ANTIBACTERIAL ACTIVITY OF OMAN’S FACULTATIVE GANODERMA APPLANATUM

* Faruck Lukmanul Hakkim 1 , Quazi Mohammad Imranul Haq 2 , Jackson Achankunju 1 and Fatema Khalifa Khalfan AlHasaniay 1 . 1. Biology Division, Department of Basic Sciences, College of Applied Sciences, A’Sharqiyah University, Ibra, Oman. 2. Department of Biological Sciences and Chemistry, College of Arts and Sciences, University of Nizwa, Nizwa, Oman. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Ganodermaapplanatum is one of the most popular medicinal mushroom due to its various biologically active components. In the present study, we explored the antibacterial effect of Oman's Ganodermaapplanatum.Methanolic extract obtained from G. Applanatum tested for growth inhibition against different bacterial strains (such as E.coli, Klebsiella,Staphylococcus and Bacillus) and further we analyzed their chemical profiling by GCMS analysis. We found appreciable anti-microbial activity with different concentration of extract tested. To the best our knowledge this is the first report on Oman's Ganodermaapplanatum for its antibacterial and chemical profiling.

…………………………………………………………………………………………………….... Introduction:-
In different parts of the world, Mushrooms have long been used in traditional medicines and are well known due to their medicinal importance. Around 700 species of Mushroom out of 14000 to 15000 species of mushrooms in the world, have known medicinal properties. However, there are about 1800 species have potential medicinal attributes (Neha et al., 2012;Hawksworth, 2001;Chang and Miles, 2004).In addition, it is also a good source of mineral nutrients. Bacteria are the smallest, unicellular organisms, equipped with all the machinery for growth and selfreplication at the expense of foodstuffs. Only a few species of bacteria cause disease, often due to the cell's ability to produce specific poisons, toxins or aggresins (Jain, 2008). Antibacterials are the substances produced by microorganisms, which selectively suppress the growth of or kill other microorganisms at very low concentrations (Neha et al., 2012).Infectious diseases are the major threats to human health. Bacterial infection and reactive oxygen species mediated biological complications remains unresolvable challenges for the past many decades although the effective drugs are available in the market. Commercial antibacterial and antioxidant agents elicit significant toxicity in long term use in particular new bacterial mutants are emerging in response to therapy. These hurdles are major concern for researchers. Finding a new molecules or extract formulation to alleviate this problem is highly warranted. In these scenario, the facultative mushrooms have attracted much attention for the past many decades due their chemical diversity, less toxic nature and abundant source of medicinally important compounds. In particular, a well knownGonaderma species has been well enumerated for numerous biological activities.
Although, natural and synthetic antimicrobial agents have been reported to completely restrict the growth of many pathogenic microorganisms. Increasing development of antimicrobial resistance in many pathogens is a danger for public health. Therefore, effective antimicrobial agents are continuously being investigated from different biological ISSN: 2320-5407 Int. J. Adv. Res. 4 (11), 780-783 781 sources. Many clinically important antibiotics were isolated from fungi belongs to order-Actinomycetales. Although, well known examples of antibiotics such as, griseofulvin, cephalosporin and penicillin is produced from different fungi. Since Greek and Roman antiquity, it has been known that macro fungi may be a new source of a useful bioactive compounds (Anke et al., 1989).
In this context,Ganodermaapplanatum cultivated in different parts of the world has been shown to be an effective antioxidant and antibacterial agents. However climatic conditions and sessional variation leads to bio synthesis of different molecules with high chemical diversity within them. Bracket shape and reddish brown colourfruit bodies are the characteristic features Ganodermaapplanatum (artist's fungus) (Halliwell, 1994). The hard waxy crust has a rough surface (Langseth, 1993), and the dark reddish-brown flesh with a fibrous texture (Halliwell, 1994). Spores are released from pores which are initially white but later turns brownish (Halliwell, 1994). Oman is tropical country with high temperature and we hypothesize that this tropical condition facilitates high accumulation of medicinally important compounds. To address this issue we tested the extract derived from Oman's Ganodermaapplanatum on different bacterial strains such as E.coli, Klebsiella, Bacillus and Staphylococcus. Similarly, these microorganisms were also tested for antimicrobial activity against the heavy oil derived from Oman's Frankincense, showed strong antimicrobial activity (Hakkim et al., 2015). To the best of our knowledge this the first study of antimicrobial activity and chemical profiling ofOman's G. applanatum.

Materials and Methods:-
Extraction of Bio active principles:-G. applanatum was collected and shade dried for seven days and pulverized by mechanical grinder. Methanolic extract obtained by soxhlet extraction procedure briefly, 20 gm of G. applanatum powder was packed in filter paper and placed in soxhlet extractor and 500 ml of methanol (Analytical grade) was used for extraction. Obtained methanolic extract dried at room temperature in fume hood and dried extract store at -20 °C until use for experiments.

Drug preparation:-
Stock solution of G. applanatum dried methanolic extract prepared in dimethylsulfoxide (DMSO). Different concentrations such as 10, 25, 50, 75 and 100 mg/ml of C. schoenanthusmethanolic extract prepared from stock using DMSO and store in -20 °C until use for experiments.

Preparation of test organisms:-
Both gram positive and gram negative bacterial strains: namely E.coli, Klebsiella, staphylococcus and Bacillus were used. The test microorganisms were grown on nutrient agar by following standard procedure as described elsewhere.

Antibacterial activity assay:-
The antibacterial activity of G. applanatummethanolic extract was determined using agar well diffusion method (Taye et al., 2011). The inoculums were prepared by taking overnight bacterial culture. For sensitivity assay test 38 g of Muller Hinton agar was dissolved in 1000 ml distilled water and autoclaved at 121 °C for 15 min. The media was then poured into sterilized petri-dishes with uniform thickness and the agar was allowed to set at ambient temperature under laminar hood until solidification. These inoculums were spread evenly on the surface of solidified Muller Hinton agar with the help of sterilized spreader. On each plate equidistant wells were made with a 6mm diameter sterilized cork borer. Then 60 µl of different concentration of G. applanatummethanolic extract was aseptically added to the respective well. This was followed by allowing the agar plate to stay for 30 min under laminar hood and then incubated at 37 °C for 24 hrs. The formations of clear inhibition zone around the wells were taken as susceptibility measurement.

Gas chromatography mass spectrometry (GCMS) analysis:-
GCMS analysis was done as described earlier (Hakkim et al., 2016). Briefly, GCMS analysis was performed on a Perkin Elmer Clarus 680 GC System, fitted with a Rtx®-5MScapillary column (30m×0.25mm i.d. × 0.25μm film thickness; maximum temperature, 250 ℃), coupled to a Perkin Elmer Clarus SQ8S MS. Ultra-high purity helium (99.9999%) was used as carrier gas at a constant flow of 1 ml/min. The injection, transfer line and ion source temperatures were 270, 240 and 240 ℃, respectively. The ionizing energy was 70 eV. Electron multiplier (EM) voltage was obtained from autotune. All data were obtained by collecting the full-scan mass spectra within the scan range 40-550 amu. The injected sample volume was 1μl with a split ratio of 50:1. The oven temperature 782 program was 60 o C and accelerated at a rate of 3 o C/minute-240 o C. The unknown compounds were identified by comparing the spectra obtained with mass spectrum libraries (NIST MS20 2011).

Results and Discussion:-
Different types of solvents were used to estimate antimicrobial activities, since they have different polarities in plants. Many antimicrobial compounds such as terpenes, lectins, polysaccharides etc. act on the bacterial cytoplasmic membrane (Lin & Chou, 1984;Yang et al., 2002;Sadaf et al., 2010). The common solvents used for extraction processes are dimethyl sulfoxide (DMSO), acetone, methanol, ethanol and water. Yield and antimicrobial activities of extract are mainly depend on the type of solvent selected (Kagen et al., 1992;Packer and Witt, 1995).Ganodermasp provides bioactive compounds that claim to posses antibacterial activity (Djide et al., 2014). In our study, antimicrobial activity of the extract of Oman's G.applanatum was determined.
G.applanatumand several other medicinaly important mushrooms have the prospective to turn into regular drugs. To investigate this potential, defined production processes, standardised quality and methods for its control and legal clinical trials are mandatory (Chang and Wasser, 2012;Lindequist, 2013;Wasser, 2010). These processes require a very close interdisciplinary collaboration, money and time (Ulrike et al., 2015).