Indian Journal of Traditional Knowledge Vol. 15(1), January 2016, pp. 143-148

Nutritional and antioxidant properties of wild edible macrofungi from North-Eastern Uttar Pradesh, India

Pratima Vishwakarma, Pooja Singh* & N N Tripathi Bacteriology and Natural Pesticide Laboratory, Department of Botany, DDU Gorakhpur University, Gorakhpur 273009, India E-mail: [email protected]

Received 24 July 2014, revised 29 July 2015

Eastern part of Uttar Pradesh, India is home to diverse form of macrofungi some of which are excellently edible and commonly used by local peoples as food and medicine. The nutritional composition and antioxidant activity of four wild edible macrofungi, viz. gambosa, , procera and Tuber aestivum collected from different areas of Gorakhpur were evaluated. The total phenol content of each along with some bioactive compounds was analysed. The nutrient composition of these macrofungi showed that they are rich source of protein and carbohydrate. Protein, carbohydrate, lipid, fibre, moisture and ash ranged from 31.40-49.05%, 41.25-65.00%, 0.27-1.08%, 6.41-17.97%, 54.75-76.97%, 2.64-10.34%, respectively on dry weight basis. Total phenol contents (major antioxidants components) ranged from 23.89-38.37GAEmg/gm in different macrofungi. Current study confirms that macrofungi are healthy source of food and medicine can also be use as alternative source of protein in human diet.

Keywords: Edible , Antioxidant, Bioactive compounds, DPPH, Tharu, Bhar, Kewat, Bhuj tribes IPC Int. Cl.8: A01G 1/04, C09K 15/00, C07G, C01, C07

Traditional medicine or Ethnomedicine is a healthcare integration into mainstream society and urbanization, practice that has been transmitted orally from they are rapidly losing their traditional knowledge and generation to generation through traditional healers culture4. have a long association with with an aim to cure different ailments, and is strongly mankind and provide profound biological and associated to religious beliefs and practices of the economical benefit. The wild mushrooms have been indigenous people1. Since the beginning of human traditionally consumed by man with delicacy civilization man has been using many herbs and probably, for their taste and pleasing flavour. They herbal extracts as medicine. The classical Indian texts have rich nutritional value with high content of Rig-Veda, Atharvaveda, Charak samhita and Sushruta proteins, vitamins, minerals, fibres, trace elements samhita are the evidence of the use of plants by our and low calories and cholesterol. ancestors. It indicates that the herbal medicines have In order to assess the consumption of traditionally been derived from rich traditions of ancient important and indigenous medicinal macrofungi civilization and scientific heritage. Among the ancient survey was carried out during January to December civilizations, India has been known to be rich (2011-2014) in the different areas of Gorakhpur storehouse of medicinal plants and macrofungi. The district and its adjoining areas. These medicinal forests in India are the principal repository of large macrofungi were used by Bhar, Kewat, Bhuj, Tharu number of medicinal macrofungi, which are largely and local villagers of this region. The ethanobotanical collected as raw materials for manufacture of drugs2. information was gathered through several visits, The local tribes are largely self contained, ritually questionnaire, group discussion with various groups sanctioned way of life where they practice utilization and local peoples. plants and macrofungi for curing many diseases3. Traditionally the tribes lived in and subsisted on the Methodology forests, but with increasing loss of forest areas, Sample collection, identification and processing —————— Samples of four wild edible macrofungi, viz. *Corresponding author , Calocybe indica, Macrolepiota 144 INDIAN J TRADIT KNOWLE, VOL 15, NO. 1, JANUARY 2016

procera and Tuber aestivum were collected from contents of the phenolic compound were expressed as forest regions of Gorakhpur during the months of July mg Gallic acid equivalent/gm dry weight. to August in 2012-13. The macrofungal samples were identified on the basis of their macro and microscopic Statistical analysis characteristic and following several authors5,6,7,8 and Experimental values are given as mean ± standard was also confirmed by Prof. Kamal (Retired Prof. deviation (SD). Statistical significance was determined Dept. of Botany, DDU Gorakhpur University). by one way variance analysis (ANOVA). Difference Collected samples were cleaned for dust and foreign at p<0.05 were considered to be significant. materials, dried in shadow at room temperature for 14 days. Samples were then made into fine powder Results with help of grinder (0.5 mm sieve) for their Table 1 clearly indicate the morphological biochemical assessment. characteristics, date and place of collection of macrofungi and voucher number of each macrofungi. Biochemical assay It is very much clear from table that two macrofungi, Moisture content was determined by direct oven viz. Calocybe gambosa and Tuber aestivum was drying method. The loss in weight after oven drying mycorrhizal in nature while Calocybe indica and 2 gm of the sample at 110°C to constant weight was Macrolepiota procera was saprobic (Fig. 1 (a-d). expressed as percent moisture content9. Protein Table 2 represents various uses of collected content of macrofungal samples were determined by macrofungi. Each macrofungi collected had its own the method of Adedayo & Rachel10. Carbohydrate important medicinal property. These macrofungi were was estimated by Anthrone method11. Crude fibre used by different tribes and many villagers for food as content was evaluated following Alam et al.12 while well as to cure various ailments, like C. gambosa used lipid was estimated by the method of Gbolagade by Kewat and Tharu tribes to increase immunity, et al.13. Ash of the sample was estimated by the C.indica by Kewat tribes to relief from stomach pain, method of Gang et al. 14. M. procera by Bhuj and Bhar to cure diabetes and also to relief from blood pressure and T. aestivum by Antioxidant activity Bhar, Kewat, Bhuj, Tharu and local villagers for DPPH (2, 2'- diphenyl-1-picrylhydazyl) and proper healthcare. reducing power of macrofungi was calculated 15 In present study some species of mushrooms such according to the method adopted by Barros et al. . as C. gambosa, C. indica, M. procera and T. aestivum were investigated for different nutrient content. Total bioactive compounds β- Carotene and lycopene was determined by the Comparative nutritional value of all collected process described by Loganathan et al.16. Contents of macrofungi is listed in Table 3. Protein content β-Carotene and lycopene were calculated according to showed great variation in all tested mushroom. the following equations: Lycopene (mg/100mL) = 0.0458 A663 + 0.372 A505 - 0.0806 A453 β- Carotene (mg/100mL) = 0.216 A663 - 0.304 A505 + 0.452 A453. The ascorbic acid content was determined titrametrically using 2, 6 Dichlorophenol Indophenol15 methods. The amount of ascorbic acid in each extract was calculated from the equation: mg of ascorbic acid per 100gm = titre x Dye factor x volume made × 100 Aliquot of extract x weight.

Total phenolic compounds in the ethanol extracts Fig. 1—Macrofungi picture a: Calocybe gambosa, b: Calocybe 17 were determined using Folin–Ciocalteu method . The indica, c: Macrolepiota procera, d: Tuber aestivum VISHWAKARMA et al.: NUTRITIONAL AND ANTIOXIDANT PROPERTIES OF MUSHROOMS 145

Table 1—Family, Ecology, Fruiting body characteristic and Ethnomycological use of wild edible macrofungi Macrofungi Voucher Family Date and place of Ecology Fruiting body number collection Calocybe gambosa DDUNPL259 7/7/12, Symbiotic, in group, Cap convex, bare, (Fr.) Donk Sahjanwan tehsil, in association with Gill free, white Piprauli block, Azadirachta indica Spore smooth, ellipsoid, Jaitpur village and Ficus bengalensis 4.3-5.9 × 3.5-4 µm Calocybe indica DDUNPL260 Lyophyllaceae 18/6/13, Sadar tehsil, Saprobic, in group Cap convex, Stipe bare, Gill Purkay. & Jangle Kaudia block, on manure and husk free, Spore print white, A. Chandra Rasoolpur village residue Spore smooth, ellipsoid, 4-6 × 3-3.9 µm Macrolepiota DDUNPL223 23/7/12, Sadar tehsil, Saprobic, in group in Cap umbonate, Stipe with procera (Scop.) Khorabar block, forest, on decaying ring, Gills free, Spore print Singer Kushmi Jungle litter white, spore smooth, ellipsoid 15-18 × 9-11 µm Tuber aestivum DDUNPL161 Tuberaceae 10/7/13, Sadar tehsil, Saprobic, under Truffle fungi, Brown to black Vittad. Khorabar block, broad leaf trees on colour, form ascocarp, Spore Kushmi Jungle, calcareous soil print black, spore reticulate, ellipsoid, non septate 24-30 × 16-25 µm, paraphyses absent

Table 2—Ethnomedical uses of mushroom Mushroom Tribes Parts use Uses Calocybe gambosa Kewat Fruiting body To increase immunity Calocybe indica Kewat Fruiting body Relief from stomach pain Macrolepiota procera Bhuj, Bhar Fruiting body Used in diabetes and blood pressure Tuber aestivum Bhar, Kewat, Bhuj, local villagers Gleba Commonly used as healthcare

Table 3—Biochemical assay of macrofungi (%) Macrofungi Protein (%) Carbohydrate (%) Lipid (%) Fibre (%) Moisture (%) Ash (%) C. gambosa 31.44±0.22 55.12±0.66 0.29±0.32 7.15±0.11 74.90±0.09 9.20±0.19 C. indica 33.25±0.35 52.13±0.54 0.27±0.07 13.55±0.37 69.45±0.03 10.34±0.11 M. procera 49.05±0.90 65.00±0.44 1.08±0.91 6.41±0.07 76.19±0.06 7.14±0.22 T. aestivum 31.40±0.81 41.25±0.09 0.48±0.39 17.97±0.13 54.75±0.23 2.64±0.05 Values are means ± SEM for groups of 3 observations with their standard errors

Protein content ranged from 31.40-49.05%, lowest in more in M. procera (0.098 mg/gm) followed by C. indica while highest in M. procera. In present C. indica, (0.056mg/gm), T. aestivum (0.042 mg/gm) work carbohydrate content ranges from 41.25-65.00% and C. gambosa (0.014mg/gm). Phenolic compounds on dry weight basis. Highest carbohydrate content are found to be major bioactive compound found was found in M. procera while lowest in T. aestivum. in food products which ranged from 23.89 to Tested samples contain low lipid content. In present 38.379 GAEmg/gm. C.indica extracts showed study lipid content ranged from 0.27-1.08%. Fibre the highest phenolic content (38.379 GAE/gm), content also ranged from 6.41 (M. procera)-17.97 while M. procera showed lowest phenol content (T. aestivum) % while ash was in range of 2.64% (23.89 GAE/gm). β carotene and lycopene were (T. aestivum) - 10.34% (C. indica). Moisture content found in very small amount. of sample ranged from 54.75-76.19%. M. procera Table 5 shows the result of DPPH scavenging shows highest moisture content (76.19%) as activity of different edible mushrooms tested. Result compared with other species. revealed that C. indica extract showed the highest The amount of ascorbic acid, total phenol and total DPPH radical scavenging activity (lowest EC50 value), carotenoids expressed as β-carotene and lycopene followed by C. gambosa, M. procera and T. aestivum, were considerably different among three studied respectively. β carotene bleaching activity of different samples (Table 4). The Ascorbic acid was found to be mushroom species are presented in Table 5. C. indica 146 INDIAN J TRADIT KNOWLE, VOL 15, NO. 1, JANUARY 2016

Table 4—Bioactive compound analysis of collected macrofungi Sample β carotene µg/mg Lycopene µg/mg Phenol GAEmg /gm Ascorbic acid mg/gm C. gambosa 1.279±0.32 0.088±0.13 36.282±0.28 0.014±0.88 C. indica 1.939±0.44 0.098±0.43 38.379±0.65 0.056±0.92 M. procera 0.025±0.61 0.650±0.58 23.890±0.81 0.098±0.98 T. aestivum 0.093±0.48 0.160±0.60 30.861±0.94 0.042±0.59 Values are means ± SEM for groups of 3 observations with their standard errors

Table 5—EC50 of collected macrofungi for their antioxidant great variation. Similar results were also reported by activity Akyüz & Kirbaǧ 23. In present study tested samples Sample DPPH (mg/ml) Reducing power (mg/ml) contain low lipid. These results were similar with 22 EC50 EC50 those obtained by Barros et al. for few species of C. gambosa 1.883±0.17 2.590±0.95 macrofungi like , Boletus, Calocybe, C. indica 0.741±0.45 1.939±0.54 , Craterellus and Marasmius. Low M. procera 2.523±0.90 3.516±0.43 amount of lipid clearly indicate its high medicinal T. aestivum 3.916±0.60 3.395±0.73 importance that it can be included in daily human diet BHT 0.052±0.41 - and also it can also referred to heart patients. Fresh Values are means ± SEM for groups of 3 observations with their macrofungi contain high amount of fibre which may standard errors be responsible for its relatively high amount of ash. showed strong reducing power activity. C. gambosa, Biochemical analysis of few macrofungi, viz. M. T. aestivum and M. procera extract showed procera and T. aestivum have been carried out for the first time. The relative high protein and carbohydrate weak reducing power (EC50= 2.590, 3.395, 3.516, content recorded in the samples is proof of its being respectively) with high EC50 value as in comparison with C. indica. highly nutritious and fit for human consumption. Nutritional value of mushrooms is generally very high Discussion due to its high protein and other nutrient content. Macrofungi are used by peoples from time immoral Nutritional value of mushrooms depends largely on as food and medicine to cure various diseases18. Now the chemical composition of the compost on which it a days with increasing modernization and is growing. Present studies reveal that these industrialization lots of new synthetic drugs are macrofungi are good sources of protein and available in markets for various disease but they carbohydrate and low in fat hence can be shows many adverse effects. Natural products being recommended for good health diet. highly effective in treating various ailments shows no Antioxidant property of mushrooms largely side effects hence these natural products must be use depends upon the amount of bioactive compounds for the extraction of compounds for the synthesis of present in the samples24,25. β carotene, lycopene, drugs or they can be directly use as whole for phenol and ascorbic acid are important bioactive rehabilitation19,20,21. compounds which imparts significant role in It was concluded from the present study that antioxidant property. Especially phenol content, larger protein, carbohydrate, lipid, fibre, moisture and ash the amount of phenol larger its antioxidant showed variation in all the evaluated samples. The property26,27. The antioxidant activity of macrofungi result of the nutritional analysis of the macrofungi increased with the increased in the concentration of (Table 3) shows great variation in nutrient content. samples, higher the antioxidant property lower the Edible macrofungi are highly valued as good source EC50 values. A lower EC50 value means better radical of carbohydrate. Carbohydrate content of C. gambosa scavenging activity. DPPH is a stable free radical (55.00%) was found to be more than amount of deep purple colour which absorption maximum reported by Barros et al.22 C. indica contain 52.13% at 570 nm. When, antioxidant is present in the sample of carbohydrate which is same as data obtained by purple colour of DPPH fades28. Hence, absorbance Alam et al.12 for dried sample. Edible macrofungi are of sample decrease due to the quenching effect of highly valued for its high protein content. In present DPPH free radical which result in the release of study, protein contents of four mushrooms show a electron. Table 5 shows the antioxidant activity of the VISHWAKARMA et al.: NUTRITIONAL AND ANTIOXIDANT PROPERTIES OF MUSHROOMS 147

mushroom extracts as measured by the bleaching of Khangchendzonga Biosphere Reserve, Sikkim, India, β carotene. The absorbance of sample decreased Indian J Tradit Knowle, 12(3) (2013) 355-369. 29 2 Lachure PS, Exploration of some Medicinal Plants used rapidly in absence of antioxidant . When antioxidant by tribals from Digras region of District - Yavatmal, is present in the sample it retains its colour and thus Maharashtra, India, Int J Sci Res Pub, 2(3) (2012) 1-4. gives absorbance for longer time. When concentration 3 Sachan SKS, Patra JK & Thatoi HN, Indigenous knowledge of sample increases antioxidant property of it also of ethnic tribes for utilization of wild mushrooms as food increases. C. indica revealed better antioxidant and medicine in similipal biosphere reserve, Odisha, India, properties (lower EC values) than other samples J Agric Technol, 9(2) (2013) 403-416. 50 4 Khongsai M, Saikia SP & Kayang H, Ethnomedicinal tested and agreed with the statement of higher content plants used by different tribes of Arunachal Pradesh, of phenols present in it. Synthetic antioxidants such as Indian J Tradit Knowle, 10(3) (2011) 541-546. BHT, BHA, TBHQ at higher level shows many toxic 5 Roger Phillips, Mushrooms, (Pan Macmillan U.K.), 2006. effect hence natural antioxidants from plants and 6 Ruhul Amin, Khair Abul, Alam Nuhu & Tae Soo Lee, macrofungi have been receiving greater attention for Effect of different substrates and casing materials on the growth and yield of Calocybe indica, Mycobiology, 38 (2) future use. All the tested samples in present study also (2010) 97-101. contain significant amount of antioxidant activity with 7 Soosairaj S, Raja P, Kala A & Raj PK, The survey of higher amount of phenol in them. It proves its macroscopic fungi from a few district of Tamil Nadu, correlation between the amount of phenol and its The Bioscan, 7(4) (2012) 669-671. antioxidant property. Higher the amount of phenol 8 Srivastava AK & Soreng PK, An effort to domesticate wild edible mushrooms growing in the forest of Jharkhand, higher its antioxidant property. Int J Recent Trends Sci Technol, 3(3) (2012) 88-92. 9 Oyetayo FL, Akindahunsi AA & Oyetayo VO, Chemical Conclusion profile and amino acids composition of edible mushrooms Now a day mushrooms are also taking important Pleurotus sajor caju, Nutr Health, 18(2007) 383-389. position with plants in treating many ailments. The 10 Adedayo & Rachel Majekodunmi, Proximate analysis on present study shows that mushrooms can be use as four edible mushrooms, J Appl Sci Environ Manage, 15(1) (2011) 9 – 11. medicine to prevent various diseases. This type of 11 Thimmaiah SK, Standard methods of biochemical analysis, knowledge can open new field for researchers to work (Kalyani Publishers. New Delhi), 1999. out and find new type of drug to prevent various 12 Alam Nuhu, Amin Ruhul, Khan Asaduzzaman, Ara Ismot, ailments. In Gorakhpur many macrofungi are widely Shim Mi Ja, Woong Lee Min & Soo Lee Tae, Nutritional used as food during the rainy season by local peoples analysis of cultivated mushrooms in Bangladesh - Pleurotus ostreatus, Pleurotus sajor-caju, Pleurotus florida and and plays vital role in socio economic life of the tribal Calocybe indica, Mycobiology, 36(4) (2008) 228-232. peoples. Beside their use as food it is highly used as 13 Gbolagade Jonathan, Ajayi Adetolu, Oku Ikpebivie & folk medicine. Some of the macrofungi are even Wankasi Donbebe, Nutritive Value of Common Wild Edible locally marketed at high rate. Present study clearly Mushrooms from Southern Nigeria, Global J Biotechnol showed that all the tested mushroom species Biochem, 1 (1) (2006) 16-21. contained considerable amount of protein which can 14 Gang Liu, Hui Wang, Benhong Zhou, Xianxi Guo & Xianming Hu, Compositional analysis and nutritional be used as good nutrient supplement for human being. studies of collected from Southwest The production of protein rich food is required China, J Med Plants Res 4(12) (2010) 1222-1227. to meet the demand for protein and overcome 15 Barros Lillian, Ferreira Maria-Joa˜o, Queiro´s Bruno, malnutrition in the developing countries hence Ferreira Isabel CFR & Baptista Paula, Total phenols, macrofungi are highly recommended as alternative ascorbic acid, β-carotene and lycopene in Portuguese wild edible mushrooms and their antioxidant activities, food source for providing adequate nutrition to the Food Chem, 103 (2007) 413–419. world’s increasing population. 16 Loganathan Jagadish K, Krishnan V Venkata, Shenbhagaraman R & Kaviyarasan V, Comparative study on Acknowledgement the antioxidant, anticancer and antimicrobial property of The authors wish to thank Head, Department of Agaricus bisporus (J. E. Lange) Imbach before and after Botany DDU Gorakhpur University, Gorakhpur for boiling, Afr J Biotechnol, 8(4) (2009) 654-661. 17 Loganathan Jagadish K, Gunasundari D, Hemalatha M, providing necessary Lab. Facilities Shenbhagaraman R & Kaviyarasan V, Antioxidant and phytochemical potential of wild edible mushroom References Termitomyces reticulatus: Individual cap and stipe collected 1 Badola Hemant K & Pradhan Bharat K, Plants used in from South Eastern part of India, Int J Pharmaceut Sci Res, healthcare practices by Limboo tribe in South-West of 1(7) (2010) 62-72. 148 INDIAN J TRADIT KNOWLE, VOL 15, NO. 1, JANUARY 2016

18 Sargunam S Davidson,Johnsy G, Samuel A Selvin & 25 Gupta Pooja, Mehla Jogender, Siddique Khalid, Jamil Shakir Kaviyarasan V, Mushrooms in the food culture of the Kaani & Gupta Yogendra Kumar, Anti-oxidant and anti- tribe of Kanyakumari district, Indian J Tradit Knowle, 11(1) inflammatory activity of Safoof mohazzid: A traditional (2012) 150-153. poly-herbal unani formulation for obesity, Indian J Tradit 19 Singh Ranjay K, Singh Anamika & Sureja Amish K, Knowle, 14(3) (2015) 461-465. Sustainable use of ethnobotanical resources, Indian J Tradit 26 Ӧ zcan Mehmet Musa & Ӧ zkan Gükan, Determination of Knowle, 6(3) (2007) 521-530. antioxidant activity and total phenol contents of two Salvia 20 Tanti B, Lisha G & Sarma GC, Wild edible fungal resources extracts, Indian J Tradit Knowle, 14(2) (2015) 226-230. used by ethnic tribes of Nagaland, India. Indian J Tradit 27 Malekpoor Fatemeh, Pirbalouti Abdollah Ghasemi, Knowle, 10(3) (2011) 512-515. Salimi Azam, Shabani Leila, Sharifi Majid & H 21 Vijaykumar Gitte, John Priya & Kotgire Ganesh, Selection amedi Behzad, Antimicrobial and antioxidant activities of different substrate for the cultivation of milky mushroom and total phenolic content of Tanacetum polycephalum (Calocybe indica P&C), Indian J Tradit Knowle, 13(2) Schutz. B.P. as a folkoric herb in South Western Iran, (2014) 434-436. Indian J Tradit Knowle, 14(3) (2015) 370-375. 22 Barros L, Cruz T, Baptista P, Estevinho LM & Ferreira ICF, 28 Shahat Abdelaaty, Ibrahim Abeer Y & Alsaid Mansour S, Wild and commercial mushrooms as source of nutrients and Antioxidant capacity and polyphenolic content of seven nutraceuticals, Food Chem Toxicol, 46(8) (2008) 2742–2747. Saudi Arabian medicinal herbs traditionally used in Saudi 23 Akyüz M & Kirbağ S, Nutritive value of wild edible and Arab, Indian J Tradit Knowle, 14(3) (2015) 28-35. cultured mushrooms, Turk J Biol, 34 (2010) 97-102. 29 Kumari Deepika, Sudhakara Reddy M & Upadhyay Ramesh 24 Singh Mhaveer, Khan Mohammad Ahmed, Khan Masood Chandra, Antioxidant Activity of three Species of Shan, Ansari SH & Ahmad Sayeed, Quality assessment and Wild Mushroom Genus Cantharellus Collected evaluation of in vitro antioxidant potential of Phyllanthus from North-Western Himalaya, Int J Agric Biol, 13 (2011) emblica L., Indian J Tradit Knowle, 14(2) (2015) 265-272. 415-418.