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Anti-diabetic Activity of Endophytic Fungi, Penicillium Species of Tabebuia argentea; in Silico and Experimental Analysis



Kumar Kalavathi Murugan, Chandrappa Chinna Poojari, Channabasava Ryavalad, Ramachandra Yarappa Lakshmikantha, Padmalatha Rai Satwadi, Ravishankar Rai Vittal and Govindappa Melappa
 
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ABSTRACT

Background and Objective: The plant and microbial phytochemicals possessing many biological activities with less toxic effects. Hence, present research was aimed to identify phytochemicals in Penicillium species extract and their role in diabetic activity. Materials and Methods: The methanolic extract of endophytic fungi Penicillium species of Tabebuia argentea was used to analyse phytochemical constituents by Gas Chromatography Mass Spectrometry (GC-MS). The same extract was used to evaluate the in vitro anti-diabetic activity. The phytochemicals profile obtained from GC-MS was used for in silico anti-diabetic activity against 21 different diabetic proteins/enzymes and ADMET (Absorption, Dissolution, Metabolism, Excretion and Toxicity). The analysis of variance was used to determine the significance of difference between treatment groups two-way (ANOVA) followed by SPSS2 (MRX version). Results: The methanol extract of Penicillium species consisted of 18 different phytochemicals and they inhibited the activity of α-amylase, β-glucosidase and dipeptidyl peptidase IV at maximum level. Out of 18 phytochemicals, the octadecanoic acid methyl ester and 3 phthalates have shown more interaction with all the 21 diabetic proteins/enzymes tested. The octadecanoic acid has shown more interaction with 1dhk, 1nu6, 2wy1, 4y14, 3i2m, 3k35, 4j5t and 5td4. The di-isobutyl isophthalate, dioctyl phthalate and bis-2-ethylhexyl phthalate have shown high interaction with 1m1j, 1ogs and 4acd. The overall observation of present study showed that octadecanoic acid is responsible for inducing anti-diabetic activity and the compound has the ability to interact with all the diabetic proteins and inactivate their activity. The in silico investigation clearly indicates how tested compounds interact with different diabetic proteins/enzymes, their role was identified and they were non-toxic and non-carcinogens. Conclusion: The Penicillium species represented potent bioactive compounds in their extract and are responsible for significant in vitro and in silico anti-diabetic activity.

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Kumar Kalavathi Murugan, Chandrappa Chinna Poojari, Channabasava Ryavalad, Ramachandra Yarappa Lakshmikantha, Padmalatha Rai Satwadi, Ravishankar Rai Vittal and Govindappa Melappa, 2017. Anti-diabetic Activity of Endophytic Fungi, Penicillium Species of Tabebuia argentea; in Silico and Experimental Analysis. Research Journal of Phytochemistry, 11: 90-110.

URL: https://scialert.net/abstract/?doi=rjphyto.2017.90.110
 
Received: January 17, 2017; Accepted: February 23, 2017; Published: March 15, 2017



INTRODUCTION

World is having problems in disease management in related to pathogens resistance, usage of same drugs and cost. The scientists are working on finding available drugs in cheaper cost or finding new drugs at less cost. A search for newer and more effective agents to deal with disease problems is now under way and endophytes are a novel source of potentially useful medicinal compounds. Endophytes comprise a large but little-explored share of fungal diversity1,2.

The endophytes may provide protection and survival conditions to their host plant by producing a plethora of substances which, once isolated and characterized, may also have potential for use in industry, agriculture and medicine3. At present, endophytes are producing biologically important bioactive compounds using to manage many infectious and non-infectious diseases.

Endophytic fungi are of biotechnological interest due to their potential as a source of secondary metabolites that have been proven useful for novel drug discovery4.

Endophytic fungi have been shown to produce several pharmacologically important compounds such as antimycotics steroid 22-triene-3b-ol5, anticancer cajanol6, podophyllotoxin and kaempferol7, anti-inflammatory ergoflavin8, antioxidant lectin9, insecticidal heptelidic acid10, immunosuppressive sydoxanthone A, B11 and cytotoxic radicicol12.

Plants used in traditional medicine have played a very important role in the search for new bioactive strains of endophytic fungi, as it is possible that their beneficial characteristics are a result of the metabolites produced by their endophytic community13,14.

Tabebuia argentea (Bignoniaceae) is an extensive and yellow blossoming tree and have turned out to be a rich wellspring of numerous natural mixes, particularly, of phenolic and polyphenolic nature. The plant is able to produce anticancer agent, lapachol, it has the ability to interfere with the bioactivities of enzymes known as, topoisomerases, a group of enzymes that are critical for DNA replication in cells15. The antitumor activity of Lapachol may be due to its interaction with nucleic acids and the interaction of the naphthoquinone moiety between base pairs of the DNA helix occurs with subsequent inhibition of DNA replication and RNA synthesis16. Other biological activities of Lapachol are antimetastatic activity17, anti-microbial and antifungal18, antiviral19, anti-inflammatory20, antiparasitic16, leishmanicidal21 and molluscicidal activity22. Only three reports are available in the identification of Lapachol producing endophytes of Tabebuia argentea from our lab research22-24.

Some research works believed to produce pharmacologically important bioactive compounds, in this context, the aims of the present study were to characterize the phytochemical profile of fungal endophyte, Penicillium species associated with Tabebuia argentea and to detect anti-diabetic activities and in silico prediction.

MATERIALS AND METHODS

Collection and extraction of phytochemicals from endophytic fungi, Penicillium species: The endophytic fungi, Penicillium species of Tabebuia argentea were collected from stock culture unit of Department of Biotechnology, Shridevi Institute of Engineering and Technology, Tumakuru, Karnataka, Bengaluru in September, 2016 and grown in 250 mL Erlenmeyer flask containing 100 mL of rose Bengal-yeast extract-sucrose broth for 2 weeks at 26+2°C with periodical shaking at 150 rpm. After the incubation period, the culture was separated from the broth and was extracted using methanol as organic solvent. Extraction was done using the mycelial mat for the metabolites with methanol. Added the equal volume of the solvent to the filtrate, mixed well for 10 min and kept for 5 min till the two clear immiscible layers formed. The upper layer of the solvent containing the extracted compounds was separated using separating funnel. Evaporated the solvent and the resultant compound was dried in rotator vacuum evaporator to yield the crude metabolites25. Then, the extract was dissolved with dimethyl sulphoxide at 1 mg mL–1 of concentration and kept at 4°C.

Phytochemical analysis: The preliminary phytochemical analysis of the crude extracts of Penicillium species was done to know alkaloids, flavonoids, tannins, phenols, saponins, terpenoids and carbohydrates using standard methods25,26.

Detection of bioactive compounds by GC-MS analysis: The methanol crude extract was subjected to GC-MS analysis to identify the bioactive compounds. The GS-MS analysis of the crude extract was carried out in a Shimadzu GC-MS-QP 2010 Plus fitted with RTX-5 (60 m×0-25 mm×0.25 μm) capillary column in IISc, Bengaluru. The instrument was set to an initial temperature of 70°C and maintained at this temperature for 2 min. At the end of this period, the oven temperature was rose up to 2800°C, at the rate of an increase of 50°C min–1 and maintained for 9 min. An injection port as 1 mL mi–1. The ionization voltage was 70 eV. The sample was injected as 10:1. Mass spectral scan range was set at 45-450 (m/z). The identification of bioactive compounds present in the extracts was performed by comparing the mass spectra with data from NIST05 (National Institute of Standards and Technology, US) library.

Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis
Fig. 1:
GC-MS analysis showing different phytochemicals identified based on retention time in methanol extract of endophytic fungi Penicillium species

The name, molecular weight and structure of the components of the test material were ascertained based on retention time.

Anti-diabetic activity
α-glucosidase activity:A 36 μL of phosphate buffer solution, 30 mL sample solution with various concentrations (10, 25, 50, 100 and 150 μg mL–1) and 17 μL of 4-nitrophenyl-α-D- glycopyranoside (PNPG) substrate as the concentration of 5 mM were put in 37°C for 5 min. After 5 min, 17 μL of α-glucosidase solution 0.15 U mL–1 was added to each well to obtain a total volume of 100 mL. The mixture was incubated for 15 min, the reaction was spotted by adding 100 μL of sodium carbonate 200 mM. Absorbance was measured at 405 nm using a microplate reader. Each test was repeated thrice27. The calculation was done based on Elya et al.28.

α-amylase assay: A 250 mL of 500 μg mL–1 extract, 250 μL of starch 2.0% (w.v) and 250 μL of 1 U mL–1 α-amylase solution was homogeneously mixed into a test tube. After incubated at 20°C for 3 min, 500 μL of color reagent (dinitrosalicylic acid) was added to stop the enzymatic reaction. The mixture was kept into boiled water and 250 μL of 1 U mL–1 α-amylase was added immediately. The mixture was heated up to 15 min. Further, the solution was removed from the heating process and cooked at room temperature (-26+20°C) for 3 min. A 4500 μL aqua dest was added to obtain a total volume of 6000 μL. The solution was homogenized using a vortex. The α-amylase activity was determined at 540 nm using spectrophotometry to measure product absorbance (maltose) which reduces DNS. The produced absorbance was compared with a blank. Percent inhibition was calculated using the equation of Elya et al.28.

Dipeptidyl peptidase IV assay: A 25 μL extract was added to 50 μL Dipeptidyl peptidase (DPP-IV) (500 μg mL–1). The mixture was incubated at 37°C for 5 min. A 100 μL Gly-Pro-P-Nitroanilide (GPPN) (2 mM) was added to the wells containing extract and enzyme. Incubation was contained for 15 min. The reaction was terminated by adding 25 μL glacial acetic acid (25%). The absorbance was measured at λ = 405 nm29.

In silico antidiabetic activity
Bioactive compound preparation: Most of the 3D (3 Dimensions) structures of drug molecules identified in the methanol extract of endophytic fungi, Penicillium species were downloaded from PubChem Compound section of National Center for Biotechnology Information (NCBI)30. Ligands during this process also being checked for Torsion count to detect currently active bonds with default settings. Importantly, amide bonds were checked and treated as non-rotatable. Ligands were also utilized to merge non-polar hydrogens. The 2D structures of 18 ligands are illustrated in Table 1 and Fig. 1. The 3D structures of these 18 ligands were elucidated.

Table 1:
Identified phytochemicals in Penicillium species extract and their synonymous, identified based on retention time in GC-MS
Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis
Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis

Table 2: List of enzymes selected for docking studies
Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis

Selection of receptors: The receptors were chosen in light of their capacity in the pathway of diabetes. The 3D structure of 1DHK, 1HNY, 1M1J, 1NU6, 1OGS, 1V4T, 1XU7, 1Y7V, 2JFE, 200X, 2ZJ3, 3CTT, 3K35, 3L2M, 3NO4, 3W37, 3WY1, 4ACD, 4J5T, 4Y14 and 5TD4. The receptors selected for present study have appeared in Table 2. The 3D structures of these receptors were accessible in their local shape in PDB database. The 3D directions of the receptors were obtained from PDB database. To verify the capacity of the model in reproducing experimental observation with a new ligand, all these structures were analyzed again at the binding site.

Docking simulations: The iGEMDOCKv2.1 was employed for binding affinity measurement between selected ligands and targeted proteins of diabetes. The content of configure file was determined as position of receptor file and ligand file.

ADME TEST: ADME/Toxicity parameters compliance was evaluated by screening through ADMET-SAR, a commercial tool. The ADMET-SAR is system pharmacology or system chemical biology and toxicology platform designed for the assessment of would be therapeutic indications, off target effects and potential toxic end points of natural products. In the studied work, this database/tool was used to predict and evaluate the human metabolism compliance, toxicity risk assessment and mode of action by using standard experimental data.

Statistical analysis: Analysis of variance two- way (ANOVA) of SPSS2 (Statistical Package for the Social Sciences) (MRX version) was used to determine the significance of difference between treatment groups (<0.05). Means between treatment groups were compared for significance using Duncan’s new Multiple Range post-test22.

RESULTS AND DISCUSSION

From qualitative phytochemical analysis of Penicillium species methanol extract exhibited potent bioactive compounds. The Penicillium species have shown the bioactive phytochemicals such as phenols, flavonoids, terpenoids, tannins, carbohydrates, alkaloids and saponins. Similar results were reported by Sharma et al.31 from Pestalotiopsis neglecta and Bhardwaj et al.25 from Penicillium frequentans.

The partially purified crude extract of Penicillium species was subjected to GC-MS analysis. Total 18 compounds were identified based on retention time and area percentage, molecular formula and weight were identified (Table 1, Fig. 1). The highest amount of 1,2-Benzenedicarboxylic acid, diisooctyl ester was noticed in GC-MS as a high peak. The endophytic fungi, Colletotrichum gloeosporioides of Phlogacanthus thyrsiflorus have yielded the phenol, 2,4-bis(1,1-Dimethyl ethyl), 1-Hexadecane, 1-Hexadecanol, hexadecanoic acid, octadecanoic acid methyl ester and 1-nonadecane26. Bis(2-ethylhexyl) phthalate, Pentanoic acid, Melamine, 4H-Pyran-4-one, 2,3-Dihydro-3,5-dihydroxy-6-methyl-, Dodecane, Nonadecane, 5-Hydroxymethylfurfural, 1,2,3-Propanetriol, 1-Acetate, Heptose, Triacetin, 2,3-Dihydroxypropanal, 1-Cycloheptene, D-Allose, Pentadecane, 1,5-Anhydrohexitol, 3-Deoxy-D-mannoic lactone, Tetradecane, Heneicosane, 4-Oxo-, 1,2-Benzenedicarboxylic acid and Bis (2-ethylhexyl) phthalate31. Papitha et al.32 have identified the similar bioactive compounds from the plant, Tinospora cordifolia.

Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis
Fig. 2: Enzymes inhibition assay of Penicillium species extract
 
The percent inhibition of α-amylase, α-glucosidase and DPP IV by Penicillium species methanol extract. The values followed by Mean+SEM remained significantly different at p<0.05

Many microbes have shown that the secondary metabolites have the ability to bind with active sites and enzymes, receptors and proteins. Phthalic acid, methyl- octyl ester and Bis (2-ethylhexyl) phthalate were identified in the extract and have reported as antimicrobial agents26,31,32. Authors have found Phenol, 2,4-bis(1-phenylethyl)-26 and Phenol, 2,6-bis(1,1-dimethylethyl)-4-[(4-hydroxy-3,5-dimethylphenyl)methyl]-33 from endophytic fungi, Colletotrichum gloeosporioides and Fusarium solani of different plants. The Naphthalene, decahydro-1,8a-dimethyl-7-(1-methylethyl)-, R-(1. alpha.,4a. beta.,7. beta.,8a. alpha.)]- were identified from Phoma herbarum34 and Aquilaria sinensis35. The octadecanoic acid methyl ester was identified from fungal endophytes of Ocimum sanctum and exhibiting many biological activities36. The results confirm that Penicillium species produce important secondary metabolites and which are exhibiting many biological activities. Comparing to earlier reports, the study supports the evidence that bioactive compounds produced by fungal endophytes may not be involved in the host-endophytic relationship but may also have industrial applications. The endophytic fungal species are exploiting for their important bioactive compounds and they having novel medical applications. The results of present study confirms that endophytic fungal species are able to produce biologically important medicinal bioactive compounds. Hence, further studies are required to explore the secondary metabolites of Tabebuia argentea and its endophytic fungal species and they can be used for management of different diseases.

In vitro antidiabetic activity, the extract of Penicillium species has potentially inhibited the activity of α-amylase, α-glucosidase and dipeptidyl peptidase IV listed in Fig. 2. The obtained result clearly indicates that, the inhibition of enzymes is concentration dependent. The α-glucosidase was inhibited more by bioactive compounds of endophytic fungal species and the result was less than positive control standard drug acarbose. Similar results were reported by many scientist using endophytic extracts against α-glucosidase37-39. The same extract was inhibited the activity of α-glucosidase at maximum level compared to standard drug acarbose. The extract inhibited the activity of DPP-IV and it was lower than standard drug diprotin as a positive control. No reports on endophytic fungal extracts showing the inhibitory action of dipeptidyl peptidase IV. The present study is the first study on inhibition of dipeptidyl peptidase IV using fungal extract. There are some reports say that, the plant extracts have the ability to inhibit the activity of dipeptidyl peptidase IV40-42.

The results concluded that, the endophytic fungal extract has shown potent antidiabetic activity by in silico assay. Molecular docking was performed on 21 different diabetic target proteins and with all 18 endophytic bioactive compounds using iGEMDock2.1. The binding interactions of these ligands with target proteins were selected on the basis of binding energy or total energy, VDW and hydrogen bonding interaction. These values along with the hydrogen bond forming residues are presented in Table 3. From the analysis, the pancreatic α-amylase has shown more interaction with octadecanoic acid methyl ester followed by Di-isooctyl phthalate, Bis-2 ethylhexyl phthalate, Phenol 2,6, bis-2 hydroxy-5-methyl Benzenedicarboxylic acid. The octadecanoic acid methyl ester binds with the amino acids of human pancreatic α-amylase followed by Diisooctyl phthalate, 2,4-Bis(1-phenylethyl)phenol, Phenol 2,6, bis-2 hydroxy-5-methyl. Native chicken fibrinogen amino acids more interact with Diisobutyl isophthalate followed by Methoxymellein, 5-diethylamino, Methyl palmitate. Out of 18, 7 bioactive compounds able to inhibits the Human Dipeptidyl Peptidase IV by interacting with different amino acids. The octadecanoic acid methyl ester is able to interact with DPP-IV enzyme followed Diisooctyl phthalate, Phenol 2,6, bis-2 hydroxy-5-methyl, Dimethyl phenol, Bis-2ethylexyl phthalate, Benzenedicarboxylic acid, 2,4-Bis(1-phenylethyl)phenol (Table 3) (Fig. 3).

The Diisooctyl phthalate have showed highest binding affinity with human β-glucosidase followed by octadecanoic acid methyl ester, Phenol 2,6-bis-2 hydroxy-5-methyl, Benzenedicarboxylic acid, Diisobutyl isophthalate, Dimethyl phenol, Methoxymellein and Bis-2ethylhexyl phthalate. The octadecanoic acid methyl ester is able to interact with human glucokinase enzyme with more binding energy followed by Phenol 2,6-bis-2 hydroxy-5-methyl, Dimethyl phenol, Diisooctyl phthalate and Methyl palmitate.

Table 3: In silico anti-diabetic activity of Penicillium species phytochemicals
Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis
Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis
Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis
Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis
Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis
Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis
Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis
Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis

Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis
Fig. 3(a -r):
Octadecanoic acid methyl ester showing interaction with diabetic enzymes and their binding energy, (a) 1dhk (-117.35), (b) 1hny (-102.76), (c) 1nu6 (-116.96), (d) 1v4t (-102.49), (e) 1xu7 (-104.07), (f) 1y7v (-100.91), (g) 2jfe (-104.37), (h) 200x (-105.58), (i) 2zj3 (-114.57), (j) 3ctt (-106.02), (k) 3k35 (-111.68), (l) 3l2m (-113.67), (m) 3no4 (-107.29), (n) 3w37 (-98.61), (o) 3wy1 (-118.62), (p) 4y14 (-116.56), (q) 4j5t (-113.49) and (r) 5td4 (-117.7)

The Human 11beta-hydroxysteroid dehydrogenase type I have shown more interaction with octadecanoic acid methyl ester followed by Phenol 2,6-bis-2 hydroxy-5-methyl, Benzenedicarboxylic acid, 2,4-Bis (1-phenylethyl) phenol, Methoxymellein, Diisooctyl phthalate, 5-diethylamino (Table 3, Fig. 3).

The octadecanoic acid methyl ester have shown more interaction with human acid-beta-glucosidase followed by Phenol 2,6-bis-2 hydroxy-5-methyl, Benzenedicarboxylic acid, Diisooctyl phthalate, Diisobutyl isophthalate, Methyl palmitate. Human cytosolic β-glucosidase firmly interact with octadecanoic acid methyl ester with high energy followed by Phenol 2,6-bis-2 hydroxy-5-methyl. The AMP activated protein kinase was more interact with octadecanoic acid methyl ester followed by Diisobutyl isophthalate, Phenol 2,6-bis-2 hydroxy-5-methyl, Methoxymellein, 2,4-Bis (1-phenylethyl) phenol and Diisooctyl phthalate. The fructose-6-phosphate amidotransferase was inhibited by octadecanoic acid methyl ester at highest binding energy followed by Bis-2ethylhexyl phthalate, 5-diethylamino, Phenol 2,6-bis-2 hydroxy-5-methyl. Statin HMG-coa reductase enzyme was inhibited by octadecanoic acid methyl ester, Phenol 2,6-bis-2 hydroxy-5-methyl at high binding energy followed by 2,4-Bis (1-phenylethyl) phenol, Bis-2ethylhexyl phthalate (Table 3, Fig. 3).

SIRT6 family member, NAD(+)-dependent protein deacetylases is able to control genomic stability and transcriptional control of glucose metabolism. The octadecanoic acid methyl ester inhibited this enzyme by showing highest binding energy followed by Diisooctyl phthalate, Bis-2ethylhexyl phthalate, Diisobutyl isophthalate, 2,4-Bis (1-phenylethyl) phenol, Phenol 2,6-bis-2 hydroxy-5-methyl, 5-diethylamino, Methoxymellein. The pig pancreatic α-amylase was greatly inhibited by octadecanoic acid methyl ester followed by Diisooctyl phthalate and Phenol 2,6-bis-2 hydroxy-5-methyl. The more interaction was between creatinine amidohydrolase with octadecanoic acid methyl ester by their binding energy followed by Methoxymellein, Diisobutyl isophthalate, Diisooctyl phthalate, Phenol 2,6-bis-2 hydroxy-5-methyl, 2,4-Bis(1-phenylethyl)phenol, Benzenedicarboxylic acid, Methyl Stearate, Bis-2ethylexyl phthalate (Table 3, Fig. 3).

Sugar beet α-glucosidase was inhibited by octadecanoic acid methyl ester at a maximum level compared with Diisooctyl phthalate, Phenol 2,6-bis-2 hydroxy-5-methyl, Benzenedicarboxylic acid, Methoxymellein. α-glucosidase was greatly inhibited its activity by octadecanoic acid methyl ester followed by Diisooctyl phthalate, Methyl Stearate, Benzenedicarboxylic acid, Bis-2ethylhexyl phthalate, Methoxymellein, Phenol 2,6-bis-2 hydroxy-5-methyl, Diisobutyl isophthalate. The glycogen synthase kinase-3 beta was showed more binding energy when it interact with Bis-2ethylhexyl phthalate, octadecanoic acid methyl ester, Diisobutyl isophthalate, Phenol 2,6-bis-2 hydroxy-5-methyl, Diisooctyl phthalate (Table 3, Fig. 3).

Octadecanoic acid methyl ester conjugate protein tyrosine phosphatase 1B exhibits more interaction followed by Phenol 2,6-bis-2 hydroxy-5-methyl, Diisooctyl phthalate, Benzenedicarboxylic acid, octadecanoic acid methyl ester have showed highest interaction with processing α-Glucosidase I with high binding energy followed by, Bis-2ethylhexyl phthalate, Diisooctyl phthalate, Phenol 2,6-bis-2 hydroxy-5-methyl, Benzenedicarboxylic acid. The human pancreatic α-amylase have firmly interact with octadecanoic acid methyl ester with more binding energy followed Diisooctyl phthalate, Di methyl, 2,4-Bis (1-phenylethyl) phenol, Phenol 2,6-bis-2 hydroxy-5-methyl (Table 3, Fig. 3).

From in silico anti-diabetic activity, the octadecanoic acid methyl ester able to interact with all most all diabetic proteins/enzymes with high biding energy, whereas the diisobutyl isophthalate have conjugated with 1m1j, iso-octyl isophthalate on 1ogs and bis-2-ethylhexyl phthalate on 4acd with more binding energy. The phthalates have expressed as anti-diabetic potentials in vitro and in silico screening43-45 (Table 3, Fig. 4).

The octadecanoic acid methyl ester is responsible for inducing antidiabetic activity reported by Iqbal et al.46 and Hashim et al.47 in vitro and in vivo conditions, respectively. Sasikala and Meenak48, Rajkumar et al.49 have reported that octadecanoic acid methyl ester was able to inhibit diabetic enzymes in in silico study. In vitro and in silico anti-diabetic activity of octadecanoic acid methyl ester was carried out by Raajshree and Chitra50. The present study clearly showed the presence of important phytochemicals which induced antidiabetic activity in vitro and in vivo conditions.

From online ADME test reveals that, octadecanoic acid methyl ester, dimethyl phthalate, Diisooctyl phthalate and bis-ethynyl phthalate are non-toxic AMETS test and non-carcinogens. The results confirm their degradable characters except for the octadecanoic acid methyl ester all are readily biodegradable. The online ADMET test for all the compounds was carried out but the data represented only for above mentioned four compounds are represented in Table 4.

Table 4: ADMET Predicted profile of the potent phytochemicals of Penicillium species
Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis

Image for - Anti-diabetic Activity of Endophytic Fungi, Penicillium  Species of Tabebuia argentea; in Silico and Experimental Analysis
Fig. 4:
Diisobutyl isophthalate, diisooctyl phthalate and bis-2-ethylhexyl phthalate and their conjugated with (a) 1m1j, (b) 1ogs and (c) 4acd of diabetic enzymes with high binding energy

CONCLUSION AND FUTURE RECOMMENDATIONS

Present day, the endophytes are being utilized as a source of novel drug compounds for the betterment of human health. The Penicillium species of Tabebuia argentea have shown medicinally important phytochemicals by GC-MS analysis. Anti-diabetic, endophytic fungal extract have inhibited the α-amylase, α-glucosidase, DPP IV activity strongly. The octadecanoic acid methyl ester and phthalates are responsible for inhibition of 21 diabetic proteins/ enzymes actively and they exhibited more binding energy. The present outcomes would provide alternate methods of natural product drug discovery which could be reliable, economical and environmentally safe. Using of these fungi, we can produce a high amount of bioactive compounds within short duration in laboratory conditions. In the study used almost all proteins or enzymes for in silico assay to know their activity on different enzymes and literature reveals that nobody has tried all these selected proteins for in silico anti-diabetic activity. Hence, further in vivo studies are suggested to investigate to isolate and identify pure compounds which are responsible for diabetic activity from Penicillium species.

SIGNIFICANCE STATEMENTS

The endophytic fungi, Penicillium species of Tabebuia argentea methanol extract yielded 18 different bioactive compounds. The same extract significantly reduced the activity of α-amylase, α-glucosidase and dipeptidyl peptidase IV enzymes in in vitro experiments. The molecular docking studies help to know inhibitory activity and binding mode of endophytic fungal phytochemicals with anti-diabetic target proteins. The octadecanoic acid methyl ester, dimethyl phthalate, di-iso-octyl phthalate and bis-ethylhexyl phthalate have showed the highest binding affinity and good hydrogen bond interactions with active site residues.

ACKNOWLEDGMENTS

The authors are grateful to Visvesvaraya Technological University (VTU), Belagavi, Karnataka, India for providing financial support (Ref No. VTU/Aca./2010-11/A-9/11339 dated 7 December 2010) with grant number VTU-RG:11339/2010-11 for this investigation. We would also like to thanks Dr S Lokesh, DOS in Applied Botany and Biotechnology, University of Mysore, Mysore, India for assistance in identifying endophytic fungi.

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