Sathyaprabha Govindaraj1 and Senthil Renganathan2

1Department of Microbiology, Marudupandiyar College, Thanjavur, TN, India.
2Department of Bioinformatics, School of Bioscience, Marudupandiyar College, Thanjavur, TN, India.

Abstract

Pleurotus sp is an edible mushroom, which contains essential nutrients and medicinal properties for living thing. Molecular characterization and genome differentiation of pleurotus sp.studiedusing gel electrophoresis, RAPDand PCR techniques.Separated DNAwas amplified using internal transcribed spacerprimers. 5.8s rRNA sequence submitted to Genbank for comparison and to obtain accession number. Differentiation and identification of Pleurotus sp found by similarity searches(Accession number: JQ740170 and JQ740171). Comparative studies have shownthese two Pleurotus sp (spp1 & ssp2)are closely related to species, 95% -100% identical,Pleurotus Citrinopileatus, Pleurotus populinus, Pleurotus ostreatus, Pleurotus abieticola, Pleurotus pulmonarius, Pleurotus eryngii and Pleurotus sapidus. These Pleurotus sp havebeen studiedfor exhibiting pharmacological potential activitiessuch asantioxidant, antitumor, immunomodulatory activity and hypocholesterolemic through sequence similarity,nucleotide composition, evolutionary studies, secondary structure prediction and conserved region.The study extended to identify the significance of species diversity and fungal samples among various applications.Pleurotus (sp) samplewas subjected to identification of bioactive compounds by using GC-MS. In Pleurotusspp1: Pyridine- 3-carboxamide, 4-dimethylamino-N-(2, 4-difluorophenyl), Piperidin-4-carboxylic acid, Aspidofractinine-3-methanol, (2à, 3á, 5à), Indolizine, and 2-(4-methylphenyl)-. Pleurotusspp2: Imidazolidine, 1, 3-dinitro, Phenol, 2-methyl-4-(1, 1, 3, 3-tetramethylbutyl), and Squalene. Enhancement of more entries as well as sequence comparison and virtual screening might help researchers to acknowledge real world problems between cultivatingnutritional, medicinal and poisonous mushroom.

Keywords: edible Mushroom-internal transcribed spacer-metabolites -pharmacological activities-species diversity.

Introduction

Mushrooms  are having highly nutritive and medicinal values so now a day’s peoples are readily used to  cultivate  in  controlled  biological environment  and  it  has  been  extensively  used  as  food since  ancient  time (Sathyaprabha et al. 2011). Although, mushrooms are rich in proteins and fibers itcontain low amount of calories and fat, in the presence of low fat, peoples are highly used the edible mushroom frequently(Manziet al., 2001).Cultivation of Pleurotus spp1 and spp2in different substrates. Spawn were prepared in three types of substrates such as sorghumgrain, Paddy grain and Maize grain. For fruit body cultivation Paddy straw, Sugar cane trash, Teak Leaves, Black gram Pods, and Banana Leaves are the substrates.Paddy grains substrate was good in growing and gives high yield of mycelium when compared to the sorghum grains and maize grain (Sathyaprabha et al., 2011).

In the presence of good  nutritive  value  of  mushrooms,  a remarkable  progress  required  in  the  breeding  of   its  new strains  which  results  in  difficulty  of   their  identification.Need  of  advance  techniques for species  identification  beyond  morphological  and  physiological  criteria,  because  these  characteristics highly influenced  by  cultivating  conditions were posed big challenge (Staniaszeket al., 2002; Iqbal et al., 2010). The  genetic  diversity  of   mushrooms  has  been  worked out  using  molecular markers  especially  random  amplified polymorphic  DNA (Staniaszek et al., 2002; Stajic et al., 2005; Ravashet al., 2009). RAPD  is  used  to  assess  genetic  similarity  and  phylogenetic analysis  due  to  the  simplicity  in  its  methodology and utility (Gepts 1993).

(Roet al., 2007) studied  the diversity of   Pleurotus  eryngii  using  RAPD technique and  its  sequence analysis  and  observed  that,  grouping  based  on physiological  parameters  is  closely  related  to  RAPD based  grouping.(Gepts,1993) used  randomly amplified  polymorphic  DNA  technique  to  access  the genetic  diversity  among  37  pleurotus  species  of   mushrooms.  In  another  study,  polymerase  chain reaction  (PCR)  amplification  was  used  to  evaluate  the genetic  diversity  among  45  pleurotus strains  and  found that, this technique was better than morphological analysis(Rajaratnam and Thiagarajan 2012).Identified and comparedthree medicinal mushroom (Ganodermalucidum, Coriolusversicolor, and Fomesfomentarius) species from Korea based on nuclear large subunit rDNA sequences (Dung et al., 2012). Nucleotide sequencing of Termitomycesalbuminosus(Lee et al., 2006; Yang et al., 2012)and Agaricusbisporus (Chen et al., 2011) among others was also performed and deposited into sequence database.  Available sequence entry and information ingenbank database for mushroom is not sufficient (Morin et al., 2012).A primer based on ITS region is rather useful for molecular characterization in fungi at the species level and within the species (Sudip Kumar Das et al., 2013; Williams et al., 1990; Chen et al., 2001). Keeping  in  view  the  usefulness  of   morphological  and molecular  markers,  the  present  study  was  planned  to investigate the genetic diversity among different strains of cultivated mushroom (Pleurotus sp.). Fragments of the DNA were amplified using ITS1 (Internal Transcribed Spacers) and ITS4 primers. The nucleotide sequences of mushrooms were matched from the available known sequences of genbank database.

Today, with the advancement of techniques and nutrient values mushrooms have occupied an important place in food in several parts of the world. We found that mushrooms are healthy foods, even though they are deficient in calories and fat and consist of about 90% water (Sathyaprabha et al., 2011). Mushrooms have been reported to be of therapeutic value, useful in preventing diseases such as hypertension, Hypercholesterolemia, cancer and also having antibacterial and antiviral properties. Functional characteristics are mainly due to their chemical composition.Pleurotus sp were further studied for screening bioactive compounds and vitamins using GC-MS. (Suresh Lalitharani 2010; Sathyaprabha et al., 2011).

 

Materials and Methods

DNA extraction and isolation from mushroom fruit body

 

0.01 g of mycelium was grinded with mini grinder using 75 µl of STE extraction buffer (320 mM Sucrose, 10 mM Tris-Cl, 20 mM EDTA, 75 mM NaCl and 2.5 ml of 20% SDS) along with 5 mg of Polyvinyl pyrolidone and 0.1 g of silica powder, incubated at 65°C for 10 minutes. Centrifuged the sample at 13,000rpm for 10 minutes. To the supernatant, equal volume of chloroform: isoamyl alcohol was added and repeated the centrifugation. To the aqueous layer, added 2/3 volume of isopropanol and centrifuged at 13,000 rpm for 10 min. The pellet was washed with 70% ethanol by centrifuging and the pellet was dried, dissolved in 50µl TE buffer.

The DNA stock samples was quantified using UV spectrophotometer at 260 and 280 nm using the convention that one absorbance unit at 260 nm wavelength equals 50 µg DNA per ml. The Ultra Violet (UV) absorbance was checked at 260 and 280 nm for determination of DNA concentration and purity. Purity of DNA was judged by the optical density ratio at 260:280 nm. The DNA having ratio between1.8 – 2.0 was considered to be of good purity. Concentration of DNA was estimated using the formula. Concentration of DNA (mg/ml) = OD 260 x 50 x Dilution factor Quality of DNA was again checked by Agarose gel electrophoresis. The 0.8 % Agarose was prepared (0.8 g Agarose power / 100 ml 1 X TBE), and was melted. 30 ml Agarose was poured into the casting tray. The gel was allowed to solidify and the comb and tape was removed. 1 X TBE (Tris-Borate-EDTA; electrophoresis buffer) was added to the chamber until the buffer just covers the top of the gel. The samples were loaded with Bromophenol blue loading dye, taking care not to puncture the well bottoms. The power pack was turned on and run at 100 V. The gel was viewed on a UV transilluminator after electrophoresis.

 

DNA Amplification

The DNA was used further for PCRamplification. ITS (Internal Transcribed Spacer) fragment was amplified by PCR from fungal genomic DNA using ITS-Primer “ITS 1: 5 ’-TCCGTAGGTGAACCTGCGG-3’”,” ITS4: 5’- TCCTCCGCTTATTGATATGC-3’ ”. PCR was carried out in a final reaction volume of 25 µl in 200 µl capacity thin wall PCR tube. Composition of reaction mixture for PCR is given in PCR tubes containing the mixture were tapped gently and spin briefly at 10,000 rpm. The PCR tubes with all the components were transferred to thermal cycler. The PCR protocol designed for 30 cycles for the primers used is given below.Deionized water 1 6.5 µl, Taq buffer without MgCl2 (10 X) 2.5µl, MgCl2 (15 mM) 1.0 µl, Forward Primer (10 pm/µl) 2.0 µl, Reverse Primer (10 pm/µl) 0.5 µl,Taq DNA Polymerase (5U/µl) 0.5 µl, Template DNA (20 ng/µl) 0.5 µl, Final Volume 25.0 µl.Initial Denaturation  95°C for 5 minutes, Denaturation 94°C for 30 seconds, annealing 55°C for 30 seconds, extension 72°C for 45 seconds, and final elongation 72°C for 10 minutes end.

 

Analysis of DNA Amplification by Agarose Gel Electrophoresis

 

Loaded 5µl of PCR product with 4 µl bromophenol blue (Loading Dye) in 1.5% Agarose gel and ran the gel at constant voltage of 100V and current of 45ºA for a period of 1 hr 20 min till the bromophenol blue has travelled 6 cms from the wells. Viewed the gels on UV transilluminator and photograph of the gel was taken (Sudip Kumar Das et al.,  2013).

 

 

DNA Sequencing

 

Amplified PCR product was purified using column purification as per manufacturers Guidelines, and further used for sequencing.The concentration of the purified DNA was determined and subjected to automated DNA sequencing on ABI3730 XL Genetic Analyzer (Applied Bios stems, USA).

 

ITS Sequence alignment and analysis

 

Each nucleic acid sequence was edited manually trimmed to remove unreadable sequence at the 3 ’and 5’ ends (considering peak and Quality Values for each base) using the sequence analysis tools. The edited sequences were then used for similarity searches using BLAST (Basic Local Alignment Search Tool) programmed in the NCBI Genbank (www.ncbi.nlm.nih.gov/blastn) database for identifying the fungal strains(Accession number: JQ740170 and JQ740171)(Senthil et al. 2011).

 

 

Qualitative analysis forphytochemical constituents on Pleurotus spp1and Pleurotus spp2

 

Aqueous extract of Pleurotus spp1 and Pleurotus spp2samples were used to carry out the Qualitative analysis using standard procedures to identify the phytoconstituent as described (Sofowora 1993). Test for tannins:0.5 gm of the fresh mushroom samples were taken in test tubes boiled with 20 ml of water and filtered. A few drops of 0.1% ferric chloride was added and observed for brownish green or a blue-black coloration. Test for phlobatannins:Deposition of a red precipitate, when an aqueous extract of each plant sample was boiled with 1% aqueous hydrochloric acid was taken as evidence for the presence of Phlobatannins. Test for saponin:About 2 gm of the fresh sample was boiled in 20 ml of distilled water in a water bath and filtered. 10 ml of the filtrate was mixed with 5 ml of distilled water and shaken vigorously for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigorously, then observed for the formation of emulsion. Test for flavonoids:5ml of the diluted ammonia solution was added to a portion of aqueous filtrate of plant extract followed by the addition of concentrated sulphuric acid shown formation of yellow color. Test for steroids:2 ml of acetic anhydride was added to 0.5 g ethanol extract of each sample with 2 ml H2S04. The color changed from violet to blue or green in some samples indicating the presence of steroids. Test for terpenoids:5 ml of each extract was mixed in 2 ml of chloroform, and concentrated H2S04 (3 ml) was carefully added to form a layer. A reddish brown coloration of the interface was formed to show positive results for the presence of Terpenoids. Test for cardiac glycosides:2 ml of glacial acetic acid containing one drop of ferric chloride solution was added to 5ml of the mushroom extract. Addition of1ml concentrated sulphuric acid shownformation of a brown ring at the interface indicates the presence of cardiac glycosides.

 

 

 

 

 

 

 

 

Fig. 1 Amplification of Pleurotus sp with ITS primers and separation of the PCR products by gel electrophoresis (spotted ~ 600-800 bp): Lane 1.  DNA marker; Lane 2 and 3: Pleurotus spp1&spp2. Gel electrophoresis marker ranges bands from 1500 bp to 100 bp while sample shows that approximately at 1200 bp to 1100 bp.

 

 

 

 

 

 

 

 

 

 

Fig. 2 Circular Tree View of Pleurotus sp based on Jukes-Cantor Correlated Distance model (0.004) including other species.

 

 

 

 

 

 

 

 

 

Fig. 3 a) Tree based on the rRNA-ITS sequence using maximum-likelihood phylogenies, shown relationships among Pleurotus sp (JQ740170 and JQ740171) and b) secondary structure predicted using mFOLD server for their identity (spp1 & spp2).

 

Identification Bioactive compounds by GC-MS – Perkin Elmer.

 

Sample preparation

 

Twenty five gram of smashed fresh Pleurotus spp1 and spp2sample was taken in a conical flask with 30 ml of distilled ethanol and methanol crushed with respective solvent and stored it for overnight soaking. Then filter the sample and concentrated the sample with help of nitrogen flushing. Filter the filtrate with sodium sulphate. 3μl of purely prepared sample was injected into the programme GC-MS instrument.

Gas Chromatography Programme   
     

Column:  Elite-5MS (5% Diphenyl / 95% Dimethyl poly siloxane), 30 x 0.25mm x 0.25mm df Equipment:  GC Clarus 500 Perkin Elmer, Carrier gas:  1ml per min, Split: 10:1, Detector:  Mass detector Turbo mass gold-Perkin Elmer, Software:  Turbomass 5.2, Sample injected:  3ml. Oven temperature Programme: 110° C -2 min hold ,Up to 200° C at the rate of 10 ° C/min-No hold,Up to 280 ° C at the rate of 5° C / min-9 min hold ,Injector temperature 250° C,Total GC running time 36 min. Mass Spectrum Programme: Library used NIST Version-Year 2005, Inlet line temperature 200°C, Source temperature   200°C Electron energy: 70 eV, Mass scan (m/z):  45-450,Solvent Delay:  0-2 min, Total MS running time: 36 min.

 

Virtual screening

 

Till date, virtual screening of compound aids to identify their interaction with proteins. Protein-ligand interactions found by Insilico analysis will exhibit the potential of bioactive compounds and to obtain lead compounds rather than chemically synthesis. Out of 17, which are known as antitumor and anticancer agents through existing methods, piperidin-4-carboxylic acid and squalene, were taken (Fig. 4 & 5). Phenol used to treat and aid for microbial and fungal infection was shown in fig. 6. It is noteworthy that screening all metabolites on various medicinal properties.

 

Results

Genomic DNA

 

Genomic DNA was extracted from harvested fruit body ofPleurotus sp(SPP1&SPP2). Extracted DNA molecules were performed by Agarose Gel electrophoresis techniques.Genomic DNA was isolated from the fungal sample/culture provided. Its quality was evaluated on 0.8% Agarose Gel anda single band of high-molecular weight DNA was observed. ITS fragment was amplified by PCR from the above isolated genomic DNA. The PCR amplicon was purified by column purification in order to remove contaminants.Commercially available 100 bp ladder was used as standard molecular weight DNA.A single discrete band was observed when resolved on Agarose Gel (Fig. 1).Extracted DNA from the fruit body of Pleurotus sp mushroom subjected to nucleotide sequencing using ITS1 and ITS4 conserved primer stretches. The sequence was aligned using similarity searches algorithm and aligned sequence (~800 bp) revealed 95% – 100% matched score with Pleurotus sp(Fig. 2). Characterized Pleurotusspp1(https://www.ncbi.nlm.nih.gov/nuccore/JQ740170) and Pleurotusspp2 (https://www.ncbi.nlm.nih.gov/nuccore/JQ740171) based on phenotype, genotype and molecular data found that both were unique to each other(Fig. 1 &3).In addition GC content of pleurotus sprangedfrom44 – 45% with a constant ratio (Table 1). Subsequently, pleurotus sp identical were retrieved for the phylogenetic analysis.Nine species grouped together one clad, pointing their diversificationand resemblance(Fig. 3).A phylogenetic analysis- maximum-likelihoodbased on 5.8s rRNA was conducted to determine the relationship of pleurotus sp with other medically important species. Except, sequence data of p. sapidusdue to its long order in length.

Table 1 Composition and conserved region found among Pleurotus sp.

 

 

 

 

 

 

 

 

 

 

Table 2 5.8s rRNA-ITS sequences/species of closely related to pleurotus spp1andspp2 studied for Phytoconstituent and medicinal values (Chen et al. 2011; Jose et al. 2002; Bobek and Ozdin 1996; Fu et al. 2009; Selegean et al. 2009; Daba and Ezeronye 2003; Adebayo et al. 2012

 

S. No
Name of the organism
Identity (%)
Accession No
Studied and found Medicinal Properties

 

1.
Pleurotus Citrinopileatus
98
JF736661
Anti inflammatory, Anti oxidant, and Anti tumor activities
2.
Pleurotus ostreatus
98
AY450345
Anti tumor activity , Immunomodulator activity, Anti oxidant activity , Hypocholesterolemic
3.
Pleurotus abieticola
98
AY450348
Anti inflammatory activity , Anti tumor activities and Anti oxidant  ,
4.
Pleurotus populinus
97
AYA450346
Prostate cancer and other cancers.
5.
Pleurotus pulmonarius
97
AY450349
Anti inflammatory, Anti oxidant and Anti tumor activities
6.
Pleurotus eryngii
97
AY450347
Anti inflammatory
7.
Pleurotus sapidus
96
JF736664
Anti viral, Anti bacterial, Anti diabetic, Anti arthritic, Anti tumor activities

 

Phylogenetic tree constructed shown a close relationship between Pleurotus Citrinopileatus and Pleurotus ostreatus, Pleurotus abieticola and Pleurotus pulmonarius. Pleurotus populinus and Pleurotus eryngii with Pleurotus spp1 & spp2, grouped together for evolutionary studies.The significance of the work is to gain proper identification knowledge of the edible and non-edible mushrooms, which may drive towards domestication and commercialization of the wild species for economic benefits, nutritional value and medically important fungi instead deriving molecular taxonomy (Table 2).

 

Qualitative analysis

 

Qualitative analysis of Phytochemical revealed that, Pleurotus spp1 shows the high concentration of tannin when compared to sugarcane trash and teak leaves, phlobatannins was present in paddy straw but absent in all other four substrates. Saponin were absent in paddy straw but present in all other four substrate. Flavonoid was present in all substrates steroids was shows that high concentration in all the substrates. Terpenoids were absent in paddy substrates and banana leaves substrates, but present in sugarcane trash, teak leaves and black gram pods. Cardiac glycosides were present in all the substrates while absent in black gram pods (Table 3).

 

Table 3 Pharmacological properties found in pleurotus spp1andspp2.

S. No Parameters Pleurotus spp1 Pleurotus spp2
S1 S2 S3 S4 S5 S1 S2 S3 S4 S5
1. Tannin +++ ++ ++ +++ +++ —– +++ +++ —– +++
2. Phlobatannins +++ —- —- —- —– +++ ——- —— —-
3. Saponin —– ++ +++ ++ ++ +++ —– +++ ++ ++
4. Flavonoids +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
5. Steroids +++ +++ +++ +++ +++ +++ +++ +++ +++ +++
6. Terpenoids —– +++ ++ ++ —- —– —– +++ ++ —–
7. Cardiac glycosides +++ +++ +++ —- +++ +++ —– +++ —— +++

Note    :           Present – +++: Slightly Present – ++: Absent – —–

S1: Paddy straw, S2: Sugarcane trash, S3: Teak leaves, S4: Black gram pods, S5: Banana leaves

In Pleurotusspp2 Tannin, phlobatannins and Terpenoids were absent in paddy straw substrates but shows the presence of saponin, flavonoids, steroids but absences of saponin, Terpenoids and cardiac glycosides. Phlobatannins were present in the sugarcane trash substrates but absent in all other substrates, paddy straw, teak leaves, black gram pods and banana leaves. Black gram pods show the absence of tannin, Phlobatannins and cardiac glycosides, while flavonoids and steroids were strongly present but presence of saponin and terpenoids. Terpenoids and phlobatannins were absent in banana leaves but tannin, flavonoids, steroids and cardiac glycosides were strongly present while saponin  present (Table 3).

Table 4   Bioactive Components identified in the ethanol extract of Pleurotus spp1.

No.
RT
Name of the compound
Molecular Formula
MW
Peak Area %
Group
1.
7.17
DL-Alanine, N-benzoyl-N-(3-chloro-4-fluorophenyl)-, methyl ester
 C17H15ClFNO3
335
0.76
Fatty Acid
2.
7.44
(1H)Pyrrole-2-carboxaldehyde, 4-(trichloroacetyl)-
 C7H4Cl3NO2
239
0.76
Aromatic organic compounds
3.
7.96
2-Amino-4-hydroxy-6,7,8-trimethylpteridine
 C9H11N5O
205
3.80
Heterocyclic
4.
12.49
Benzoic acid 1-methoxy-1H-tetrazol-5-ylmethyl ester
 C10H10N4O3
234
0.76
Fatty acid
5.
13.06
Pyridine-3-carboxamide, 4-dimethylamino-N-(2,4-difluorophenyl)-
 C14H13F2N3O
277
74.14
Heterocyclic
6.
13.45
à-Ethyl aspartate
 C6H11NO4
161
2.28
Esters
7.
18.80
Piperidin-4-carboxylic acid
 C6H11NO2
129
1.52
Heterocyclic
8.
20.82
Aspidofractinine-3-methanol, (2à,3á,5à)-
 C20H26N2O
310
4.94
Alkaloids
9.
24.67
Indolizine, 2-(4-methylphenyl)-
 C15H13N
207
11.03
Alkaloids

 

 

 

Table 5 BioactiveComponents identified in the ethanol extract of Pleurotus spp2 .

No.
RT
Name of the compound
Molecular Formula
MW
Peak Area %
Group
1.
7.96
5-(4-Hexyloxybenzoyloxy)-2-(4-nitrophenyl) pyrimidine
C23H23N3O5
421
0.01
Heterocyclic
2.
10.15
Imidazolidine, 1,3-dinitro-
C3H6N4O4
162
0.02
Heterocyclic
3.
12.64
dl-Alanine
C3H7NO2
89
0.01
α-amino acid
4.
16.89
Phenol, 2-methyl-4-(1,1,3,3-tetramethylbutyl)-
C15H24O
220
0.07
Phenols
5.
17.51
1H-Tetrazol-5-amine
CH3N5
85
0.02
organic compound
6.
20.90
Aspidofractinine-3-methanol, (2à,3á,5à)-
C20H26N2O
310
99.83
Alkaloids
7.
24.66
Squalene
C30H50
410
0.05
Triterpene

 

Identification of Bioactive Compounds in

Phytochemical properties found by qualitative analysis suggesting their roles in synthesis of bioactive compound and vitamins. According to the results, the Phytocomponents screenedwere shown in the (Table 4 and 5). Animals and Human might require vitamins for their routine activities, in addition to food. Disseminationof vitamins has made with its dissolving nature and storage system. (Sathyaprabha et al.,  2011).

 

 

 

 

 

 

 

 

 

Fig. 4 Structure of 4-(4-tert-butylbenzyl)-1-(7h-pyrrolo (2, 3-d) pyrimidin-4-yl) piperidin-4-amine bound to protein kinase b (2XH5).  Ball and stick representation of ligand (piperidin) shown with hydrogen donor and acceptor.  Complex structure of receptor and ligand was shown in ribbon model. Schematic diagram of bonded and non bonded interaction was shown in yellow and black dot line (ligplot and pose view).

 

 

 

 

 

 

 

 

Fig. 5 Crystal structure of supernatant protein factor bound to squalene (PDB: 4OMK).  Ball and stick representation of  ligand (squalene) shown with active site residues.

 

 

 

 

 

Fig. 6 Structure of the flavoenzyme vanillyl-alcohol oxidase in complex with phenol was shown (PDB: 1AHZ).  Schematic representation of phenol with receptor has shown for the affinity and enzyme inhibition (Pose view and ligplot).

 

 

Discussion

Pleurotus sp was described as edible mushroom but the significance of species diveristy and nutrients , clinical values  studied through  amplification and ITS region sequence comparison.The ITS fragment of the genomic DNA of two edible mushrooms, collected from TNAU, Aduthurai, Thanjavur, India, was amplified using ITS1 and ITS4 primers and subjected to nucleotide sequence determination for identification of species. The amplification and sequence comparison uncovers species diversity and other species with similar molecular information for its domestication, characterization of nutritional value and medicinal benefits. Qualitative analysis made to estimate the pharmacological properties. By ranking strongly present, present and absent in 7 groups indicate that the availability and rich properties. Comparative study of the ranking method describes the abundance of flavonoids and steroids.The results obtained from GC-MS analysis is given in the (Table 4 and 5). Both pleurotus sp contains vitamins namely A, D3, E, K, niacin, pyridoxine, thiamine and riboflavin, were analyzed. Phyto constituents screened through GC-MS werestudied for identification of compound nature and group. Chemical composition and secondary metabolites of pleurotussp were studied. Totallyseventeen metabolites screened were examined for different properties and grouped as those belonging toacids, alcohol, alkane, amides,esters, fatty acids, terpenoids, heterocyclic and phenols (Mohamed et al., 2014). Hetero cyclic compounds were appeared more in numberthan fatty acids, ester, aroma and phenols (Rivera et al.,2012).

 

Piperidin-4-carboxylic acid

Piperidin derivatives were found in GC-MS studies further investigated with virtual screening to access their potent as antitumor agent (McHardy et al.,2010).Here the complex structure demonstrated that how the piperidin derivatives bind to protein and inhibits. Interaction between the receptor and ligand studied based on substitution method (McHardy et al., 2010)  prepared piperidin moieties to verify it’s multifold with side chains (Fig.4).

 

Squalene

 

Squalene and squalene analogs studied by several techniques and found useful in cancer therapy. Still its availability and usage widens the research on theskin treatment (Huang  et al., 2009) and cholesterol endosynthesis(Christen et al., 2015).Here supernatant protein factor (SPF) has modeled and studied for cholesterol endosynthesis (Fig.5).

 

Phenol

 

Phenolic compounds have known to possess medicinal values.(Alves et al.,2013) reported that phenolic compounds could be used for antimicrobial activityas activator and inhibitor.Especially for resistance bacteria, phenolic compounds had shownthe high inhibition rate when compared to different concentration level.A Study and virtual screening carried out to negotiate the species diversity and medicinal properties among pleurotussp. wereavailable.Recent studies shown that heterocyclicand phenolic compounds,hasmore impact on cancer related studies.Sequence comparison and insilico analysis made itpossible to explore molecules interaction with metabolites.

Acknowledgements

            We thank Dr. A. Panneerselvam, Professor, Department of Botany and Microbiology, A.V.V.M Sri Pushpam College, Thanjavur, TN- India for his valuable guidance and Tamil Nadu Agricultural University, Aduthurai, Thanjavur, TN-Indiafor sample preparation.

References

  1. Adebayo EA, Oloke JK, Majolagbe ON, Ajani RA, Bora TC. Antimicrobial and anti-inflammatory potential of polysaccharide from Pleurotus pulmonarius LAU 09. Afri J Microbiol Res, 2012;6:3315-3323.
  2. Alves MJ, Ferreira IC, Froufe HJ, Abreu RM, Martins A, Pintado M. Antimicrobial activity of phenolic compounds identified in wild mushrooms, SAR analysis and docking studies. Journal of applied microbiology. 2013; 115(2):346-57.
  3. Bobek P and Ozdin L, Oyster mushroom (Pleurotus ostreatus) reduces the production and secretion of very low density lipoproteins in hyper-cholesterolemic rates. Zeitschrift für Ernährungswissenschaft, 1996; 35: 249-252.
  4. Chen JN, de Mejia EG, Wu JS, Inhibitory Effect of a Glycoprotein Isolated from Golden Oyster Mushroom (Pleurotuscitrinopileatus) on the lipopolysaccharide – induced inflammatory Reaction in RAW 264.7 Macrophage. J Agric Food Chem, 2011; 59:7092-7097.
  5. Chen YC, Eisner JD, Kattar MM, Rassoulian-Barrett SL, Lafe K, Bui U, Limaye AP, Cookson BT, Polymorphic internal transcribed spacer region 1 DNA sequences identify medically important yeasts. Journal of Clinical Microbiology, 2001; 39: 4042–4051.
  6. Chen S, Xu J, Liu C, Zhu Y, Nelson DR, Zhou S, Li C, Wang L, Guo X, Sun Y,Luo H, Li Y, Song J, HenrissatB, Levasseur A, Qian J, Li J, Luo X, Shi L, He L,Xiang L, Xu X, Niu Y, Li Q, Han MV, Yan H, Zhang J, Chen H, Lv A, Wang Z, Liu M, Schwartz DC, Sun C,  Genome sequence of the model medicinal mushroom Ganoderma lucidum. Nat Commun, 2012; 26: 913.
  7. Christen M, Marcaida MJ, Lamprakis C, Aeschimann W, Vaithilingam J, Schneider P, Hilbert M, Schneider G, Cascella M, Stocker A, Structural insights on cholesterol endosynthesis: Binding of squalene and 2, 3-oxidosqualene to supernatant protein factor. Journal of structural biology. 2015; 190(3):261-70.
  8. Daba AS, Ezeronye OU, Anti-cancer effect of polysaccharides isolated from higher Basidiomycetes African Journal of Biotechnology, 2003; 2: 672-678.
  9. Das SK, Mandal A, Datta AK, Gupta S, Paul R, Saha A, Sengupta, S, Dubey PK, Nucleotide Sequencing and Identification of Some Wild Mushrooms. The Scientific World Journal, 2013; 2013:
  10. Dung NTP, Tuyen DB, Quang PH, Morphological and genetic characteristics of oyster mushrooms and conditions effecting on its spawn growing. International Food Research Journal, 2012; 19: 347–352.
  11. Fu HY, Shieh DE, HO CT, Antioxidant and free radical scavenging activities of edible mushrooms.  Journal of Food Lipids, 2009; 9:35-43.
  12. Gepts P, The use of molecular and biochemical markers in crop Evolution studies. Evolutionary Biology, 1993; 27: 51-94.
  13. Huang ZR, Lin YK, Fang JY, Biological and pharmacological activities of squalene and related compounds: potential uses in cosmetic dermatology. Molecules. 2009; 14(1):540-54.
  14. Iqbal A, Sadia B, Khan AI, Awan FS, Kainth RA, Sadaqat HA, Biodiversity in the sorghum (Sorghum bicolor L. Moench)  germplasm of Pakistan. Genetics and Molecular Research, 2009; 9: 756-764.
  15. Jose N, Ajith TA, Janardhanan KK,Antioxidant, anti inflammatory, and antitumor activities of culinary-medicinal mushroom Pleurotus pufmonanus (Fr.) Quel. (Agaricomycetideae). International Journal of Medicinal Mushrooms2002; 4: 329-335.
  16. Lalitharani S, Mohan VR, Selial Regini G, GC-MS analysis of ethanolic extract of Zanthoxylum rhetsa (ROXB.) DC spines. Journal of Herbal Medicine and Toxicology, 2010; 4: 191-192.
  17. Lee JS, Lim MO, Cho KY, Cho JH, Chan;g SY, Nam DH, Identification of medicinal mushroom species based on nuclear large subunit rDNA sequences. Journal of Microbiology, 2006; 44: 29–34.
  18. Manzi P, Aguzzi A, Pizzoferrato L,Nutritional value of mushrooms widely consumed in Italy. Food Chemistry, 2001; 73:321-325.
  19. McHardy T, Caldwell JJ, Cheung KM, Hunter LJ, Taylor K, Rowlands M, Ruddle R, Henley A, de Haven Brandon A, Valenti M, Davies TG, Discovery of 4-amino-1-(7 H-pyrrolo [2, 3-d] pyrimidin-4-yl) piperidine-4-carboxamides as selective, orally active inhibitors of protein kinase B (Akt). Journal of medicinal chemistry 2010; 53:2239-49.
  20. Mohamed EM, Farghaly FA, Bioactive compounds of fresh and dried Pleurotus ostreatus mushroom. International journal of biotechnology for wellness industries. 2014; 3(1):4-14.
  21. Morin E, Kohler A, Baker AR, Foulongne-Oriol M, Lombard V, Nagye LG, Ohm RA., Patyshakuliyeva A, Brun A, Aerts AL, Bailey AM, Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche. Proceedings of the National Academy of Sciences, 2012; 109: 17501–17506.
  22. Rajaratnam S, Thiagarajan T, Molecular characterization of wild mushroom. European Journal of Experimental Biology2012; 2:369–373.
  23. Ravash R,  Shiran  B,  Alavi  A, Zarvagis  J, Evaluation of genetic diversity in Oyster mushroom (Pleurotus eryngii) isolates using RAPD marker. Journal of Science ;and Technology of Agriculture and Natural Resources,2009; 13: 729-739.
  24. Rivera A, Quiroga D, Rios-Motta J, Dusek M, Fejfarova K, 1, 3-Dinitrosoimidazolidine. Acta Crystallographica Section E: Structure Reports Online. 2012; 68(8):2440.
  25. Ro HS, Kim SS, San Ryu J, Jeon CO, Lee TS, Lee HS,Comparative studies on the diversity of the edible mushroom Pleurotus eryngii: ITS sequence analysis, RAPD fingerprinting, and Physiological characteristics. Mycological Research 2007; 111 : 710-715.
  26. Sathyaprabha G, Kumaravel S, Panneerselvam A, Bioactive Compounds Identification of Pleurotus platypus and Pleurotus eous by GC-MS. Adv Appl Sci Res, 2011;  2:51-54.
  27. Sathyaprabha G, Kumaravel S, Panneerselvam A, Studies on phytochemical and vitamin analysis of pleurotus platypus and pleurotus oeus by GC-MS AND HPLC technique. International Journal of Pharmaceutical Sciences and Research 2011; 2:2816-2821.
  28. Sathyaprabha G, Panneerselvam A, Kumaravel S, Cultivation of Pleurotus platypus and Pleurotus eous in different agricultural waste substrates. Journal of Pharmacy Research2011; 4: 2543-2544.
  29. Selegean M, Putz MV, Rugea T, Effect of the polysaccharide extract from the edible mushroom Pleurotus ostreatus against infectious Bursal Disease Virus, International journal of molecular sciences2009; 10: 3616-3634.
  30. Senthil R, Angel KJ, Malathi R, Venkatesan D, Isolation, identification and computational studies on Pseudomonas aeruginosa strain MPC1 in tannery effluent.Bioinformation 2011 ; 6:187-190.
  31. Sofowora A, Medicinal plants and Traditional medicine in Africa. Spectrum Books. Ltd. Ibadan, Nigeria 1993;2: 270-289.
  32. Stajic M, Sikorski J, Wasser SP, Nevo E, Genetic similarity  and taxonomic  relationships  within  the  genus  Pleurotus  (higher Basidiomycetes)  determined  by  RAPD  Mycotaxon 2005; 93: 247-255.
  33. Staniaszek M, Marczewski W, Szudyga K, Maszkiewicz J, Czaplicki A, Qian G,Genetic relationship between Polish and Chinese strains of the mushroom Agaricus bisporus (Lange) Sing., determined by the RAPD method.  Journal of applied genetics2002;43: 43-48.
  34. Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV, DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic acids research,1990;18:6531-6535.
  35. Yang F, Xu B, Zhao S, Li J, Yang Y, Tang X, Wang F, Peng M, Huang Z, De novo sequencing and analysis of the termite mushroom (Termitomyces albuminosus) transcriptome to discover putative genes involved in bioactive component biosynthesis. Journal of Bioscience and Bioengineering 2012; 114: 228–231.