BIBECHANA Vol. 20, No. 2, August 2023, 161–175 ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher:Dept. of Phys., Mahendra Morang A. M. Campus (Tribhuvan University)Biratnagar Biochemical, Antimicrobial, and Antioxidant activities of some wild Mushrooms from Nepal Pratiksha Chaudhary, Nabin Panth, Bimal Kumar Raut, Nisma Pokhrel Nita Shrestha, Sajan Shakya, Bijaya Bahadur Thapa Akkal Dev Mishra, Niranjan Parajuli∗ Biological Chemistry Lab, Central Department of Chemistry, Tribhuvan University Kirtipur, Kathmandu, 44618, Nepal ∗Corresponding author. Email: niranjan.parajuli@cdc.tu.edu.np Abstract Wild mushrooms represent a crucial dietary staple for many tribal groups throughout the world since they consist of an excellent amount of biologically active constituents includ- ing phenolic compounds, and tocopherol, and act as anti-cancer, anti-allergic, anti-obesity, anti-inflammatory compounds, etc. Wild mushrooms including Scleroderma citrinum, Heter- obasidion annosum, Coriolus hirsutus, Cavimalum indicum, Russula sanguinea, and Suillus punctatipes were studied to evaluate their phytochemicals, antimicrobial activity, antioxidant activity, toxicity and relevance as a food source along with safety concerns. Initially, the to- tal phenolic content (TPC), total tannin content (TTC), and total flavonoid content (TFC) along with antioxidant and antimicrobial activity were assessed using ethanolic extracts of mushrooms. Furthermore, a Brine shrimp bioassay was performed, the correlation of which with antioxidant activity, TPC, TFC, TTC, and lethal concentration (LC50) was shown by principal component analysis (PCA). Secondary metabolites such as glucosides, flavonoids, polyphenols, alkaloids, terpenoids, saponins, and quinones were identified using phytochemical investigations. The TPC ranged from 45.98 to 102.3 mg GAE/g for the extracts, TFC from 100 to 225 mg QE/g, and TTC from 80 to 180 mg GAE/g. The findings of the antioxidant studies demonstrated that S. punctatipes exhibited the highest antioxidant activity (IC50 = 16.95 µg/mL), followed by C. indicum (IC50 = 22.5 µg/mL), and C. hirsutus (IC50 = 35.34 µg/mL). Likewise, S. punctatipes exhibited strong antimicrobial activity as compared to other extracts. The larvicidal efficacy against Brine shrimp bioassay revealed that three mushrooms; C. hirsutus, C. indicum, and S. punctatipes—contain highly toxic chemicals while the other three are non-toxic and can be consumed to some extent. Keywords Wild mushroom, Phytochemicals, Antioxidants, Antimicrobial. Article information Manuscript received: May 15, 2023; Accepted: June 13, 2023 DOI https://doi.org/10.3126/bibechana.v20i2.54887 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 161 http://nepjol.info/index.php/BIBECHANA niranjan.parajuli@cdc.tu.edu.np https://doi.org/10.3126/bibechana.v20i2.54887 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Pratiksha Chaudhary et al./ BIBECHANA 20 (2023) 161-175 162 1 Introduction Mushrooms are fungi that develop fleshy, spore- bearing fruiting bodies and are members of the higher groups; Ascomycota and Basidiomycota [1]. An ample diversity of mushrooms has long been identified as edible fungi that possess a wealth of nutritional value, low-calorie content, and an abundance of structurally varied and beneficial sec- ondary metabolites [2]. As a beloved delicacy food source, mushrooms have long been used, and re- cent research into their antioxidant and health ad- vantages further adds to their allure [3]. Bioac- tive substances derived from fungi have long been recognized, and fungi’s secondary metabolites have been the subject of investigation [4]. Medicinal chemists have considered the potential benefits of mushrooms in humans for a variety of diseases to be beneficial in drug discovery and development by offering fundamental template structures and phar- macophores of therapeutically and economically ef- fective products [5]. The therapeutic advantages of mush- rooms, which include anti-hyperlipidemic, anti- inflammatory, anti-cancer, antioxidant, im- munoregulatory, and cardioprotective properties, are all owing to the presence of secondary metabo- lites in mushrooms [6–9]. Numerous bioactive con- stituents including tocopherols, ergosterols, lectins, ergothioneine, glutathione, vitamin D, selenium, Repandiol, Polysaccharide-K, and many more are reported as a source of antioxidant activity in vari- ous mushroom species [5,7,10]. Likewise, striatal A, B, C, and D, effective against cancer, were isolated from Cyathus striatus and showed antimicrobial properties [11]. Several triterpenoids; ganoderic acid E, lucidumol A, and ganoder-manontriol, found in Ganoderma lucidum demonstrated re- markable cytotoxicity against human cancer cells. Poisonous mushrooms that smell bad or cause se- vere gastrointestinal complications are classified as non-edible species. Wild edible mushrooms are a natural forest resource well recognized for their nutritional, medicinal, economic, and cultural worth [12]. Meanwhile, poisonous mushrooms are frequently mistaken for edible species when ingested as wild mushrooms because of their similarity in form and color. Concern is also raised about the dangerous byproducts that are produced by toxic mushrooms [13]. However, the value of wild mush- rooms is increasing as a result of their phenolic compounds, which act as potent antioxidants in addition to their pharmacological, nutritional, and sensory qualities [2]. Thus, it is equally crucial to explore secondary metabolites found in mushrooms. Mushrooms had been used as a therapeutic agent in civilizations all over the world. It has long been known as a medicinal plant in China and other Asian countries, including Japan and Korea, and has extended to countries such as the United States as well as Eastern Europe, including Russia [14]. Even though nature has endowed wild mushrooms with diverse economic and therapeutic significance, research on the bioactivity and potential medical benefits of available mushrooms has not been thor- oughly conducted extensively. The situation is even direr in developing nations like Nepal where there has only been a scant study on wild mushrooms re- gardless of the availability of 108 families, 357 gen- era, and 1291 species of mushrooms with about 159 being edible, and 74 species reported to have medic- inal properties [15]. Scleroderma citrinum (genus Scleroderma), also called Scleroderma aurantium or Scleroderma vul- gare horn, grows from late summer to early win- ter frequently seen in Europe and Nepal, has re- markable medicinal properties, and thus, used in the pharmaceutical industries, cosmetics, and nu- tritious food as well [16]. It is reported to con- tain bioactive compounds such as sclerocitrin, nor- badione A, xerocomic acid, badione A, etc [17]. Heterobasidion annosum (Agaricomycetes species), also known as Polyporus annosum, is a species of the Bondarzewiaceae family that can grow a diameter of up to 40 cm and a thickness of 3.5 cm. It is frequently found in North America and Nepal, is inedible, and has been discovered to help prevent colon cancer [18]. Likewise, Coriolus hirsutus commonly known as Rau Bhako Chyau is a common hairy beechwood-grown bracket fungus that not only serves as a food source but is also reported to cure wounds in traditional Nepalese communities [19]. Coriolus hirsutus is a common hairy bracket fungus that is grown specifically in beechwood. It persists throughout the year, has a slightly zoned crown, is occasionally white-gray with short hairs, and is tomentose and yellowish near the border. Another wild mushroom species, Cavimalum indicum mostly grows in bamboo trees and very little study has been reported to date. The Basidiomycota family includes Russula san- guinea which grows beside coniferous trees and is mycorrhizal with softwood trees. Moreover, stud- ies on R. sanguinea, a vibrantly brightly colored mushroom, exhibit strong enzyme inhibition; anti- amylase and anti-glucosidase activities along with effective antioxidant activities [20]. Bioactive con- stituents like sangusulactones A-C, blennin A, and 15-hydroxyblennin A responsible for several phar- macological activities are found in this species [21]. Suillus puntatipes, often known as puffballs, are characterized by their white-yellow cap meat Mass, a layer of grayish-red-purple skin, and pores that are greenish-yellow in color. It has been demon- strated for the production of cytokinins like zeatin and ribosylzeatin [22]. The human adenosine A2A Pratiksha Chaudhary et al./ BIBECHANA 20 (2023) 161-175 163 Figure 1: List of some collected mushrooms. A- Scleroderma citrinum, B- Suillus punctatipes, C- Coriolus hirsutus, D- Russula sanguinea, E- Heterobasidion annosum, F- Cavimalum indicum. receptor, which is widely represented by many dif- ferent types of cells and has an important function in controlling the activity of cells engaged in both adaptive and innate immunity, has recently been shown to be activated by the naturally occurring cytokinin zeatin riboside [23]. Numerous species of mushrooms have been the subject of various research throughout the world, but only a small number of studies have focused on specific wild mushrooms. The key purpose of this research is to investigate the phytochemical con- tent, evaluation of toxicity of mushroom extracts to some extent, antioxidant activity, antimicrobial activity, TPC, TFC, and TTC of six selected wild mushrooms from Nepal along with their correlation to each other have been investigated by PCA. 2 Materials and Methods 2.1 Collection and Extraction of Mush- room The six different mushroom species were primar- ily collected during the rainy season (July-August) from different locations and were identified taxo- nomically at the National Academy of Science and Technology (NAST), Khumaltar, Lalitpur. Figure 1 represents the lists of collected mushroom species and their botanical description along with phar- macological importance are mentioned in Table 1. Each mushroom species was thoroughly cleaned, freed of contaminants, and dried in the shade be- fore the ethanol-based soxhlet extraction method was used to extract the mushroom powder. The extracts were concentrated using a rotary evapora- tor, and each extract was thoroughly extracted for 12 hours. By using this formula, the percentage yield of dried mushroom extracts was calculated: Percentage Yield = Dry weight of extract Dry weight of sample × 100% (1) The metabolites found in these mushrooms were surveyed thoroughly, thereby the chemical struc- tures of some metabolites reported previously are illustrated in Figure 2. 2.2 Screening Identification of the phytochemicals found in the mushroom extracts was done by chemical method. As per the previously specified procedures [24], various tests including flavonoids, glycosides, al- kaloids, steroids, phenolic compounds, terpenoids, saponins, tannins, carbohydrates, fats, and fixed oils were carried out. Pratiksha Chaudhary et al./ BIBECHANA 20 (2023) 161-175 164 Figure 2: Some potential phytochemicals found in wild mushrooms. Table 1: Name of Mushroom, Collection site, Common Name, Family, and Pharmacological Application Name of Mushroom Collection site Common Name Family Pharmacological Application References S. citrinum Nagarkot, Kath- mandu Common earth Ball Sclerodermataceae Anti-inflammatory, antiseptic proper- ties Lopusiewicz, 2018) H. annosum Dhulikhel, Kavre Root rot fun- gus Bondarzewiaceae Used as poten- tial candidates for bioremedia- tion and act as antioxidants, and anti-inflammatories (Sadowska et al., 2020) C. hirsutus Sundarijal, Kath- mandu Turkey tail mushroom Polyporaceae Potential im- munomodulatory properties, anti- cancer properties (Adhikari et al., 2005) C. indicum Sundarijal, Kath- mandu Indian mal- low Clavicipitaceae - - R. sanguinea Godawari, Lalitpur Bloody brit- tle gill mush- room Russulaceae Anti-inflammatory, antimicrobial, antioxidant, and analgesic properties (Alkan et al., 2020) S. punc- tatipes Matatirtha, Kath- mandu Puffball mushroom Suillaceae Anti-inflammatory, antioxidant, and immune- modulating proper- ties (Crafts & Miller, 1974) Pratiksha Chaudhary et al./ BIBECHANA 20 (2023) 161-175 165 2.3 Phenolic Content The total phenolic content (TPC) present in the mushroom extracts was assessed by the Folin- Ciocalteu colorimetric method [25]. Initially, the gallic acid stock solution was made by dissolving 10 mg of the acid in 10 mL of ethanol (1 mg/mL). Gallic acid was produced in different quantities, in- cluding 125, 250, 500, and 1000 g/mL. The stock so- lution was made by dissolving 10 mg of each extract in 1 mL ethanol. The concentrations of 125, 250, 500, and 1000 g/mL were obtained by sequential dilutions. Then, 1 mL sample solution was taken in a test tube, to which 4 mL of 7% Na2CO3 and 5 mL of 10% FCR were added, shaken well, and kept at ambient temperature in the dark for half an hour. The absorbance of this blue mixture was then taken at 760 nm against control, with each experiment being carried out in a triplicate manner. To plot calibration curves at various concentrations, Gallic acid was employed as the reference and written as Gallic acid equivalent (mg GAE/g). 2.4 Flavonoid Content An aluminum chloride colorimetric test was used to assess the total flavonoid content (TFC) of mush- room extracts [26]. To make a quercetin stock solu- tion, quercetin stock solution was made by dissolv- ing its 10 mg in 10 mL ethanol (1 mg/mL). Different quercetin concentrations, including 125, 250, 500, and 1000 g/mL, were produced. Ten milligrams of each extract were dissolved in one mL of ethanol to create the stock solutions. To attain the concentra- tion of 500 g/mL, several dilutions were performed. The sample solution of concentration 10 mg/mL in ethanol was diluted to 1 mL, and successively 0.3 mL of 5% NaNO3, 0.3 mL of 10% AlCl3, and 2 mL of 1 M NaOH were added. Five minutes later, 0.3 mL of 10% AlCl3 was introduced after the addition of double-distilled water to the mixture. The ab- sorbance of the pink color was then compared to the control (quercetin) at 510 nm. The experiment for each concentration was carried out thrice. 2.5 Tannin Content The total tannin content (TTC) was assessed by the Folin and Ciocalteu technique based on the work carried out by this procedure [27]. A gallic acid stock solution was prepared by dissolving 10 mg of gallic acid in 10 mL ethanol (1 mg/mL). Gallic acid was produced in a variety of quantities, in- cluding 125, 250, 500, and 1000 g/mL. Following the initial mixing of 0.1 mL sample solution with 7.5 mL distilled water, 0.5 mL of 10% FCR and 1 mL of 35% Na2CO3 were added and diluted to 10 mL by distilled water. After a thorough shaking, the blue-colored mixture was maintained at room temperature for 30 minutes in the dark. Then, the absorbance at 725 nm was compared to the control. Each experiment was performed three times. 2.6 Activity on DPPH for Antioxi- dant Assay The DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical test was performed to determine the antioxidant ac- tivities of six different mushrooms employing the earlier-mentioned approach [28]. The molecular weight of 2, 2-diphenyl-1 picrylhydrazyl (DPPH) is 394.32 Da. As a result, 100 mL of 0.1 mM so- lution of DPPH was produced by precisely weigh- ing 0.4 mg of DPPH, and dissolving it in ethanol, and was then maintained the volume at 100 mL. At first, a 100 mL solution of 0.1 mM DPPH was made in ethanol along with ethanolic solutions of Ascor- bic acid and mushroom extract were prepared in a range of concentrations (15-500 g/mL). After thor- ough vortexing, 1 mL of DPPH solution and 1 mL of sample were combined and left at ambient tem- perature for roughly 30 minutes in the dark. The absorbance was compared to the control at 517 nm. The experiment for each concentration was carried out thrice. Finally, the determination of antioxi- dant activity was done by the equation below: DPPH free radical scavenging (%) =[ A517 of control −A517 of sample A517 of control ] × 100 (2) 2.7 Antimicrobial Activity The antibacterial activity of the mushroom extracts was assessed by the Agar well diffusion method following the standard protocol [29]. Salmonella typhimurium, Escherichia coli, Staphylococcus au- reus, and Bacillus cereus, the four tested bacterial strains, were cultured in MHB media with turbid- ity matched with 0.5 McFarland and swabbed in the MHA plates with 1.5 x 108 CFU/mL bacterial suspension for 24 hours before the antibacterial test at 37 0C incubator. Using a 6 mm cork borer, wells form in these plates, and 100 mg/mL of each extract in DMSO is prepared. About 40 µL of extracts are subsequently kept in the well, along with DMSO as a negative control and Neomycin as a positive con- trol. After incubating overnight at 37 0C, the plates were observed as well as the zone of inhibition was determined. 2.8 Brine Shrimp Lethality Assay The brine shrimp bioassay, to assess the cytotoxi- city, was performed on each ethanolic extract fol- lowing the procedure [30]. Brine shrimps (Artemia Pratiksha Chaudhary et al./ BIBECHANA 20 (2023) 161-175 166 Salina) were incubated by spreading brine shrimp eggs ( 50mg) on top of the artificial seawater-filled beaker and illuminated with a table lamp (100 watts) at a temperature of 30 0C. In Brine shrimp bioassay, freshly hatched brine shrimp nauplii—the egg of Artimia salina—are exposed to various solu- tions of certain mushroom extracts. To carry out the brine shrimp bioassay, the shrimp’s egg must hatch, which required precise equipment steriliza- tion, the making of artificial seawater, and sample processing. The artificial seawater was prepared by following protocol [31].Thus, newly hatched brine shrimp nauplii were subjected to a categorized so- lution of mushroom extracts, and based on their cytotoxicity against the nauplii, the bioactivity was assessed. Probit Analysis was used to evaluate the LC50 value (g/mL) at 95% confidence intervals. Meyer and others regarded the substance with LC50 of 100 ppm to be effective (potent). The percentage mortality (%M) was also computed to confirm that the bioactive substances in the mushroom extracts were responsible for nauplii’s demise. Percentage mortality (%M) = Number of dead nauplii Total number of nauplii × 100 (3) 2.9 Statistical Analysis The statistical data were analyzed by imple- menting R (version 4.2.2) and RStudio (version 2022.10.31). In general, we used antioxidant activ- ity (IC50 value), TFC, TPC, TTC, and LC50 value (for cytotoxicity) for the analysis using this soft- ware, along with the correlation of these data to- gether with principal components analysis. To determine an appropriate correlation method, it was necessary to conduct a normal- ity test. The parameters DPPH, TFC, TPC, TTC, and IC50 were subjected to the Shapiro-Wilk nor- mality test to validate their skewness, normality, and kurtosis. In case the data were not found to be normally distributed, Kendall rank correlation was analyzed. The coefficient of determination (R2) ranged between 0.9701 and 0.9753 after the data were linearly fitted. 2.9.1 Principal Components Analysis High-dimensional data can be simplified using prin- cipal component analysis (PCA) while maintain- ing the integrity of underlying trends and patterns. This is accomplished through the compression of obtained data into limited dimensions that serve as feature summaries [32]. Principal components are used to deal with correlated predictors and show data in a two-dimensional space. 3 Results and Discussion 3.1 Phytochemical Screening The respective percentage yield of ethanolic ex- tract of mushrooms is displayed in Table 2, with R. sanguinea having the highest percentage yield (31.90%) and C. indium demonstrating the lowest (5.10%). The differences in the amounts of mush- room extracts may be due to the solvent’s abil- ity, which relies on the chemical composition of the mushroom, the extraction technique, and the sol- vents’ polarity utilized for extraction. Phytochemical investigation of mushroom ex- tracts showed the existence of polyphenols, quinones, terpenoids, saponins, and flavonoids. All mushroom extracts except for S. citrinum consist of reducing chemicals, whereas alkaloids predomi- nate in the extracts of S. citrinum, R. sanguinea, S. punctatipes, and C. indicum. On the contrary, only two mushroom species, namely H. annosum and R. sanguinea, demonstrated the presence of glycosides. Preliminary screening of R. sanguinea indicates the presence of every phytochemical examined. The list of the phytochemicals contained in the ethanolic ex- tract of the studied mushrooms is represented in Table 3. Studies on the ethanol extracts of the edible mushrooms Pleutorus ostearus and Coprinus co- matus, by high-performance liquid chromatography (HPLC), reported the existence of bioactive sub- stances such as flavonoids, alkaloids, terpenes, gly- cosides, and saponins, which is consistent with the current result [33,34]. Table 2: Percentage yield of ethanolic extract of different mushrooms S.N. Name of the mush- room species Dry weight of the sample (g) Dry weight of extract (g) Percentage yield (%) 1 C. indicum 40 2.041 5.10 2 C. hirsutus 40 3.979 9.94 3 H. annosum 40 4.897 12.24 4 S. citrinum 40 5.232 13.08 5 S. punctatipes 40 6.235 15.58 6 R. sanguinea 40 12.76 31.90 Pratiksha Chaudhary et al./ BIBECHANA 20 (2023) 161-175 167 Table 3: Phytochemical investigation of six wild mushroom species (Note: (+) present and (-) absent) Group of compounds S. cit- rinum H. an- nosum C. hir- sutus C. in- dicum R. san- guinea S. punc- tatipes Polyphenols + + + + + + Reducing compounds - + + + + + Glycosides - + - - + - Quinones + + + + + + Saponins + + + + + + Alkaloids + - - + + + Terpenoids + + + + + + Flavonoids + + + + + + Several investigations have indicated that ex- tracts from a wide variety of other mushroom species contain significant amounts of phenols, flavonoids, alkaloids, terpenoids, tannins, steroids, and cardiac glycosides [35–37]. These bioac- tive substances have been attributed to various biological activities, including anti-diabetic and anti-inflammatory properties by saponins, anti- cancer and antimalarial properties by terpenoids [38], and scavenging, antiallergenic, antiviral, anti- inflammatory, and vasodilating effects by flavonoids [39]. Moreover, phenolic compounds have also been acclaimed with considerable restorative ben- efits [34]. Hence, the sufficient utilization of potent compounds could provide an important base for the study of the bioactivity of the mushrooms. 3.2 Total Phenolic Content Antioxidant activity in plants is mostly attributed to phenolic compounds, which are crucial elements with redox characteristics since hydroxyl groups help to neutralize free radicals [40]. The total phe- nolic contents of different mushroom extracts, ex- pressed as mg GAEs/g of the extract, are listed in Table 4. Among the examined mushrooms, S. punctatipes exhibited the highest phenolic content (102.3 mg GAE/g), and S. citrinum was the lowest (45.98 mg GAE/g). This is in contrast to a study that showed S. citrinum melanin having a greater polyphenol content responsible for increased antiox- idant activity [16]. Such variations in total phenolic content present in the mushrooms could be resolved by various parameters, such as harvest time, growth conditions, and environment. Numerous investiga- tions have demonstrated that phenolic compounds vary in quantity and composition at the subcellular level throughout tissues [41]. Moreover, the content of simple phenolic acids such as Caffeic acid, Fer- ulic acid, etc., is typically higher in younger tissues since many phenolic acids condense later into com- plex phenolic compounds like flavonoids, tannins, and lignins [42]. 3.3 Total Flavonoid Content Flavonoids, possibly the most significant natural phenols, are among the most varied and common groups of natural compounds being beneficial in the context of human health [43]. The total flavonoid content was determined using the aluminum chlo- ride colorimetric technique with Quercetin as a ref- erence and indicated as mg QEs/g of extract. With S. punctatipes having the maximum flavonoid con- tent and S. citrinum the minimum, Table 4 below shows that the total flavonoid content of six mush- room extracts varied from 100 to 225 mg QE/g. It was determined that S. punctatipes with the maxi- mum total phenolic content also contained the max- imum flavonoid content (225 mg QE/g). This il- lustrates the positive correlation between TPC and TFC implying that there is some relationship con- necting TFC and antioxidant potential [44]. Our results can therefore be connected with the previ- ous work showing the positive impact of flavonoid and phenolic content in the free radical scavenging activity [45]. 3.4 Total Tannin Content Tannins are a unique group of water-soluble polyphenols, most commonly present in various mushrooms, and are regarded as good supplies of bi- ologically active substances in the human diet [46]. The total tannin content present in the ethanolic mushroom extracts was quantified by the Folin- Ciocalteu technique, where Gallic acid was used as a reference based on the previously described pro- cedure [47]. Table 4 shows that S. punctatipes had the maximum total tannin concentration (180 mg GAE/g), and it gradually decreased from C. in- dicum, C. hirsutus, and H. annosum, to the lowest S. citrinum (80 mg GAE/g), respectively. Pratiksha Chaudhary et al./ BIBECHANA 20 (2023) 161-175 168 Table 4: Comparison of IC50, TFC, TPC, and TTC content in various mushroom extracts Mushroom extracts Phenolic contents(mg GAE/g TPC) Flavonoids con- tents (mg QE/g TFC) Tannin contents (mg GAE/g TTC) Antioxidant ac- tivity IC50 Val- ues (µg/mL) S. punctatipes 102.30 225 180 16.95 C. indicum 82.76 200 160 22.50 C. hirsutus 74.71 175 140 35.34 H. annosum 63.23 175 140 39.89 R. sanguinea 51.73 125 100 53.40 S. citrinum 45.98 100 80 138.00 3.5 Antioxidant Activity The DPPH free radical scavenging technique was employed to assess the antioxidant activity of sev- eral ethanolic extracts of mushrooms in which the decolorization of an ethanol solution of 2,2- diphenyl-1-picrylhydrazyl was observed to evaluate the mushroom extract’s ability to donate hydro- gen atoms (DPPH). When DPPH is dissolved in ethanol, it gives a violet or purple tint that, when present with antioxidants, fade to varying degrees of yellow (DPPH-H). Antioxidant activity was deter- mined in terms of IC50 value in which antioxidant activity increases as the IC50 value decreases [6]. The antioxidant property of certain prevalent edi- ble wild mushrooms resembles their total phenolic content [48]. Since, phenolic compounds in mush- rooms, which include free hydrogen, contribute to the antioxidant capacity of mushroom extracts, many species of wild mushrooms demonstrated an- tioxidant activity [49]. The IC50 value of Ascorbic acid was low i.e, 15.62 µg/mL, indicating that it exhibits the highest antioxidant activity (Table 4) which implies that, in comparison to other mush- rooms, S.punctatipes had the greatest antioxidant activity since its IC50 value is close to that of Ascor- bic acid. The Suillus species are reported to have strong antioxidant activity, and thereby prevent ox- idative stress in humans [50]. The mushrooms—H. annosum, C.hirsutus, and C.indicum having less than 50 µg/mL IC50 possess significant antioxidant properties, in contrast to R. sanguinea’s that exhib- ited greater than 50 µg/mL IC50 value. S. citrinum demonstrated the least level of antioxidant activity. As per the study, the mushroom extracts’ varying antioxidant activity was likely caused by the inter- action of several phenolic chemicals found in the plant itself [51]. Boletus, a wild culinary mush- room, was shown to correlate with phenolic com- pounds, the main group of phytocomponents that contribute to the antioxidant properties of mush- room species [52]. A recent study found that con- suming 18 g of mushrooms daily might boost cellu- lar antioxidant functions and reduce the likelihood of cancer [53]. Several antioxidant components are frequently used in different foods to defend against oxidative damage caused by free-radical molecules. Since the beginning of time, people have consumed wild mushrooms as a part of their diets and as a source of nutritional supplements as they exhibit good antioxidant effects [54]. Moreover, the antiox- idant property of mushrooms has been reported to prevent oxidative damage of lipids, proteins, and nucleic acids by neutralizing free radicals. The level of oxidative stress could be lowered by supplement- ing the diet with antioxidant-rich edible mushrooms [3]. Due to these attributes, the use of mushrooms in several nutraceutical products is rising in popu- larity [55,56]. Likewise, antioxidant activity, nutri- tional value, and healthy properties of food sources are interrelated to each other [57]. According to this, S. punctatipes which exhibited good antioxi- dant activity could be a potential nutrient dietary source. 3.6 Antimicrobial Activity The antibacterial activity of six different species of mushroom was analyzed by the Agar-well dif- fusion technique with Neomycin as standard. Ta- ble 5 shows the antimicrobial activity of six mush- rooms towards all four test pathogens comprising two gram-positive (S. aureus and B. cereus) and two gram-negative (E. coli and S. typhimurium) bacteria. In our experiments, the S. punctatipes demonstrated the greatest antimicrobial activity with a zone of inhibition of 12 mm and 11 mm against gram-positive bacteria while B. cereus and S. aureus, along with a zone of inhibition of 9 mm and 10 mm against gram-negative bacteria S. typhimurium and E. coli. Likewise, S.citrinum, H.annosum, C.indicum, and R. sanguinea showed good antimicrobial efficacy against gram-positive bacteria, while C.hirsutus, and C.indicum, dis- played antimicrobial activity against gram-negative bacteria. The variation in the outcomes of an- timicrobial activity could be attributed to several variables, such as the environmental and climatic conditions in which the mushroom was grown, the choice of mushroom extracts, the choice of extrac- tion techniques, the antimicrobial test method, and the test microorganisms. As per a previous study, Pratiksha Chaudhary et al./ BIBECHANA 20 (2023) 161-175 169 the pigment β-carotene, most commonly found in Suillus sp., exhibited significant antimicrobial prop- erties towards S. aureus and E. coli [50]. Nu- merous secondary metabolites including alkaloids, flavonoids, tannins, and other phytochemicals have been documented to affect antimicrobial activity [58]. As per the prior study, the melanin of Scle- roderma citrinum showed no antimicrobial effec- tiveness towards B. cereus, E. coli, and S. au- reus, which in contrast to our present study, exhib- ited good antimicrobial effectiveness towards both gram-positive strains [16]. There’s a probability that some of the tested extracts from this inves- tigation will succeed as antimicrobial medicines in the future. Table 5: In vitro antimicrobial activity of mushroom extracts against selected bacterial strains Zone of Inhibition(mm) Mushroom extract (100 mg/mL) Gram-positive bacteria Gram-negative bacteria S. aureus B. cereus E. coli S. typhimorium S. citrinum 9 9 6 7 H. annosum 10 9 6 7 C. hirsutus 7 7 10 9 C. indicum 10 10 9 9 R. sanguinea 11 10 7 7 S. punctatipes 12 11 9 10 Neomycin 34 35 24 23 DMSO solution 0 0 0 0 Table 6: LC50 values of different mushroom extracts Mushroom extracts LC50 (g/mL) S. citrinum 4265.8 H. annosum 14791 C. hirsutus 338.8 C. indicum 338.8 R. sanguinea 16596 S. punctatipes 257.0 Table 7: Kendall rank correlation coefficient DPPH TPC TFC TTC LC50 DPPH 1 -1 -0.96609 -0.96609 0.69007 TPC -1 1 0.96609 0.96609 -0.69007 TFC -0.96609 0.96609 1 1 -0.64286 TTC -0.96609 0.96609 1 1 -0.64286 LC50 0.69007 -0.69007 -0.64286 -0.64286 1 3.7 Brine Shrimp Assay The brine shrimp assay is a preliminary screening method used to assess the bioactivity of crude ex- tracts by measuring their toxicity. It has been em- ployed to identify fungal toxins, plant extract tox- icity, and cytotoxicity [59–61]. The LC50values, which represent the concentration of the extract that causes 50% mortality in the brine shrimp, are calculated using this method [62]. A lower LC50 value indicates higher toxicity. According to Meyer et al.,LC50 values of less than 100 ppm are consid- ered potent, values less than 1000 ppm are toxic, and values greater than 1000 ppm are non-toxic [63]. This study demonstrates the larvicidal activ- ity of ethanolic extracts from different mushrooms against brine shrimp. The calculated LC50 values of the mushroom extracts analyzed in this study are listed in Table 6. The concentration of these mushroom extracts and their efficiency were directly correlated. At a concentration of 1000 ppm, all mushroom ex- tracts demonstrated high toxicity with the high- Pratiksha Chaudhary et al./ BIBECHANA 20 (2023) 161-175 170 est mortality rates. This suggests that three of the six mushrooms examined, Coriolus hirsutus, Cavi- malum indicum, and Suillus punctatipes, might con- tain highly potent toxic substances, while the other three, S. citrinum, H. annosum, and R. sanguinea, were found to be non-toxic. According to our study, the three mushrooms S. citrinum, H. annosum, and R. sanguinea were found to be non-toxic, with an LC50 value greater than 1000, which is considered non-toxic according to Meyer et al. (1982). This finding of H. annosum being moderately or mildly toxic corresponds to previous findings [18]. The non-toxicity or low cy- totoxicity of these mushroom species suggests that they are potentially suitable for consumption. However, when it comes to safety concerns, the edibility of the six mushrooms depends on proper examination. All mushrooms can be consumed, but one mushroom, namely S. punctatipes, should only be consumed if the cap is removed. Therefore, it is important to be aware of this and conduct suf- ficient surveys and examinations when consuming such mushrooms. Additionally, the study, classification, and pro- duction of medicinal mushrooms could benefit from the application of cutting-edge technologies such as metabolomics, proteomics, transcriptomics, and genome sequencing [64]. Further studies on the tox- icity and nutritional value of these mushrooms will likely reveal whether they could be a valuable ad- dition to our diets in the near future. 3.8 Statistical Analyses 3.8.1 Correlation According to the Shapiro-Wilk test, the W and p- value were found to be 0.98676 and 0.9672, respec- tively. Since the p-value was greater than 0.05, the data are not normally distributed. Therefore, the Kendall rank correlation coefficient was performed, and the results are shown in Table 7. 3.8.2 Principal Components Analysis of Different Variables The scree plot of the Principal Component Analy- sis (PCA) (Figure 3) displayed two principal com- ponents that accounted for 97.7% of the overall variation. The variables TPC, TFC, and TTC showed a strong correlation with the formation of axis 1 (79.4%), while DPPH and LC50 strongly contributed to the development of axis 2 (18.3%) (Figure 4). This plot indicates that variables such as TFC, TPC, and TTC are negatively correlated with LC50, while variables are positively correlated with LC50 values. This means that an increase in the amount of phenolic, flavonoid, and tannin con- tent leads to a decrease in toxicity (lethal concen- tration). Figure 3: Scree plot of Principal component analysis. Pratiksha Chaudhary et al./ BIBECHANA 20 (2023) 161-175 171 Figure 4: Principal component plot of variables. 4 Conclusion Mushrooms contain a variety of phytochemicals, protein, dietary fiber, vitamins, and minerals, mak- ing them suitable as food supplements for all age groups. This research focused on analyzing the phytochemicals and evaluating the biological prop- erties of the selected wild mushrooms. The pre- liminary assessment effectively demonstrated their cytotoxic, antibacterial, and antioxidant character- istics. The presence of various phytochemicals can be attributed to the potential antibacterial and an- tioxidant properties of the examined samples. All mushroom extracts exhibited at least one phyto- chemical, while only R. sanguinea exhibited all tested phytochemicals. The brine shrimp bioas- say demonstrated the potential toxicity of S. punc- tatipes, C. indicum, and C. hirsutus against brine shrimp nauplii, while S. citrinum, H. annosum, and R. sanguinea were found to be non-toxic. Further studies should be conducted to analyze their bioac- tivity and potential toxicity. S. punctatipes exhib- ited the most notable antibacterial and antioxidant activity among all the mushrooms studied. Addi- tionally, S. punctatipes and C. indicum contained the highest levels of flavonoids, phenolics, and tan- nins. Further in-depth research may lead to the discovery of substances that could serve as poten- tial drugs or templates for new drug development. Mushrooms can be a great source of antioxidants due to their high phenolic content and can be con- sumed when handled properly. 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[64] Uwe Groß, Elzbieta Brzuszkiewicz, Katrin Gunka, Julia Starke, Thomas Riedel, Boyke Bunk, Cathrin Spröer, Dietmar Wetzel, Anja Poehlein, Cynthia Chibani, Wiebke Bohne, Jörg Overmann, Oliver Zimmermann, Rolf Daniel, and Heiko Liesegang. Compara- tive genome and phenotypic analysis of three Clostridioides difficile strains isolated from a single patient provide insight into multiple in- fection of C. difficile. BMC Genomics, 19(1):1, 2018. Introduction Materials and Methods Collection and Extraction of Mushroom Screening Phenolic Content Flavonoid Content Tannin Content Activity on DPPH for Antioxidant Assay Antimicrobial Activity Brine Shrimp Lethality Assay Statistical Analysis Principal Components Analysis Results and Discussion Phytochemical Screening Total Phenolic Content Total Flavonoid Content Total Tannin Content Antioxidant Activity Antimicrobial Activity Brine Shrimp Assay Statistical Analyses Correlation Principal Components Analysis of Different Variables Conclusion