In Vitro and In Silico Antioxidant, Anti-Diabetic, Anti-HIV and Anti-Alzheimer Activity of Endophytic Fungi, Cladosporium uredinicola Phytochemicals

The present work was aimed to identify phytochemicals in C. uredinicola methanol extract from qualitative, TLC and GC-MS method and evaluated for antioxidant, anti-HIV, anti-diabetes, anti-cholinesterase activity in vitro and in silico. The C. uredinicola extract showed flavonoids, tannins, alkaloids, glycosides, phenols, terpenoids, and coumarins presence in qualitative method. From GC-MS analysis, identified seven different phytochemicals and out of seven, four (coumarin, coumarilic acid, hymecromone, alloisoimperatorin) are coumarins. The C. uredinicola extract has shown significant antioxidant activity in DPPH (73) and FRAP (1359) method. The HIV-1 RT (83.81+2.14), gp 120 (80.24+2.31), integrase (79.43+3.14) and protease (77.63+2.14), DPPIV, βglucosidase and acetyl cholinesterase activity was significantly reduced by the extract. The 2-diphenylmethyleneamino methyl ester had shown significant interaction with oxidant and HIV-1 proteins whereas alloisoimperatorin have interacted with diabetes and cholinesterase proteins followed by hymecromone with high binding energy. These three phytochemicals are noncarcinogens, non-toxic, readily degradable and have drug likeliness properties. The C. uredinicola phytochemicals are responsible for management of diabetes, HIV-1 and Alzheimer. Further in vivo work is needed to justify our research.


Introduction
Plant drugs constitute 25% of the total drugs and have no or minimal side effects. If we use these plant-based drugs continuously we need plants in large quantity and they may vanish on the earth in the future. So, to save plants, exploiting the endophytes to obtain plant-based drugs is practicing nowadays. Endophytes are endosymbionts resides in plant tissues, they either bacteria or fungi but they are not causing any diseases to host. The endophytic fungi are able or capable to produce what host is producing, by using these endophytes we can produce large quantity of the drug within short period by applying biotechnological aspects to meet public demand.
Calophyllum tomentosum (Calophyllacace) is endemic plant commonly known as bintangur grows in Sri Lanka and Western Ghats regions of Karnataka, India. In Ayurveda, the extracts are being practiced to treat ulcers, snake bites and eye diseases. Xnthones and triterpenes were identified from bark of C. tomentosum [1] and flavonoids, saponins and terpenoids from leaf part [2] exhibited strong α-glucosidase inhibitory activity. The C. tomentosum shown alkaloid, flavonoid, terpenoid, tannin, glycoside, saponin [3] are responsible for inhibition of α-glucosidase activity. The literature survey indicates that no reports on endophytic fungal species from C. tomentosum plant. In our lab, we have isolated three fungal endophytes from (different parts of C. tomentosum), analysed their phytochemicals through GC-MS and identified by molecular level using 18S rRNA (unpublished data). The present research was aimed to identify phytochemicals using qualitative, TLC, GC-MS The extract of C. uredinicola was used for DPPH activity [8]. The freshly prepared samples were dissolved in 24 mg DPPH in 100 ml ethanol and stored at −20 °C. The sample solution of 150 μl (10 μl of sample and 140 μl of distilled water) was mixed with 2850 μl of sample containing 190 μl of reagent and 2660 μl of distilled water and allowed 24 h for reaction each in dark condition. The reaction was measured at 515 nm. The standard ascorbic acid curve was range of 25 to 800 μM used to analyse test sample. To get absorbance of 1.1±0.02 units at 517 nm, the 45 ml of methanol was added to stock solution (10 ml) [9]. Triplicate was maintained to all the experiments. The per cent inhibition of DPPH due to sample was measured and used the standard formula for calculation as mentioned below; Inhibition (%) = AC -AS / AC X 100, where AC-absorbance of DPPH activity with ethanol, AS-absorbance of DPPH activity with sample or absorbance of DPPH activity with standard.

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In vitro anti-diabetic activity of C. uredinicola extract

Activity of inhibition of α-amylase
The working solution contains 250 µL of 2% (w/v) starch (250 µL), α-amylase solution (250 µL of 1 U/mL) and C. uredinicola extract (250 mL of 500 µg/mL) was incubated for 3 min at 20 °C. To stop the reaction, dinitrosalicylic acid (500 µL) was added to the reaction mixer, subjected to boiled water immediately added α-amylase (250 µL) and heated the solution for 15 min. After heating, the reaction mixer was kept at 26+2 °C for 3 min. To get total volume of 6000 µl, 4500 µL of aqua dest was mixed and homogenized the mixer in vortex. At 540 nm, the activity of α-amylase was measured in with sample or standard or without sample with the help of spectrophotometer and triplicate was maintained for each experiment. Inhibition of α-amylase activity was calculated using standard equation [11].

Inhibitory activity of α-glucosidase
The reaction mixer contains phosphate buffer solution (36 µL), C. uredinicola solution (30 mL) at different concentrations of 10, 25, 50, 100 and 150 µg/ml and 4-nitrophenyl--α-D glycopyranoside (PNPG) substrate (17 µl) was allowed to reaction for 5 min at 37 °C. Added the α-glucosidase solution (17 µl of 0.15 U/mL) to each well to get 100 mL of volume after 5 min of incubation. The reaction solution allowed for 15 min and added sodium carbonate (100 µl of 200 mM). The reaction was observed at 405 nm in micro plate reader and repeated the each experiment thrice and calculated the reaction [11].

Inhibitory activity of dipeptidyl peptidase IV
Incubated the reaction mixture of 50 µL dipeptidyl peptidase (DPP-IV) was mixed with 25 µL C. uredinicola extract for 5 min at 37 °C. Added the 100 µL Gly-Pro-P-Nitroanilide (GPPN) (2 mM) to the reaction mixture and enzyme activity allowed for 15 min. The reaction was terminated by adding 25 µL of acetic acid glacial (25%) and reaction activity was measured at 405 nm [11].

Inhibition of activity of HIV-1 reverse transcriptase (RT)
Using 5mM MgCl2, 150 mM KCl, 0.05% NP-40, 5mM DTT, 0.5 mM EGTA, 0.3M Glutathione, 2.5 µg/ml BSA, 2.5µg/ml Poly(rA).p(dT), 20 µM dTTP, 0.5µCi (microcurie) of [3H]TTP, 50mM Tris (pH 7.8), the 100 µl of reaction mixture was prepared. To the reaction mixture added the 0.5 units RT enzyme and incubated for 3 h at 37 °C. By adding 0.1M EGTA (25µl) the enzyme activity was terminated later incubated the reaction mixer on ice for chilling. 100 µl of C. uredinicola was spotted on 2.5cm Whatman filter paper (circular) and was incubated for 15 min at 26+2° C to dry. By using 5% aqueous NaHPO4.7H2O, washed the filters four times later two times with double distilled water. The filters were subjected to dry and to scintillation counting. To analysis, used azidothymidine as positive control and without sample considered as negative control.
The percentage of inhibition calculated as, Per cent inhibition = Negative control -Test sample/ Negative control X 100 Inhibitory activity of HIV-1 gp120 binding ELISA kit was used to study binding of CD4 with gp120 [12]. We have studied our extract could interfere with biding of gp120 with CD4. The 5mg/ml of extract was added to gp120 (25ng) at 50 µl of equal concentration of 50 µl and was subjected to incubation at 26+2°C. Then transformed the reaction mixture to CD4 ligand containing microtiter plate wells and was subjected to incubation at 26+2 °C incubated for 1h. By using buffer washed the reaction mixer three times. Through detector reagent, analysed the gp120 binding. For positive control, the heparin was considered as standard control and with sample used as negative control.

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Vol. 13 Calculated the percentage of inhibition as mentioned below, A is optical density.

HIV-1 protease inhibition assay
The assay was performed based on standard procedure of Narayan and Rai [13]. Using buffer (50 mM of sodium acetate (pH 5.0), 1 mM ethylenediamine disodium (EDTA.2Na) and 2 mM 2-mercaptoethanol (2-ME), the HIV-1 PR solution was diluted and added the glycerol in the ratio 3:1. The Arg-ValNle-NH2 (substrate peptides) was diluted with 50 mM of sodium acetate (pH 5.0). Two µl of extract, C. uredinicola and HIV-1 PR (4µl) was mixed with substrate solution (2µl, 2 mg/ml) and 10 μl of reaction mixture was incubated for 1h at 37°C. Without endophytic extract was used as control and terminated the reaction by keeping the reaction mixture for 1 min at 90 °C. Later, added the 20 μl of sterile water and an aliquot of 10 μl was analyzed by HPLC using RP-18 column (4.6 mm X 150 mm). The reaction mixture of 10 µl was injected to the column and eluted gradient by using 15-40 % of acetonitrile and trifluoroacetic acid (TFA) (0.2%) in water with 1.0 ml/min flow rate. Monitored the elution profile at 280 nm. The HIV-1 PR inhibitory assay was analysed using following formula: % inhibition = (AC-AS)/AC X100, for positive control the acetyl pepstatin was used.

Inhibition of protease enzyme
In a 500µl of reaction mixture, 800µg haemoglobin, C. redinicola extract and 50µg pepsin was incubated for 20 min at 37° C to allow proper mixing and to stop the reaction added the 5% of TCA. Centrifuged the reaction for 5 min at 14000 g and the supernatant OD was recorded at 280 nm. To compare the reaction effect of our sample, both negative (pepstatin A, a protease inhibitor) and positive controls (enzyme and standard substrate) were used. Triplicate was maintained for each sample.

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In vitro anti-Alzheimer activity Acetyl and butyryl cholinesterase inhibition assay 96-well microplate reader was used to carry out acetylcholinesterase assay. The ChE enzyme (10 mL volume, diluted 100 times in phosphate buffer, pH 7.4) was mixed with DTNB (5,50dithiobis-(2-nitro-benzoic acid)) (104 M concentration), 70 mM of phosphate buffer (Na2HPO4/NaH2PO4, pH 7.4) and ATCh (1.35 X 10-4 M concentration) in plate wells and allowed for reaction at 37° C and reaction was measured for 5 min at 412 nm. For each experiment, three replicates were maintained. The enzyme inhibition percentage was calculated by comparing with negative and positive control [14].

Preparation of ligands
All the seven phytochemicals of C. uredinicola structures were obtained from NCBI PubChem database and their canonical smiles were used to generate 3D structure from www.mnam.com/online_demos/corina_demo. The pharmacokinetic properties such as carcinogenicity, toxicity, inhibitory properties and various other properties were screened using admet-SAR device. Sedate likeliness, Adsorption, Dissolution, Metabolism, Excretion profile, toxicity and adverse factors of the ligand was anticipated. The ADME incorporates rate of retention, metabolism, digestion system and excretion. The admet-SAR employs Caco-2-cell (human epithelial colorectal adenocarcinoma cell lines) and MDCK (Madin-Darby Canine Kidney) cell models for oral medication, retention, skin porousness and human intestinal absorption to demonstrate oral and transdermal medical assimilation. Pre-ADMET predicts poisonous quality in view of the ADMET parameters and Rat acute toxicity [15][16].

Docking studies/Virtual screening
Molecular docking is the study utilized to predict the binding interaction of a molecule (ligands/drug candidates) to target proteins/receptors to discern the fitness of the ligand in the active site of the receptor. Consequently, the knowledge about the affinity and activity of the ligand can be determined. The 3D structure of all the seven ligands obtained from GC-MS study were developed using the CORINA tool (http:/mn-am.com/online-demos/corina-demo), by entering the chemical structures as SMILES strings. CORINA is a fast and powerful tool which generates single, highquality and low energy 3D structures of drug like molecules used for in silico profiling. The receptors were prepared by eliminating the water molecules from the PDB structures. Docking was carried out using iGEMDOCKv2.1. It is a tool used to study the interactions of pharmacologically important drugs. It provides basic idea on interactive or binding sites of receptor or proteins. After docking is completed a protein-ligand complex is generated along with the interaction profile which is used to rank the ligands based on the pharmacological energy. We have used two different methods to evaluate the docking scores, which accounts for biasing by dividing by molecular weight and nonhydrogen atoms [17].

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AdmetSAR test
The ADMET (Absorption Distribution Metabolism Excretion Toxicity) profiling describes the disposition of pharmaceutical compound within an organism. The inhibitory properties of phytochemicals were studied through ADMET profile by submitting the canonical smiles downloaded from PubChem in admetSAR (admetSAR@LMMD). Human intestinal absorption, human oral bioavailability, penetration of blood-brain barrier, binding of plasma protein, volume of distribution, cytochrome P450 substrate, inhibitor, inducer, activator, half time (t1/2), renal clearance, drug induced toxicity, genomic toxicity, aquatic and terrestrial toxicity, reproductive toxicity, environmental factor -biodegradability.

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These endophytic fungal phytochemicals are responsible for in vitro antioxidant activity [31][32]. The in vitro antioxidant method, DPPH is universally considered to screen the antioxidant compounds and it is causing any effect on enzyme inhibition and metal [33]. The methanol extract of C. uredinicola was studied free radical scavenging activity using DPPH method and standard ascorbic acid was used. The C. uredinicola extract had shown significant antioxidant activity and it was concentration dependent. Fig. 2 represents the antioxidant activity of endophytic activity was compared with ascorbic acid. The phytochemicals present in extract of C. uredinicola are responsible for antioxidant properties. The endophytic fungal phytochemicals donate electrons to DPPH and they are responsible for reduction of purple coloured DPPH to colourless solution [34]. Our findings are agreement with results of Manjunath et al. [35], Hulikere et al. [31]. The methanol extract of C. uredinicola phytochemicals reduced the Fe3+ TPTZ complex to Fe2+-tripyridytriazine (blue coloured complex) by donating electron at low pH and the reaction was observed and measured in absorbance at 593 nm. The C. uredinicola phytochemicals are acts strong as antioxidants agents in reducing power potential (Fig. 3) [36][37][38].  The C. uredinicola methanol extract significantly reduced the activity of α-amylase, αglucosidase and dipeptidyl peptidase IV activity in vitro condition (Fig. 4). The result confirms that inhibitory activities of diabetic enzymes are dependent on concentration of the sample. The C. uredinicola phytochemicals strongly inhibited the activity of α-amylase and it was significant when compared to positive control standard drug acarbose. Our results are confirmation with the findings of endophytic fungal α-glucosidase inhibitory activity [39][40]. The C. uredinicola extract inhibited the α-glucosidase activity at maximum level. The activity of DPP-IV significantly inhibited by C. uredinicola extract and it was compared with standard drug diprotin. The literature survey indicates that no results were found in dipeptidyl peptidase IV inhibitory activity using fungal extracts. The obtained results proving significant inhibition of dipeptidyl peptidase IV by C. uredinicola extract and are confirmation with the results of Kumar et al. [41]. The plant extracts also exhibited the dipeptidyl peptidase IV inhibitory activity [41][42]. The C. uredinicola extract had shown inhibition of HIV-1 proteins viz., protease, RT, protease and gp120. The C. uredinicola was inhibited RT activity strongly (83.81+2.14) and it was high compared to standard AZT (74.36+1.89) (Table 3) [27,43]. Similarly, the gp120 (80.24+2.4) ( Table  4), protease (77.63+2.14) (Table 5), integrase (79.43+2.14) ( Table 6) proteins activity was decreased due to C. uredinicola extract. The HIV-1 proteins activity was inhibited due to potent phytochemicals of C. uredinicola and the activity may in combination of all the phytochemicals or any single potent phytochemical [41][42].      (Table  8). No reports on 2-diphenylmethyleneamino methyl ester antioxidant activity but alloisoimperatorin [46] and hymecromone [47] had shown strong antioxidant activity in vitro condition but no reports on in silico antioxidant activity.    (Table 9) (Fig. 5) with high binding energy. The diphenylmethyleneamino methyl has exhibited strong in vitro anti-HIV activity [48]. There is no report on in vitro and in silico anti-HIV activity of alloisoimperatorin and hymecromone. The diphenylmethyleneamino methyl ester had ability to interact with HIV-1 integrase, protease, RT, gp120 proteins with high binding energy leads to confirmation changes to inhibit their functions.

1-Binding energy (kj/mol), 2-VDW, 3-H-bond
The alloisoimperatorin was shows highest interaction with all the three diabetic enzymes (α-amylase, β-glucosidase and DPPIV) by showing highest binding energy compared to diphenylmethylene amino methyl ester and hymecromone (Table 10) (Fig. 6). The alloisoimperatorin have firmly interacted with α-amylase and DPPIV followed by β-glucosidase. The alloisoimperatorin had interacted with val234, glu233 of 4x9y and showed highest binding energy. No reports are available on these compounds as antidiabetic activity from in vitro and in silico assays.  The alloisoimperatorin had shown strong interaction with AChE and BChE proteins with biding energy and results shows that the compounds have firm interaction with AChE. The alloisoimperatorin have ability to bind with tyr124, aer125, thr83, gly82, tyr337 of 4pqe. No reports on in vitro and in silico acetycholinesterase activity of alloisoimperatorin (Fig. 6) (Table 11). From in slico results, we have discussed only three best phytochemicals possessing antioxidant, antidiabetic, anti-HIV and anti-acetycholinesterase activity. Out of seven phytochemicals of C. uredinicola, the diphenylmethyleneamino methyl ester showed strong anti-oxidant and anti-HIV

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activity and alloisoimperatorin shows potent anti-diabetic and anti-cholinesterase activity in both in vitro and in silico experimental analysis.
International Journal of Pharmacology, Phytochemistry and Ethnomedicine Vol. 13    The C. uredinicola extract biologically phytochemicals are responsible for strong antioxidant, anti-HIV, anti-diabetes and anti-cholinesterase activity. The in silico experiment clearly understands that the three phytochemicals (diphenylmethyleneamino methyl ester, alloisoimperatorin, hymecromone) have the ability to interact with oxidant, HIV-1, diabetic and cholinesterase proteins with highest binding energy. The admetSAR and Molsoft proven that these three compounds are nontoxic, non-carcinogens, easily biodegradable and having drug likeliness properties.
The oxidant proteins SOD, catalase, gpx, glutathione peroxidase 7, human oxidant enzyme are inhibited by endophytic fungal extract. All these enzymes break down potentially harmful molecules in cells and these oxygen molecules play a role in disease or cell damage.
The diabetic proteins, α-amylase, β-glucosidase and DPP-IV are strongly inhibited by the C. uredinicola extract. The α-amylase and β-glucosidase are involved in digestion of carbohydrates lead to increase of blood glucose level in diabetes -2. The DPP-IV increases glucagon and blood glucose level. These enzymes play vital role progression of diabetes-2. HIV-1 RT (essential step in retroviral replication), protease (play crucial role HIV life cycle and it cleaves the newly synthesized polyproteins to obtain mature components of protein of an infective HIV), integrase (requires for multidomain enzyme essential for the viral DNA in the host genome), gp120 (essential for virus entry into cells and helps in attachment to specific cell surface receptors) are greatly inhibited by the endophytic fungal extract. The AChE and BChE are pathogenesis of Alzheimer disease and progression.

Conclusion
Based on outcomes of our in vitro and in silico research clearly indicates that the endophytic fungi C. uredinicola have shown biologically important phytochemicals in methanol extract and these are responsible for antioxidant, anti-HIV, anti-diabetes and anticholinesterase activity and suggested their possible role. Further in vivo work is needed to justify our research.