by a colorimetric assay (^{Singleton & Rossi, 1965}) with some minor modifications. For this, 1 mL of Folin Ciocalteu's phenol reagent was added to 1 mL of sample and after 3 min, 1 mL of saturated sodium carbonate solution was added to the mixture, and the volume was made to 10 mL with double distilled water. The reaction was kept in the dark for 90 min, after which the absorbance was read at 725 nm against ethanol as blank UV/Visible Spectrophotometer (Cecil, England). Gallic acid (0.01-1 mg/mL) was used as standard.

Total flavonoids: Flavonoid concentration was determined using standard procedures (^{Park et al., 1997}). The mushroom extract (1 mL) was diluted with 4.3 mL of 80 % aqueous ethanol and 0.1 mL of 10 % aluminum nitrate and 0.1 mL of 1 M aqueous potassium acetate were added. After 40 min at room temperature, the absorbance was measured at 415 nm with a UV/ Visible Spectrophotometer (Cecil, England) against ethanol as blank. Total flavonoid concentration was calculated using quercetin as standard (0.01-0.5 mg/mL).

Ascorbic acid: Ascorbic acid was determined according to the method of ^{Klein & Perry (1982}). The dried ethanolic extract (100 mg) was extracted with 10 mL of 1% metaphosphoric acid for 45 min at room temperature and filtered through Whatman No.4 filter paper. The filtrate (1 mL) was mixed with 9 mL of 2, 6-dichloroindophenol, and the absorbance was measured within 30 min at 515 nm with a UV/Visible Spectrophotometer (Cecil, England) against a double distilled water blank. Content of ascorbic acid was calculated using L-ascorbic acid as standard (0.01-0.5 mg/mL).

β-carotene and lycopene:β-carotene and lycopene were determined according to the method of ^{Nagata & Yamashita (1992}). The dried ethanolic extract (100 mg) was vigorously shaken with 10 mL of acetone-hexane mixture (4:6) for 1min and filtered through Whatman No.4 filter paper. The absorbance of the filtrate was measured at 453, 505, 645 and 663 nm with a UV/Visible Spectrophotometer (Cecil, England) against double distilled water blank. Content of β-carotene and lycopene was calculated according to the following equations:

Lycopene (mg/100mL) =

-0.0458A663 + 0.372A505 - 0.0806A453;

β-carotene (mg/100mL) =

0.216A663 - 0.304A505 + 0.452A453.

added. The mixture was shaken vigorously, left to stand for 30 minutes.

Anti-oxidant potential of the fungal extract by radical scavenging activity analysis: The capacity of the crude macrofungal extract to scavenge the 'stable' free radical DPPH was monitored according to the method of ^{Blois (1958}) with some modifications. To 100 µL of the extract in different concentrations, 5 mL of 0.1 mM ethanolic solution of DPPH was min in the dark, and the decrease in absorbance was measured at 517 nm using a UV/ Visible Spectrophotometer (Cecil, England). BHT was used as positive control and the blank used was ethanol. The radical scavenging activity was measured as a decrease in absorbance of DPPH and calculated as:

DPPH scavenging effect (%) = (Ad - As/Ad) × 100,where Ad is the absorbance of the control reaction containing DPPH solution, and As, the absorbance in presence of the extract or BHT.

Nutritional composition analysis: The mushroom sample was analysed for chemical composition (moisture, protein, fat, carbohydrates and ash) using the AOAC procedures (¹⁹⁹⁵). The crude protein content (N* 4.38) of the samples was estimated by the macro Kjeldahl method; the crude fat was determined by extracting a known weight of powdered mushroom sample with petroleum ether, using a Soxhlet apparatus; the ash content was determined by incineration at 600±15 °C; reducing sugars were determined by DNS (dinitrosalicylic acid) method. Total carbohydrates were calculated as follows:

Total carbohydrates = 100 - (g moisture + g protein + g fat + g ash).

Total energy was calculated according to the following equation:

Energy (kcal) = 4 × (g of protein + g of carbohydrate) + 9 × (g of lipid).

Evaluation of anti-inflammatory properties through the human Red Blood Cell (HRBC) membrane stabilization test: Institutional ethical guidelines of North-Eastern Hill University, Shillong, Meghalaya, India, were followed. Fresh whole human blood (10 mL) was collected from a healthy human volunteer who had not taken any anti-inflammatory drugs for two weeks prior to the experiment; sample was transferred to heparinised centrifuge tubes. The tubes were centrifuged at 3 000 rpm for 10 min and were washed three times with equal volume of normal saline. The volume of blood was measured and reconstituted as 10% v/v suspension with normal saline (^{Govindapppa et al., 2011}). The reaction mixture consisted of 2 mL of hypotonic saline (0.25 % w/v NaCl), 1 mL of 0.15 M phosphate buffer (pH 7.4), 0.5 mL of test solution (10 mg/mL) in normal saline and 0.5 mL of 10 % HRBC in normal saline. For control, 0.5 mL of normal saline was used instead of test solution. Aspirin was taken as a standard drug (1 mg/ mL). All the centrifuge tubes containing reaction mixture were incubated in water bath at 56 °C for 30 min. At the end of the incubation, the tubes were cooled under running tap water. The reaction mixture was centrifuged at 3 000 rpm for 5 min and the absorbance of the supernatants was taken at 560 nm with a UV/Visible Spectrophotometer (Cecil, England) against buffer as blank. The experiment was performed in triplicates for all the test samples. Percent membrane stabilization activity was calculated by the formula given below:

% Inhibition of haemolysis = (Ac - As/Ac) × 100

where Ac is the absorbance of the reaction mixture without the extract or Aspirin, and As is the absorbance in the presence of extract or Aspirin.

Results

Habitat search of wild edible mushrooms using GIS applications: The observations of the Land Use and Land Cover datasets of the study area i.e. Shillong region in the East Khasi Hills District of the state of Meghalaya in Bhuvan for two timeframes i.e. 2005-06 and 2011-12, reveals that the area classified as Urban has increased from 2005 to 2012 (http://bhuvan-noeda.nrsc.gov.in/theme/thematic/theme.php, accessed 04/01/2013). There is also a reduction and change in the areas classified as Evergreen and Semi-evergreen forests during the same time span (http://bhuvan-noeda.nrsc.gov.in/theme/thematic/theme. php Dataset: Meghalaya District East Khasi Hills). Correlation of the habitat information of ecto-mycorrhizal or saprotrophic wild edible mushrooms (WEM) using Lentinula edodes as a representative genus with the forest cover patterns available in the LULC datasets helped in the selection of the forest areas likely to harbour WEM (Table 1). The annual rainfall distribution pattern of Meghalaya reveals the present study area (Table 1; http://bhuvan-noeda.nrsc. gov.in/theme/thematic/theme.php) to be having low to medium rainfall annually (APIB, 2013 http://megapib.nic.in/agro_atlas_rd.htm ). The study area is also shown to be characterized into the Cold, Moisture (C, M) surrounded by the Cold, Wet (C, W) agro-climatic zone in its periphery (APIB, 2013 http://megapib.nic.in/ agro_atlas_azm.htm). This additional information obtained from the Agriculture Planning and Information Bank of the East Khasi Hills District of Meghalaya was in agreement with the type of climatic condition that favoured the growth of ecto-mycorrhizal and saprotrophic mushrooms in the presence of evergreen tree cover.

Morphological characteristics: Based on the comparison of the morphological features of the collected specimens with standard mushroom identification manuals and mycokeys, the specimens were tentatively identified (Table 2). Out of the ten different specimens, the sample PKSR14 (Lentinula edodes)was further subjected to biochemical analysis based on its demand and availability in the forest and local markets.

Content of bioactive compounds: The major bioactive components of the extract were the total phenolic content (TPC) which was 10.08±0.13 mg/g. The total flavonoid content was found to be 0.91±0.16 mg/g. Ascorbic acid was found in small amounts (0.72±0.04 mg/g), and β-carotene (6.00±0.37 μg/g) and lycopene (2.12±0.27 μg/g) were found in vestigial amounts (<10 μg/g).

Estimation of anti-oxidant potential: Scavenging effects on DPPH radicals increased with the increase in concentrations of the extract and BHT (Fig. 2). At 10 mg/mL the sample extract of the isolate Lentinula edodes PKSR14 scavenged up to 61.33 % of the free radicals showing effective radical scavenging capacity (Fig. 2).

Table 2: Identity of the collected wild edible mushroom (WEM) specimens 

Nutrient composition: The results of the nutrient composition assays of Lentinula edodes PKSR14 revealed it to be rich in carbohydrates and total energy. The carbohydrate content was determined to be 58.69±4.35 g/100 g, while the total energy value was estimated to be 298.63±17.92 Kcal. The total fat and crude protein content were found to be 3.54±0.06 g/100g and 8.00±0.57 g/100 g, respectively. The moisture content was found to be 23.33±4.37 g/100, while the ash content was determined to be 6.43±0.23 g/100 g.

Fig. 2: Scavenging activity (%) on DPPH radicals of ethanolic extracts from Lentinula edodes (PKSR14). Each value is expressed as mean ± SEM (n = 3). 

Evaluation of anti-inflammatory properties: The inhibition of HRBC membrane lysis i.e, stabilization of HRBC membrane was taken as a measure of the anti inflammatory activity of the extract. Ethanolic extract of Lentinula edodes PKSR14 showed effective anti-inflammatory activity of 50±4.40 % at 10 mg/mL concentration. The standard drug Aspirin showed higher anti-inflammatory activity of 81.66±0.57 % at 10 mg/mL concentration.

Discussion

The habitats of most wild edible mushrooms in Meghalaya are undergoing changes due to urbanization, deforestation and climate change leading to a reduced availability of these mushrooms in the traditional rural markets (Khaund &^{Joshi, 2013}). These observations are corroborated by visualization of the land use and land cover patterns of the region in Bhuvan. Although a variety of spatial distribution models have been applied to map many different plants (^{Franklin, 1998}; ^{Iverson et al., 1999}; ^{Austin, 2002}; ^{Zaniewski et al., 2002}; ^{Peltoniemi, 2005}), similar reports for mushrooms have been very scarce. In the present study, the principles of the GIS based expert system was used to successfully predict the habitats of the wild edible ecto-mycorrhizal and saprotrophic mushrooms sold extensively in the rural markets (Khaund & Joshi, 2013). The prediction ability of the GIS based approach was validated by field surveys for collection of specimens for the present study.

^{Kranabetter et al., (2002}) using a similar approach estimated the habitat of American matsutake (Tricholoma magnivelare) using aerial photos in collaboration with summarized ecological descriptions. ^{Yang et al., (2006}), attempted to model the spatial distribution of matsutake mushrooms at a fine scale in a more complex environment i.e., mountainous terrains of Yunnan in China, using a logistic regression and GIS expert system. They reported that the overall mapping accuracy of their GIS expert system was slightly better than the logistic regression approach (70.37 % versus 65.43 %). Further, they observed that, unlike the logistic regression model, developing the GIS expert system required no field-based samples. Presently, efforts are on to incorporate local mycological knowledge as gathered in the previous study (Khaund &^{Joshi, 2013}) in decision making process of a GIS expert system (^{Skidmore et al., 1996}) for creating a parsimonious model of mushroom distribution that will form the part of a mushroom specific non-wood forest resource mapping and search system for Meghalaya, India.

Phenolics exhibit a plethora of antibacterial, anti-inflammatory, antihyperglycemic and antioxidant actions (^{Yaltirak et al., 2009}; ^{Palanisamy et al., 2011}). The total phenolics act as strong antioxidants and free radical scavengers that help in chelation of heavy metals along with interaction with enzymes, adenosine receptors and biomembranes (^{Saija et al., 1995}). There are reports on the total phenolic content (TPC) and total flavonoid content (TFC) in the crude extract of Lentinula edodes (^{Cheung et al., 2003}; ^{Reis et al., 2012}). In the present study, the TPC (10.08 mg GAE/g extract) of Lentinula edodes PKSR14 was higher than that obtained for methanolic extracts of Chinese samples (8.51 mg GAE/g extract) (Cheung et al., 2003). Ascorbic acid and carotenoids such as β-carotene and lycopene are known to be potent naturally occurring antioxidants (^{Sies & Stahl, 1995}; ^{Rao & Agarwal, 1999}; ^{Wilson, 2005}). Similar concentrations of ascorbic acid, β-carotene and lycopene in wild edible mushrooms have been reported previously (^{Barros et al., 2007}; ^{Barros et al., 2008a}, ^b). The high content of total phenols in the L. edodes PKSR14 extracts might account for the better results found in their radical scavenging effect on DPPH radicals(^{Ferreira et al., 2007}).

In a similar study on the antioxidant properties of edible mushroom extracts from China, ^{Cheung et al. (2003}) reported the DPPH radical scavenging activity of Lentinula edodes to be 29.40 % at a concentration of 9 mg/mL. In another study it was reported that alcoholic extracts of Lentinula edodes scavenged up to 50 % at 6.43 mg/mL (^{Reis et al., 2012}). The present findings indicate that Lentinula edodes PKSR14 has high radical scavenging activity (61.33 %) which may be primarily due to the presence of various natural antioxidants in the crude extracts.

Moisture content and water activity affect the texture and participate in the short shelf life of fruiting bodies (^{Kalač, 2009}). The macronutrient profile, in general, revealed that Lentinula edodes PKSR14 has higher amount of protein and carbohydrate than fat. This high protein and carbohydrate with a low fat characteristic of the wild edible mushrooms has been reported previously (^{Diez and Alvarez, 2001}; ^{Agrahar-Murugkar and Subbulakshmi, 2005}) which makes them suitable as health promoting foods. However, the differences between the nutrient composition of Lentinula edodes PKSR14 as revealed in the present study andthat of other Lentinula edodes reported across the world (^{Manzi et al., 1999}) can probably be attributed to a number of environmental and genetic conditions that guide the growth of this mushroom species (^{Manzi et al., 2001}).

Anti-inflammatory activity of metabolite extracts of medicinal plants has been studied widely (^{Leelaprakash & Mohan Dass, 2011}). It has been reported that the presence of tannins (^{Mota et al., 1985}), flavonoids (^{Huang et al., 2009}), triterpenoids (^{Della et al., 1994}) and phenolic compounds (Song et al., 2008) in the extracts are responsible for anti-inflammatory activity. In the present investigation, it was seen that the extracts of Lentinula edodes PKSR14 showed potential anti-inflammatory capacity in-vitro, suggesting that it could be regarded as a potential source of natural antiinflammatory agents.

The unique and novel mushroom specimens from the selected locations, which are yet to be fully explored, have been shown to be rich sources of bioactive compounds, antioxidants, anti-inflammatory substances and nutrients that make them potential candidates for further development as functional foods. Technological advancements such as GIS and remote sensing based open access platforms like Bhuvan and Agriculture Planning and Information Bank (APIB), developed under the Space Research programme have been successfully tested in the present investigation to locate the natural habitats of the wild edible mushrooms which are sold extensively in the rural markets of Meghalaya. The explored technology offers scope to map the wild non-woody forest resource which can have application in efforts designed for conservation, bioprospection and cultivation of such resources.