The Journal of Phytochemistry. 118 (2017) 417-422 https://sites.google.com/site/photonfoundationorganization/home/the-journal-of-phytochemistry Original Research Article. ISJN: 4172-8352: Impact Index: 4.90

The Journal of Phytochemistry Ph ton

Evaluation of bioactive components and bactericidal action of tropical mushrooms Ganoderma tsugae and Dadaelea quercina

Shweta Panchala, Joanna Dcostaa, Dr. Shubhada Walvekarb*

Biotechnology Department, SVKM’s Mithibai College Mumbai, India

Article history: Abstract Received: 17 June, 2017 The study aims at the study of two species of tropical Accepted: 20 June, 2017 mushrooms for the phytochemical profile and Available online: 13 September, 2017 bactericidal action. Ganoderma tsugae and Dadaelea

Keywords: quercina, the Basidiomycetes representatives showed the Ganoderma tsugae, Dadaelea quercina, phenols, antioxidant presence of phenolic compounds, steroids, alkaloids, activity, bactericidal activity. glycosides in them with high free radical scavenging activity. The two mushrooms showed bactericidal Corresponding Author: activity against selected clinical Gram positive as well as Dr. Walvekar S.* Gram negative organisms. The synergistic antibacterial Assistant Professor action was not shown by the mushrooms instead the Email: shubhadaw ( at ) gmail ( dot ) com extracts were antagonistic to each other.

Panchal S. Citation: Dr. Walvekar S.*, Panchal S., Dcosta J., 2017. Student Evaluation of bioactive components and bactericidal action of

tropical mushrooms Ganoderma tsugae and Dadaelea quercina. Dcosta J. The Journal of Phytochemistry. Photon 108, 135-138 Student

All Rights Reserved with Photon. Photon Ignitor: ISJN41728352D869913092017

1. Introduction

Indian subcontinent hosts a vast variety of cardiovascular diseases. It is also used in treating mushrooms belonging to various genera having an immune disorder and has been used in association established history of use in traditional oriental with cancer chemotherapy treatments and surgery medicine. Many traditionally used mushrooms (Hobbs, 1995). from genera, Auricularia, Flammulina, Ganoderma, Grifola, Lentinus, Pharmacological research has now isolated (Coriolus) and Tremella have been demonstrated to antifungal, antiviral, and anti- protozoan, isolates possess significant medicinal properties. (Wasser, from fungi. Medicinal mushrooms are widely used 2002). Of the 14,000 to 15,000 species of as traditional medicinal ingredients for the mushrooms in the world, around 700 have been treatment of various diseases and related health known for having medicinal properties. (Lucas, problems. Most of the medicinal extracts from 1957). Auricularia auricula and A. polytricha have mushroom are different forms of polysaccharides been used in folk medicine in for thousands of which strengthens the immune system with little or years in China and has traditionally been used to no side effect. Medicinal mushroom research has treat hemorrhoids and as a stomach tonic. focused on discovery of compounds that can edulis is the that has been found to have modulate positively or negatively the biological antitumor properties. In traditional folk medicine, it response of immune cells (Usman et al., 2012). is also used in treating lumbago, leg pains, and numbness in limbs and tendon discomfort to name The tropical forest may have different species of a few. Cordyceps sinensis commonly called the saprophytic mushrooms in close vicinity of each Caterpillar is an antidote for opium other. Ganoderma tsugae (Phylum-, poisoning. In traditional Chinese medicine, Family-Ganodermataceae) and Dadaelea quercina Cordyceps has been used to treat respiratory and (Phylum-Basidiomycota, Family-) pulmonary diseases, e.g. renal, liver and are two common representatives of the mushroom

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flora of the Indian forests. These mushrooms were Botanical classification selected for the present study of phytochemical Division : Basidiomycota profile and antimicrobial potential and to check if Class : Basidiomycetes any synergistic activity exists since the class Order : (Ganoderma spp) are mutual Family : Fomitopsidaceae symbionts for both plants as well as animals and Genus : the two mushrooms were growing together in the Species : quercina same place from where these samples were Commonly known as the mazegill or maze-gill collected. fungus, the specific epithet refers to the oak genus Quercus, upon which it frequently grows, causing a 1.1 Ganoderma tsugae: brown rot. characteristic maze-like (daedaloid or labyrinthine/labyrinth form) This mushroom is inedible due to its cork-like texture. It is a saprophytic fungus on hardwoods causing a brown rot of it. Fruiting body is 4-20 cm in size, fan- shaped in outline, dry, smooth or finely fuzzy (generally smoother toward the margin); whitish when fresh, but grayish, brown, or black in age (often darker toward the point of attachment in specimens that are several years old).

2. Objective of Research: The study aims at the study of biochemical Ganoderma tsugae - Fruiting Body components present in the extracts of the two tropical mushrooms selected by qualitative and Botanical Classification quantitative methods as well as to examine the Phylum : Basidiomycota independent and synergistic bactericidal activity of Class : Agaricomycetes their extracts against clinical bacterial isolates and Order : Polyporales therefore check whether can be used as a potential Family : Ganodermataceae antibacterial agent. Genus : Ganoderma Species : tsugae 2.1 Materials and Methods

Ganoderma tsugae, also known as Hemlock Isolates of of the mushrooms varnish shelf, is a flat mushroom of the (G.tsugae and D.quercina) were collected. The genus Ganoderma. Because of its solid woody samples were sliced and oven dried at up to 500C nature, G. tsugae which is non-poisonous is for 24 hours. Dried material was ground to fine generally considered inedible. The spread of powder. 1 gram of the powdered materials was fruiting body is from 4cm to 24cm with kidney extracted separately with 10 ml of 80% Ethanol in shaped or fan shaped with a shiny or lustrous conical flasks with occasional shaking for 24 hours. varnished surface. The stem is 3 to 14cm long and The extract was filtered through a muslin cloth and up to 3cm thick angled away from the side of the stored under refrigeration. This extract was used cap. for qualitative analysis, quantitative analysis and Antimicrobial Sensitivity Testing. 1.2 2.2 Qualitative analysis of the extracts: Qualitative tests for phytochemicals: Qualitative tests for proteins fats, oils and steroids, glycosides alkaloids phenolic compounds terpenoids and tannins were carried out according to standard methods.

TLC analysis: TLC analysis was carried out using ethanolic extract of the mushrooms. The solvent system and developing reagent for various phytochemicals are described in the Table 1.

Daedalea quercina fruiting body

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TLC table for the analysis of phytochemicals: Active Solvent system Proportion Developing reagent Obserevations Principle Alkaloids Ethyl Acetate: Methanol: Water 100:13.5 :10 Draggondorff's reagent Brown spots Anthraquinone Quenching under UV derivatives Ethyl Acetate: Methanol: Water 100:13.5 :10 5% - 10% KOH Bitter Principle Ethyl Acetate: Methanol: Water 17:15:08 -- Quenching under UV

2.3 Quantitive analysis of phytochemicals: Where A is absorbance of DPPH and B is 1. Determination of total phenolic content: absorbance of DPPH and extract combination Total phenolic content of plant extracts are (McCune and Johns. 2002). determined by the Folin-Ciocalteu method 2.4 Analysis of variations in the bactericidal (Sadasivam and Manickam. 2008) with certain activity by agar cup diffusion method: modifications. The method involves the use of Folin- Ciocalteau reagent which reacts with the 1. Microbial strains and culture conditions reducing compounds i.e. polyphenols in the a. Test organisms: extracts under alkaline conditions there by Clinical strains of Gram negative bacteria - producing a blue coloured complex. This is Escherichia coli, Klebsiella pneumoniae, and quantitatively estimated at 720nm against 100 to Gram positive bacteria - Staphilococcus. aureus, 1000 µg/ml of standard gallic acid solution. were used for the test. The diluted extracts (100 µl) were pipetted into different test tubes to which 0.2 mL of Folin- b. Preparation of inoculums: Ciocalteau reagent and 1 mL of 20 % (w/v) The inoculums for each bacterial culture was Na2CO3 solution were added. The tubes placed in a prepared from broth cultures grown in sterile boiling water bath for exactly 1 min and then Mueller-Hinton broth at 370C for 18 hours Saline were cooled under running tap water. The suspensions of the test organisms were prepared absorbance of the resulting blue solution was and the cell density was adjusted to 108 cfu/ml by measured at 720 nm with a spectrophotometer. The comparison with the 0.5 McFarland tube. experiment was carried out in triplicates. The Standard control drug – Ciprofloxacin (50 µg/ml) amount of phenolics present in the sample was was used as a positive control for all the determined from a standard curve of Gallic acid microorganism strains. and was expressed in mg per gram of the extract. 2. Determination of inhibitory effect: 2. Determination of antioxidant activity by The antimicrobial activity was determined by agar DPPH Radical Scavenging Assay: cup diffusion method. Clear inhibition zones The DPPH (2, 2-diphenyl-1- picrylhydrazyl) assay around the wells indicated the presence of is based on the capability of stable free radical 2, 2- antimicrobial activity for the extracts of the diphenyl-1-picrylhydrazyl to react with H-donors. mushrooms singly and in synergistic manner. DPPH, when acted upon by an antioxidant, is converted into diphenylpicryl hydrazine. The 3. Results and Discussion: degree of stable DPPH decolourization to DPPHH 3.1 Qualitative analysis: (reduced form of DPPH) which is yellow, indicated The biochemical components of ethanol extracts of the scavenging efficiency. This method was non edible mushrooms Ganoderma tsugae and reported by McCune and Johns (2002). Dadaelea quercina were studied. The extract of G.tsugae showed presence of Flavonoids, DPPH free radical scavenging assay was performed Alkaloids (Dragondroff’s test), Phenolic to determine the antioxidant activity of different compounds, Terpenoids and Tannins. extracts. DPPH (0.002%) was used as free radical Extract of D.quercina showed presence of Steroids donor, with standard ascorbic acid solution as the (Salkowski reaction), Glycosides particularly control. Equal volume of extracts (1mg/ml) and cardiac glycosides (Keller–Killani test), DPPH were mixed and the tubes were incubated at Flavonoids, Alkaloids (Dragondroff’s test), room temperature in dark for 10 minutes. The Phenolic compounds and Tannins. optical density was measured at 517nm using UV- Phytochemical analysis of methanol and n-butanol Vis Spectrophotometer. The experiment was fraction extracts of Ganoderma lucidium was carried out in triplicates. The scavenging activity of performed by Shamaki et al, (2012) showing the extract against the stable DPPH was calculated presence of flavonoids, carbohydrates, tanins, using the following equation - cardiac glycosides, saponins and terpenoids. Phytochemical examination of solvent extracts of Free radical Scavenging activity (%) = (A – B) / A the -rotting fungi Daedalea quercina and x 100 confragosa var. tricolor was

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performed by Rosecke et al, (2000) which led to The results showed that the extracts were rich in the isolation of five new triterpene derivatives and phenolic compounds wherein the concentrations in some known fungal constituents and all structures the extracts of G. tsugae and D. quercina were 580 were identified by one- and two-dimensional NMR µg/ml and 600 µg/ml respectively. These findings spectroscopy and mass spectrometry. TLC analysis indicated that the antioxidant activity of might be was performed which confirmed the presence of attributed to the phenolic contents. The DPPH the phytochemicals. radical scavenging activity was estimated to be 72.22% and 66.67% in G. tsugae and D. quercina 3.2. Quantitive analysis of phytochemicals: respectively. Estimation of total phenols were determined in the present study. The results are presented in Table 1.

Table 2: Quantitative analysis of Phytochemicals in Ganoderma tsugae and Daedalea quercina

Sample Ganoderma tsugae Daedelea quercina Total Phenolic compounds 580µg/ml 600µg/ml DPPH radical scavenging 72.22 % 66.67 % assay

3.3 bactericidal activity of independent and produces an effect greater than the sum of their combined extracts of the two mushrooms: individual effects. In toxicology, synergism refers In the present study it was found that the two to the effect caused when the exposure of two or extracts exhibited antimicrobial activity against all more chemicals at the same time results in health the three clinical isolates used (Escherichia coli, effect that are greater than the sum of the effects of Staphylococcus aureus and Klebsiella the individual chemicals. The combination of the pneumoniae). The results are presented in Table 2. two extracts did not produce any inhibitory effect The zones of inhibition of the ethanol extract of on the tested microorganisms. The reason for this G.tsugae are 20mm against all the three bacteria. might be because the two extracts may have an This shows that E. coli, S. aureus and K. antagonistic effect on each other thereby reducing pneumonia showed equal sensitivity to G. tsugae their overall effect as compared to when they are extract. Similar work was performed by Nagraj et used separately. Synergistic studies were presented al, (2013) which tested the antimicrobial activity of by Udu- Ebiam et al, (2015), wherein synergistic G.applanatum where methanolic extracts exhibited antimicrobial activity of the extracts of Pleurotus antimicrobial activity against all of the tested species and Psychotria microphylla against five bacteria. clinical bacterial isolates: Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, The zones of inhibition of the ethanol extracts of D. Klebsiella pneumoniae and Staphylococcus aureus quercina are 12mm, 13mm and 15mm respectively. were studied and the herb-mushroom extract This shows that E. coli was least sensitive and combination produced a better antimicrobial K.pneumonia was most sensitive to the extract of activity against the isolates with highiest sensitivity D.quercina. of 16.0 mm recorded against S.aureus.

Synergistic effect is the interaction between two or more agents, entities, factors, or substances that

Table 2: Antimicrobial activity and Synergistic effect of Phytochemicals in Ganoderma tsugae and Daedelea quercina Test Zone of inhibition(mm) micro-organism Ganoderma tsugae Daedelea quercina Synergistic effect Escherishia coli 20 12 No inhibition Staphylococcus 20 13 No inhibition aureus Klebsiella 20 15 No inhibition pneumoniae

Conclusion Daedalea quercina. Ganoderma tsugae extract showed presence of Flavonoids, Alkaloids, In the present study the biochemical components Phenolic compounds, Terpenoids and Tannins. were detected in the ethanol extracts of two non Extract of D.quercina showed presence of Steroids, edible mushrooms Ganoderma tsugae and Glycosides particularly cardiac glycosides,

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Flavonoids, Alkaloids, Phenolic compounds and Tannins. TLC analysis was performed which Adam, H.K., Campbell, I.M., McCorkindale, N.J., confirmed the presence of the phytochemicals. 1967b. Ergosterol peroxide: a fungal artefact. Nature The two tropical mushrooms showed high 216, 397 antioxidant activity which might be attributed to Asatiani, M et al., 2007 Free-Radical Scavenging the presence of natural antioxidants such as Activity of Submerged Mycelium Extracts from Higher phenolic compounds in due to the presence of Basidiomycetes Mushrooms, Bioscience, Biotechnology, phenolic compounds in them. and Biochemistry, 71:12, 3090-3092,

Both the extracts showed antimicrobial activity Baldrian, P. 2004, Purification and characterization of against selected Gram positive and Gram negtive laccase from the white rot fungus Daedalea quercina and organisms G.tsugae and D.quercina. The ganoderic decolorization of synthetic by the enzyme. Journal acids present in G.tsugae may play key roles in its of Microbiology and Biotechnology 63 (5), 560-568. antioxidant and antimicrobial activities. These Chung Shu, S et al. 2008. Ganoderma tsugae extracts findings indicate that the extracts from G.tsugae inhibit colorectal cancer cell growth via G2/M cell cycle and D.quercina could be used as natural additives arrest. 120(3), 394-401. in many foods. There was no synergistic effect of the two extracts on the tested microorganisms. This Gebhardt,P et al 2007, “Quercinol, an anti-inflammatory may be due to the antagonistic effect of the extracts chromene from the wood-rotting fungus Daedalea against each other. Further studies will be required quercina (Oak Mazegill)” Institute for Natural Product to isolate and purify the extracts to determine the Research and Infection Biology, HKI, Beutenbergstr. components responsible for bioactivity of these 11a, 07745 Jena, Germany Friedrich-Schiller-University, Jena, Germany. extracts.

Gray et al.1996. Oxidative quality and shelf life of meats. Acknowledgements Meat Science. 43. pp 111–123.

The authors thank the teaching and non teaching Hai Chen, D. and Dee Chen, W. 2002. Determination of staff of Biotechnology Department for their Ganoderic acids in Triterpenoid Constituents of assistance in carrying out this research work. Ganoderma tsugae. Journal of Food and Drug Analysis. 11(3), pp. 195-201. Research Highlights Hobbs, C.H. 1995. Medicinal Mushrooms: An Exploration of Tradition, Healing and Culture. Botanica Study of bioactive compounds of the tropical wild Press, Summertown, Tennessee. mushrooms, Study of antimicrobial activity of mushroom Joachim RoÈ secke, Wilfried A. KoÈ nig* “Constituents extracts and the possibility of synergistic of the fungi Daedalea quercina and Daedaleopsis bactericidal action. confragosa var. tricolor” Institut fuÈr Organische Chemie, UniversitaÈt Hamburg, Martin-Luther-King- Limitations Platz 6, D-20146 Hamburg, Germany April 2000

Kamaraj, C. 2012. Evaluation of antibacterial activity of Study of fresh fungal sample could not be carried selected medicinal plant extracts from south India against out due to unavailablilty of the sample human pathogens. Asian Pacific Journal of Tropical Synergistic bactericidal action needs to be studied Disease. 2(1). S296-S301. with the change in certain parameters. Kenneth, J. 1990, Reishi, ancient herb for modern times, Declaration Sylvan pres,. 6 The research work is the own contribution of the authors and there are no conflicts of interest with Lucas E.H et al. 1957. Tumor inhibitors in Boletus anyone else. edulis and other holobasidiomycetes. Antibiot. Chemother. 7, 1–4.

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