4.4.1 Isolation of genomic DNA from fungi
18S rRNA sequencing for fungi was performed using universal primers. DNA isolation was done with NucleoSpin® Plant II Kit (Macherey-Nagel) (Andrade-Monteiro et al., 2000).
About 25 mg of the fungal mycelium was homogenized using liquid nitrogen and the powdered tissue was transferred to a microcentrifuge tube. Four hundred microlitres of buffer PL1 was added and vortexed for 1 minute. Ten microlitres of RNase A solution was added and inverted to mix. The homogenate was incubated at 65oC for 10 minutes. The lysate was transferred to a Nucleospin filter and centrifuged at 11000 x g for 2 minutes. The flow through liquid was collected and the filter was discarded. Four hundred and fifty microlitres of buffer PC was added and mixed well. The solution was transferred to a Nucleospin Plant II column, centrifuged for 1 minute and the flow through liquid was discarded. Four hundred microlitre buffer PW1 was added to the column, centrifuged at 11000 x g for 1 minute and flow though liquid was discarded. Then 700 µl PW2 was added, centrifuged at 11000 x g and flow through liquid was discarded. Finally 200 µl of PW2 was added and centrifuged at 11000 x g for 2 minutes to dry the silica membrane. The column was transferred to a new 1.7 ml tube and 50 µl of buffer PE was added and incubated at 65oC for 5 minutes. The column was then centrifuged at 11000 x g for 1 minute to elute the DNA. The eluted DNA was stored at 4oC.
4.4.2 Agarose Gel Electrophoresis for DNA Quality and Quantity check
The quality of the DNA isolated was checked using agarose gel electrophoresis. 1µl of 6X gel-loading buffer (0.25% bromophenol blue, 30% sucrose in TE buffer pH-8.0) was added to 5µl of DNA. The samples were loaded to 0.8% agarose gel prepared in 0.5X TBE (Tris-Borate-EDTA) buffer containing 0.5 µg/ml ethidium bromide. Electrophoresis was performed with 0.5X TBE as electrophoresis buffer at 75 V until bromophenol dye front has migrated to the bottom of the gel. The gels were visualized in a UV transilluminator (Genei) and the image was captured under UV light using Gel documentation system (Bio-Rad)
4.4.3 PCR analysis
PCR amplification reactions were carried out in a 20 µl reaction volume which contained 1X PCR buffer (100mM Tris HCl , pH-8.3; 500mM KCl), 0.2mM each dNTPs (dATP, dGTP, dCTP and dTTP), 2.5mM MgCl2, 1 unit of AmpliTaq Gold DNA polymerase enzyme, 0.1 mg/ml BSA, 4% DMSO, 5pM of forward and reverse primers and FTA disc as template.
|Target||Primer name||Direction||Sequence (5’ 3’)|
The PCR amplification was carried out in a PCR thermal cycler (GeneAmp PCR System 9700, Applied Biosystems).
95 oC – 5.00 min
95 oC – 30 sec
54 oC – 40 sec 40 cycles
72 oC – 60 sec
72 oC – 7.00 min
4 oC – ∞
4.4.4 Agarose Gel electrophoresis of PCR products
The PCR products were checked in 1.2% agarose gel prepared in 0.5X TBE buffer containing 0.5 µg/ml ethidium bromide. 1 µl of 6X loading dye was mixed with 5 µl of PCR products and was loaded and electrophoresis was performed at 75V power supply with 0.5X TBE as electrophoresis buffer for about 1 to 2 hours, until the bromophenol blue front had migrated to almost the bottom of the gel. The molecular standard used was a 2-log DNA ladder (NEB). The gels were visualized in a UV transilluminator (Genei) and the image was captured under UV light using Gel documentation system (Bio-Rad)
4.4.5 ExoSAP-IT Treatment
ExoSAP-IT (USB) consists of two hydrolytic enzymes, Exonuclease I and Shrimp Alkaline Phosphatase (SAP), in a specially formulated buffer for the removal of unwanted primers and dNTPs from a PCR product mixture with no interference in downstream applications.
Five micro litres of PCR product is mixed with 2 µl of ExoSAP-IT and incubated at 37oC for 15 minutes followed by enzyme inactivation at 80oC for 15 minutes.
4.4.6 Sequencing using Big Dye Terminator v3.1
Sequencing reaction was done in a PCR thermal cycler (GeneAmp PCR System 9700, Applied Biosystems) using the BigDye Terminator v3.1 Cycle sequencing Kit (Applied Biosystems, USA) following manufactures protocol.
The PCR mix consisted of the following components:
PCR Product (ExoSAP treated) -10-20 ng
Primer -3.2 pM (either Forward or Reverse)
Sequencing Mix -0.28 µl
Reaction buffer -1.86 µl
Sterile distilled water -make up to 10µl
The sequencing PCR temperature profile consisted of a 1st cycle at 96oC for 2 minutes followed by 30 cycles at 96oC for 30 sec, 50oC for 40 sec and 60oC for 4 minutes.
4.4.7 Post sequencing PCR clean up
- Master mix I of 10µl milli Q and 2µl 125mM EDTA per reaction and master mix II of 2µl of 3M sodium acetate pH 4.6 and 50 µl of ethanol were prepared.
- 12µl of master mix I was added to each reaction containing 10µl of reaction contents and was properly mixed.
- 52 µl of master mix II was added to each reaction.
- Contents were mixed by inverting and incubated at room temperature for 30 minutes
- Spun at 14,000 rpm for 30 minutes
- Decanted the supernatant and added 100µl of 70% ethanol
- Spun at 14,000 rpm for 20 minutes.
- Decanted the supernatant and repeated 70% ethanol wash
- Decanted the supernatant and air dried the pellet.
The cleaned up air dried product was sequenced in ABI 3730 DNA Analyzer (Applied Biosystems).
4.4.8 Sequence analysis
The sequence quality was checked using Sequence Scanner Software v1 (Applied Biosystems). Sequence alignment and required editing of the obtained sequences were carried out using Geneious Pro v5.6 (Drummond et al., 2012).
4.5 IDENTIFICATION OF BIOACTIVE COMPOUNDS BY GAS CHROMATOGRAPHY- MASS SPECTRUM (GC-MS) ANALYSIS
GC-MS technique was used to identify the phytochemicals in the solvent extracts of leaves and stem of Hypericum hookerianum and Hypericum mysorense. The solvent extracts of endophytic fungi isolated from H. hookerianum and H. mysorense were also analysed using GC-MS (Susmita Mondal et al., 2011)
The extracts were separated on an Elite-5MS capillary column fitted to a Perkin Elmer Clarus 680 GC was used in the analysis employed a fused silica column, packed with Elite-5MS (5% biphenyl 95% dimethyl polysiloxane, 30 m × 0.25 mm ID × 250μm df) and the components were separated using Helium as carrier gas at a constant flow of 1 ml/min. The injector temperature was set at 260°C during the chromatographic run. The 1μL of extract sample injected into the instrument the oven temperature was as follows: 60°C (2 min); followed by 300°C at the rate of 10°C min−1; and 300°C, where it was held for 6 min. The mass detector conditions were transfer line temperature 240°C; ion source temperature 240°C; and ionization mode electron impact at 70 eV, a scan time 0.2 sec and scan interval of 0.1 sec. The fragment from 40 to 600Da. Turbo Mass version 5.4.2 software was used for the spectral analysis.
4.5.1 Identification of compounds
The compounds were identified by interpreting mass spectrum of GC-MS using the database of National Institute Standard and Technology (NIST). The spectrums of the unknown components were compared with the database of spectrum of known components stored in the NIST (2008) library. The molecular weight and structure of the compounds in the test materials were ascertained.
4.6 EVALUATION OF IN VITRO CYTOTOXIC ACTIVITY OF THE PLANTS AND FUNGAL EXTRACTS AGAINST HEp-2 CELL LINE BY MTT ASSAY.
The cytotoxicity activity of the leaves, stem of H. hookerianum and H. mysorense and their fungal extracts were carried out against HEp-2 cell line using MTT assay [(3-(4, 5-dimethylthazol-2-yl)-2, 5-diphenyl tetrazolium bromide) (Mosman, 1983).
The MTT assay depends on the number of cells present, and on the mitochondrial activity per each cell. The tetrazolium salt MTT was turned into a blue coloured derivative (FORMAZAN) by living cells is clearly a very effective principle on which the assay is based.
4.6.1 Preparation of test samples
10 mg of ethanol, hexane extracts leaves, stem of H. hookerianum and H. mysorense and ethyl acetate, chloroform extracts of fungi isolated from H. hookerianum and H. mysorense, were weighed separately and dissolved in 0.5ml of Dimethyl sulphoxide (DMSO). The volume was made up to 10 ml with maintenance medium (MEM with 2% FBS) to obtain a stock solution of 1 mg/ml concentration, sterilized with Millipore® 0.22 μm, Riviera Cellulose Nitrate Membrane Filter (Catalog No. 754004722) and stored at -20°C. The stock solutions of plants and fungal extracts were diluted with Eagle’s Minimum Essential Medium (MEM) to desired concentrations ranging from 100μg, 50μg, 25μg, 12.5μg, 6.25μg, 3.12μg, 1.56μg/0.1ml. The final concentration of DMSO in each sample did not exceed 0.1% v/v
4.6.2 Cell Culture
Human epithelial type 2 (HEp-2) cells were procured from National Centre of Cell Sciences (NCCS, Pune, India). The HEp-2 cell lines were cultured in rectangular canted neck flasks (Fig-4.10) using Eagle’s Minimum Essential Medium (MEM) supplemented with 10% FBS (Fetal Bovine Serum), Earle′s salts, L-glutamine, Sodium bicarbonate, HEPES buffer, Penicillin (100U/ml,), Streptomycin (100μg/ml), and Amphotericin-B (2.5μg/ml). The pH was adjusted to 7.1 to 7.4
4.6.3 Sub culturing of cells
When the HEp-2 cells attained a confluent monolayer (Fig-4.11), separation of the cell line was carried out using TPVG (Trypsin, Phosphate buffered saline, Versene (EDTA) and Glucose) and Phosphate buffered saline (PBS) under room temperature. The medium over the cell line was gently poured out from the culture flask. The confluent cell line was gently washed with 2ml of PBS twice in order to remove the remaining serum from the cell line. 4 ml of TPVG solution was delivered over the cell line, by a micropipette. The flask was incubated at 37ºC in a 5% CO2 atmosphere for 4 minutes until cell line becomes opaque. At this stage cells had undergone rounding without detachment which was observed under Inverted Tissue Culture Microscope. The TPVG solution, applied over the cell line was carefully pipette out from the culture flask. Then 5 ml of 5% MEM growth medium was added into the flask and the content was aspirated several times with a Pasteur pipette in order to suspend and separate the cell line into individual cells.
4.6.4 Cell counting (Haemocytometer)
Cells were counted using haemocytometer. Dilute 0.1 ml of the cell suspension was added in 0.1 ml of trypan blue ( 100 µl cell suspension : 100 µl trypan blue), mixed well and loaded to the haemocytometer using the improved neubauer capillary tube, taking care to avoid overflow. Cells in each of the four corner of the counting chamber were counted under the microscope; Cells in the upper and left border were counted. Dead cells and cells on lower and right border were omitted. The following formula was used for cell counts
Number of cells counted X dilution factor X 104 Number of squares counted
4.6.5 In vitro cytotoxicity assay by MTT method
0.1 ml of HEp-2 cell suspension was seeded at density of 1.4×105 cell/ml cells to each well of a 96 well microtitre plate (Fig-4.12) and incubated at 37ºC in a 5% CO2 atmosphere for 24 hrs. Then the medium was removed and 0.1μl of varying concentrations (100μg, 50μg, 25μg, 12.5μg, 6.25μg, 3.12μg, 1.56μg/0.1ml) of each extracts prepared by two fold serial dilutions with 2% MEM were transferred into the respective wells. Further 200μl of 2% MEM was added in to all the experimental wells. 0.1% DMSO added to separate well was taken as negative control and well with positive control Acyclovir [9-(2-hydroxyethoxymethyl) guanosine, were also maintained. The microtitre plate was then incubated at 37ºC atmosphere in a 5% CO2 for 72 hrs (Denis Mabeya Ogato et al., 2015) (Fig-4.13 & 4.14).
184.108.40.206 MTT assay
After 72 hrs, the cells were treated with 20 μl MTT (5mg/ml in PBS) and incubated at 37ºC in a 5% CO2 for 4 hrs. The MTT solution was removed without disturbing the Formosan crystals and 150 μl of DMSO was further added in to all the wells and kept at 37ºC for 10 min, where by the crystals were completely dissolved. The plate was read on a microtitre plate reader (Biotek USA) at 540nm. All experiments were performed in triplicate. Cytotoxicity was calculated based on the viability of HEp-2 cell lines using the following formula.
% Cell viability = [O.D of test compound)/ (O.D. of control)] X 100
Concentrations with viable cells and intact cell lines in the experiment were considered as non toxic concentrations and the one detected with morphologically changed cells will not be considered for the antiviral studies. The readings obtained for each extracts were statistically analysed by SPSS software.
Fig- 4.13: Experimental design for in vitro cytotoxicity assay of leaves and stem extracts of Hypericum species in the 96 well plate.
|Concentration (µg/0.1ml)||Ethanol extracts of leaves and stem||Hexane extracts of leaves and stem||Acyclovir||Cell control|
Fig- 4.14: Experimental design for in vitro cytotoxicity assay of fungal extracts in the 96 well plate.
|Concentration (µg/0.1ml)||Ethyl acetate extracts||Chloroform extracts||Acyclovir||Cell control|
4.7 EVALUATION OF IN VITRO ANTIVIRAL ACTIVITY OF THE PLANTS AND FUNGAL EXTRACTS AGAINST HSV-2 USING HEP-2 CELL LINE BY MTT ASSAY.
4.7.1 Virus stock
HSV-2 (Strain no. 753167) of National Institute of Virology (NIV), Pune, India, was propagated in HEp-2 cell line and incubated at 37ºC for 96 hrs. Complete cytopathic effect virus stock was used for the estimation of TCID50 by end point dilution assay and 10-6.5 TCID50/ml virus stock concentration was used for the antiviral study (Reed and Muench,1938).
4.7.2 Estimation of TCID50
Preparation of virus dilution
The confluent monolayer of HEp-2 cell line grown in culture flask was used for the estimation of TCID50 assay of HSV-2.
The monolayer of HEp-2 in tissue culture flask was tripsinized. 0.1ml of cell suspension was seeded into 96 well plates with 5% MEM. Thus six vertical rows of seven wells and seventh row of three wells with cell suspension and medium was prepared. The set up was incubated at 37ºC in a 5% CO2 for 24 hrs. When the cells in each well attained confluent monolayer, the growth medium was carefully pipette out and 0.1 ml of 2% MEM was added on the cell line in each well. 0.9 ml of 2% MEM was transferred each of the seven 5 ml sterile glass vial, 0.1 ml of virus stock was added into first vial, tenfold serial dilution was done from first vial to seventh vial (10-1 dilution to 10-7 dilution). New sterile tips were fitted to the micropipette for each dilution step.
0.1 ml of 10-1 dilution was transferred in to each of the six wells of first horizontal row. Horizontal row of 2, 3, 4, 5, 6 and 7 were meant for 0.1 ml of 10-2, 10-3, 10-4, 10-5, 10-6 and 10-7 dilutions. 0.1 ml of 2% MEM alone was transferred into three wells of seventh vertical row for cell control. The entire set up was incubated under 37ºC with 5% CO2 atmosphere for optimum growth of the virus. The microtitre plate wells were observed under inverted tissue culture microscope for development of CPE. The medium in the wells were removed and fresh growth medium was provided on the 4th day and further incubated till complete CPE occurred to the cell line(Fig-4.15). On 7th day, the individual wells in the 96 well plates were score as infected if CPE is more than 2/3 of cells and uninfected less than 2/3 by adopting the method of (Reed and Muench,1938). To calculate the TCID50 by 0.1 ml of virus stock
Fig-4.15: A schematic presentation of TCID50 estimation of HSV-2 in a 96 well microtitre plate
|Dilution of virus inoculums||1||2||3||4||5||6||7||8||9||10||11||12|
+ — Complete CPE
— Absence of CPE
— Cell control
After the observation of results based upon CPE the TCID50of HSV-2 was estimated (Fig-4.16).
Table- 4.1: Data on estimation of TCID50 for HSV-2
|Virus concentration||Effective cell culture||Accumulative of effects|
|CPE negative||Positive total||Positive||Negative||Ratio: Positive control||% number positive|
50% end point was between the dilutions of one in 10-6 (37.5) and 10-7. The proportional distance of the 50% endpoint from these dilutions can be calculated by using following formula.
(%CPE at dilution next above 50%) – 50
TCID50 = ——————————————————————————————–
(%CPE at dilution next above 50%) – (% CPE at dilution next below 50%)
= = = 0.42
Negative logarithm of the lowest dilution (next above-50%CPE) = -6.0 and proportionate distance (0.4) x log dilution factor = -0.4
TCID50 titer = -5.4 /0.1ml
Log TCID50 titer = 10-6.4 / 1 ml
1 TCID50 titer of given passage of Herpes simplex virus is approximately
= 10-6.4/ 1 ml
4.7.3 In vitro Antiviral assay by MTT method
The in vitro antiviral activity of leaves, stem extracts of H. hookerianum and H. mysorense and their fungal extracts were performed by MTT assay (Muller et al., 2007).
Monolayer of HEp-2 cells was grown in 96 well microtitre plates as described earlier. The growth medium was carefully removed by micropipette from the monolayer. 100 μl ml of 10-6.5 TCID50/ml dose of viral suspension, obtained by seven consecutive tenfold dilution in 2% MEM, were added into eight wells containing the confluent monolayer of HEp-2 cells. Then the 96 well microtitre plates were kept for virus adsorption to the cell line at 37ºC in a 5% CO2 atmosphere for 90 min.
The highest non toxic concentrations of the leaves, stem and fungal extract were diluted by serial two fold dilutions with 2% MEM and 100μl of each extracts were added on the virus inoculated monolayer of cell. The non toxic concentration of Acyclovir was used as positive control for HSV-2 inhibition and DMSO (0.1%) was used as negative control. To act as a virus control and cell control, the virus suspension and 2% MEM maintenance medium without samples were added. The entire setup was incubated at 37ºC in a 5% CO2 atmosphere for 72 hrs (3 to 4 days) to allow multiplication of virus and subsequent development of cytopathic effect (CPE). Each well was observed under Inverted Tissue Culture Microscope every day for presence or absence of cytopathic effect (Fig-4.17 & 4.18).
Gross absence of cytopathic effect was inferred by intact of cell layer, without distortion in the morphology of cells or nucleus and syncytial cells in comparison with the control rows and thus wells with or without CPE were marked. Important observations of the incubated wells, both experimental and control were recorded by microphotography through Inverted Tissue Culture Microscope
After 72 hrs, cells in each well were treated with 20μl MTT (5mg/ml) and incubated at 37ºC in a 5% CO2 atmosphere for 4 hrs. Further 150μl of DMSO was added to all the wells and kept at 37ºC in a 5% CO2 atmosphere for 10 min. The plate was read on a microtitre reader (Biotek USA) at 540nm. All experiments were performed in triplicate. The percentage of cytopathic effect (CPE) was compared with virus control.
Fig,4.17: Experimental design for in vitro antiviral assay of leaves and stem extracts of Hypericum species on HSV-2 in the 96 well plates.
|Concentration (µg/0.1ml)||Ethanol extracts of leaves and stem||Hexane extracts of leaves and stem||Acyclovir||Cell control|
Fig 4.18: Experimental design for in vitro antiviral assay of fungal extracts on HSV-2 in the 96 well plate.
|Concentration (µg/0.1ml)||Ethyl acetate extracts||Chloroform extracts||Acyclovir||Cell control|
4.8 IN SILICO ANTIVIRAL ACTIVITY OF PLANTS AND FUNGAL EXTRACTS AGAINST HSV-2.
4.8.1 DOCKING ANALYSIS-AN IN SILICO APPROCH
Database and Tools
PROTEIN DATA BANK (PDB)
Accelyr’s Discovery Studio 4.0
4.8.2 PREPARATION OF LIGANDS.
PubChem and Chemspider.
2D and 3D structure of the compound identified from leaves and stem extracts of Hypericum mysorense and Hypericum hookerium and the fungal compounds of Alternaria alternate, Phomopsis sp. Curvularia spicifera, Chaetomium globosum were retrieved from online data base PubChem and chemspider. All the ligands were saved in .sdf format.
4.8.3 RETRIEVAL OF TARGET PROTEIN
220.127.116.11Protein Data Bank (PDB)
The protein data bank (PDB) is a repository for the three – dimensional structural data of large biological molecules, such as protein and nucleic acid. The 3D structure of HSV-2 protein or receptor (PDB ID: 1AT3) was retrieved from online data base in www.rcsb.org/pdb (Research Collaboratory for Structural Bioinformatics) and saved in .pdb file format. Since the 3D structure of HSV-2 whole proteomic sequences (DNA polymerase) is unavailable in online data base RCSB, molecular modelling was preferred to obtain the protein structure and saved in .pdb file format.
18.104.22.168 Molecular modeling
Structure-based drug design method involves bioinformatics, proteomics, biochemistry, and computer modelling of 3-dimensional protein structures. Whole proteomic sequences (DNA polymerase) were downloaded for Human herpes virus 2 from NCBI in FASTA format. The Gene bank number of possible protein is KM068900.1. The modelling of the three dimensional structure of the protein was performed by SWISSMODEL 2.0. The overall stereochemical property of the protein was assessed by Ramchandran plot analysis. The validation for structure models obtained from the three software tools was performed by using PROCHECK. The models were further checked with WHAT IF. The docking of the ligands with the target protein modelled basis of DOPE Score and RC Plot analysis performs using Accelyr’s Discovery Studio 4.0.
4.8.4 Pharmacokinetics and phamacodynamics profiling of screened analogs.
Pre clinical ADME/TOX studies help in ruling out false positives and identify the most potential drug candidates with appropriate kinetic and dynamic properties. For this reason, compound screened from PubChem compound database were subjected to ADME/TOX property calculations using PreADMET and Accelyr’s Discovery Studio 4.0.
PreADMET is a web-based application used for predicting ADME data and building drug like library using in silico method.
Druglikness is a complex of various molecular properties and features which determines whether a particular molecule is similar to the known drugs. The rules involve are Lipinki’s Rule
Lipinski’s rule of five also known as Rules of five (RO5) is used thumb to evaluate druglikeness or determine if a chemical compound is an orally active drug in humans. These are the rules to be followed
- Not more than 5 hydrogen bond donors (OH and NH groups)
- Not more than 10 hydrogen bond acceptors (notably N and O)
- A molecular weight under 500g/mol
- A partition co-efficient log P less than 5
- Rotable bonds less than 10.
4.8.5 ADME PREDICTION
A drug requires suitable pharmacokinetics profile to be efficacious in vivo in humans the relevant pharmacokinetics properties involve Absorption, Distribution, Metabolism, and Excretion (ADME) profile of drug.
4.8.6 PLASMA PROTEIN BINDING
Generally, only the unbound drug is available for diffusion or transport across cell membranes, and also for interaction with a pharmacological target. As a result, a degree of plasma protein binding of a drug influences not only the drugs action but also its disposition and efficacy in PreADMET can predict percent drug bound protein as in vitro data on human (Table – 4.2).
|Classification||Plasma Protein Binding (%PPB)|
|Chemials stringly bound||More than 90%|
|Chemicals weakly bound||Less than 90%|
Table- 4.2: PreADMET-General classification of plasma protein binding of compounds
22.214.171.124 Blood Brain Barrier (BBB) Penetration
Predicting BBB penetration means predicting whether compounds pass across the blood brain barrier. This is crucial in pharmaceutical sphere because CNS-active compound must pass across it and CNS-inactive compounds mustn’t pass across it in order to avoid of CNS side effects. PreADMET can predict in vivo data on rates for BBB penetration (Table – 4.3).
Table- 4.3: PreADMET-General classification of Blood Brain Barrier of compounds
|CNS-Active compounds (+)||More than 1.0||More than 0|
|CNS-Inactive compounds (-)||Less than 1.0||Less than 0|
4.8.7 SKIN PERMEABILITY
In the pharmaceutical, cosmetic and agrochemical field, it is important to predict the skin permeability rate for a crucial parameter for the trans dermal delivery of drugs and for the risk assessment of all chemicals that come into contact with the skin either accidently or by design. PreADMET can predict in vitro data on human for skin permeability PreADMET predicts in vitro skin permeability and the result value is given as logKp.Kp(cm/hour) is defined as
where Km = distribution coefficient between stratum corneum and vehicle
D = average diffusion coefficient (cm2/h) h = thickness of skin (cm).
4.8.8 HUMAN INTESTINAL ABSORPTION
Human intestinal absorption data are the sum of bioavailability and absorption, evaluated from ratio of excretion or cumulative excretion in urine, bile and faeces. For prediction of HIA in PreADMET, chemical structure at pH 7.4 are applied, because HIA is measured by in vivo test (Table – 4.4).
Table-4.4: PreADMET-General classification of Human intestinal Absorption of compounds
|Classification||HIA(Human Intestinal Absorption)|
|Poorly absorbed compounds||0-20%|
|Moderately absorbed compounds||20-70%|
|Well absorbed compounds||70-100%|
126.96.36.199Caco-2 cell permeability
Caco2 cell model has been recommended as a reliable in vitro model for the prediction of oral drug absorption in thedrug selection process for assessing the intestinal absorption of drug candidates. For prediction of Caco2 cell permeability in PreADMET, chemical structure at pH 7.4 was used (Table – 4.5).
Table-4.5: PreADMET-General classification of Caco2 permeability of compounds
|Low permeability||Less than 4|
|High permeability||More than 70|
4.8.9 TOXICITY PREDICTION
Toxicity profile was computationally predicted based on ames mutagenicity tests. DICOVERY STUDIO TOPKAT (Toxicity Predtion Komputer Assisted Technology) was used to study the toxicity of identified compounds.
Ames Mutagenicity studio predicts toxicity TA 100, TA 1535, TA 1537, TA 1538, TA 98 which are often used in Ames test. A compounds is considered a mutagen if a positive response.
188.8.131.52 PASS (Prediction of Activity Spectra for Substance)
PASS online provides a possibility of simultaneous prediction of about 3,600 kinds of biological activity for drug-like organic compound. Input data represents a structural formula of compounds in MOL file format. The input file represent a list of activities with two probabilities Pa (Probability to be active) and Pi (probability to inactive).
4.8.10 MOLECULAR DOCKING (ACCELRY’S DISCOVERY STUDIO 4.0)
Docking is a technique where protein and ligands are interacted. This kind of interaction is possible through Accelry’s Discovery Studio 4.0 software. Through this technique, binding site of the target/receptor can be identified. The target was viewed in discovery studio and observed that 1AT3 and DNA polymerase receptor has binding sites by removing the complexes and water molecules from them. Then the graphical views of all the ligands were visualized individually and docked. By using ligand fit algorithm the interaction of a target molecules and a particular ligand was made possible. After interaction the results can be inferred through dock score. The maximum dock score represents the maximum binding of ligand with the target and PMF value was calculated.
4.9 SATISTICAL ANALYSIS
The statistical significance of in vitro cytotoxic activity and in vitro antiviral activity was performed using Statistical Package for Social Science (SPSS) version 22.0. The triplicate values were expressed as Mean ± Standard Deviation (SD). P value considered as significant level at < 0.5.
5.1 Identification of plants samples
The collected plants samples of Hypericum mysorense F. Heyne and Hypericum hookerianum Wight and Arn, were identified and authenticated by Botanical Survey of India (BSI), Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, with the voucher specimen (No.BSI/SRC/5/23/1 5/Tech-1145).
5.2 Molecular identification of endophytic fungi
5.2.1 18SrRNA sequencing of HM1
Sequence of HM1 was isolated from Hypericum mysorense showed 1020 base pairs (Fig -5.1). Query sequence showed 100 blast hits that represented highest alignment score (Fig -5.2). The sequences were subjected to NCBI GeneBank and the identification was based on closely related sequences. The 18S (NS1 & NS2) sequence of HM1 resulted 100% similar with GeneBank sequences of Alternaria alternata (KX609769) (Table-5.1). The sequences submitted to the National Center for Biotechnology Information (NCBI) Genbank with accession no. KF493862.
184.108.40.206 Sequence of HM1
Fig 5.1: PCR amplification of 18SrRNA sequences
Fig 5.2: Graphical representation of HM1 sequence alignment
Table 5.1: Sequence alignment representation of HM1
5.2.2 18SrRNA sequencing of HM3
Sequence of HM3 isolated from Hypericum mysorense showed 1021 base pairs (Fig-5.1). Query sequence showed 100 blast hits that represented highest alignment score (Fig-5.3). The sequences were subjected to NCBI GeneBank and the identification was based on closely related sequences. The 18S (NS1 & NS2) sequence of HM3 resulted 100% similar with GeneBank sequences of Phomopsis sp (KT824640.1) (Table-5.2). The sequence submitted to the National Center for Biotechnology Information (NCBI) Genbank with accession no. KF493862.
220.127.116.11 Sequence of HM3
Fig 5.3: Graphical representation of HM3 sequence alignment
Table 5.2: Sequence alignment representation of HM 3
5.2.3 18SrRNA sequencing of HK1
Sequence of HK1 isolated from Hypericum hookerianum showed 869 base pairs (Fig- 5). Query sequence showed 100 blast hits that represented highest alignment score (Fig- 5.4). The sequences were subjected to NCBI GeneBank and the identification was based on closely related sequences. The 18S (NS1 & NS2) sequence of HK1 resulted 100% similar with GeneBank sequences of Curvularia spicifera (KM111208.1) (Table-5.4). submitted to the National Center for Biotechnology Information (NCBI) Genbank with accession no. KF493862.
18.104.22.168 Sequence of HK1
Fig 5.4: Graphical representation of MK1 sequence alignment
Table 5.3: Sequence alignment representation of HK1
5.2.4 18SrRNA sequencing of HK2
Sequence of HK2 isolated from Hypericum hookerianum showed 1021 base pairs (Fig- 5.1). Query sequence showed 103 blast hits that represented highest alignment score (Fig- 5.4). The sequences were subjected to NCBI GeneBank and the identification was based on closely related sequences. The 18S (NS1 & NS2) sequence of HK2 resulted 100% similar with GeneBank sequences of Chaetomium globosum (JQ964323.1) (Table-5.5). submitted to the National Center for Biotechnology Information (NCBI) Genbank with accession no. KF493862.
22.214.171.124 Sequence of HK2
Fig 5.5: Graphical representation of HK2 sequence alignment
Table 5.4: Sequence alignment representation of HK 2
5.2.5 18SrRNA sequencing of HK3
Sequence of HM3 isolated from Hypericum Hookerianum showed 1028 base pairs (Fig- 5.1). Query sequence showed 100 blast hits that represented highest alignment score (Fig-5.6). The sequences were subjected to NCBI GeneBank and the identification was based on closely related sequences. The 18S (NS1 & NS2) sequence of HK3 resulted 100% similar submitted to the National Center for Biotechnology Information (NCBI) Genbank with accession no. KF493862.
126.96.36.199Sequence of HK3
Fig 5.6: Graphical representation of HK3 sequence alignment
Table 5.5: Sequence alignment representation of HK3
5.3 GC-MS analysis
5.3.1 GC-MS analysis of ethanol leaves extract from Hypericum mysorense
The GC-MS chromatogram of ethanol leavesextract of Hypericum mysorense was shown in the Fig 5.7. The GC-MS spectral studies revealed the presence of thirteen compounds namely Beta. -d-glucopyranose, 1,6-anhydro; 3,7,11,15-Tetramethyl- 2-hexadecen-1- ol; Hexadecanoic acid, ethyl ester; 9,12,15-Octadecatrienoic acid, ethyl ester, (Z, Z, Z)-; 19-Norpregn-4-ene-3, 20-dione, 17-hydroxy-1-methyl-; 1H-1,2,3,4-tetrazole-1-acetamide, n-[5-(trifluoromethoxy)- 1,3-benzothiazol-2-yl]-; 2- Allyl- 5- ethoxy-4 methoxy phenol; 3-(3,4-Dimethoxy- phenyl) -3-hydroxy-4-; 2-Benzofurancarboxylic acid, 2,4,5,6,7,7a-hexahydro-4,4,7a-trimethyl-, methyl ester, cis-; 6-Pentyl-4-nor-3,5-secoandrostane-3,5.beta.,17.beta.-triol; .Beta.-sitosterol acetate; Dl-.alpha.-tocopherol; 3,3-Diisopropoxy-1,1,1,5,5,5-hexamethyltrisiloxane. Compounds with their retention time (RT), molecular formula (MF), molecular weight (MW) and peak area (%) and molecular structure were presented in the Table 5.6 & 5.7.
Fig 5.7: GC-MS chromatogram of ethanol leaf extract of Hypericum mysorense
Table 5.6: Identified phytoconstituents of ethanol leaves extract of Hypericum mysorense
|Molecular formula||Molecular weight||Peak area (%)|
|3||19.78||Hexadecanoic acid, ethyl ester||C18H36O2||284.48||9.59|
|4||22.57||9,12,15-Octadeca trienoic acid, ethyl ester, (Z,Z,Z)-||C20H34O2||306.49||4.21|
|9||24.94||2-Benzofuran carboxylic acid, 2,4,5,6,7,7a-hexahydro-4,4,7a-trimethyl-, methyl ester, cis-||C13H20O3||224.3||5.36|
Table 5.7: Molecular structure of phytoconstituents identified in ethanol leaves extract of Hypericum mysorense
|S.No.||Compound name||Molecular structure|
|3||Hexadecanoic acid, ethyl ester|
|4||9,12,15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z)-|
|9||2-Benzofuran carboxylic acid, 2,4,5,6,7,7a-hexahydro-4,4,7a-trimethyl-, methyl ester, cis-|
5.3.2 GC-MS analysis of hexane leaves extract from Hypericum mysorense
The GC-MS chromatogram of hexane extract of leaves of Hypericum mysorense was shown in the Fig 5.8. The GC-MS spectral studies revealed the presence of sixteen compounds namely Caryophyllenyl alcohol; 3,7,11,15-Tetramethyl-2-hexadecen-1-ol; 9,12,15-Octadecatrienoic acid, (Z,Z,Z)-; Silane, dimethylhexyloxynonyloxy; 2-Methyl-1,1-bis(3,4-dimetoxyphenyl)propane; 1H-1,2,3,4-tetrazole- 1-acetamide, n-[5-(trifluoromethoxy)- 1,3-benzothiazol-2-; Heptacosane; 3-(3,4-Dimethoxy-phenyl)-3-hydroxy-4-nitrocyclohexanone; 2,6-Lutidine 3,5-dichloro-4-dodecylthio; (1-Ethyl-3,7-dimethylocta-2,6 dienylthio) benzene; 2-Benzofurancarboxylic acid, 2,4,5,6,7,7a-hexahydro- 4,4,7a-trimethyl-, methylester,cis-; Di-n-decylsulfone; Dl-.alpha.-tocopherol; 1,2,4-Cyclopentane trione, 3,3-bis(3-methyl-2-butenyl)-5-(3-methyl-1-oxobutyl)-; 17-Pentatriacontene; Cholest-5-en-3-ol, 6-nitro-, acetate (ester), (3.beta.)-. Compounds with their retention time (RT), molecular formula (MF), molecular weight (MW) and peak area (%) and molecular structure were presented in the Table 5.8 & 5.9.
Fig 5.8: GC-MS chromatogram of hexane leaves extract of Hypericum mysorense
Table 5.8: Identified phytoconstituents of hexane leaves extract of Hypericum mysorense
Peak area (%)
|3||19.61||9,12,15-Octadecatrienoic acid, (Z,Z,Z)-||C18H30O2||278.43||2.18|
|9||24.57||(1-Ethyl-3,7-dimethylocta-2,6 dienylthio) benzene||C20H42O2S||346.64||3.90|
|11||24.83||2-Benzofurancarboxylic acid,2,4,5,6,7,7a-hexahydro-4,4,7a-trimethyl-, methyl ester, cis-||C13H20O3||224.30||2.10|
|16||29.05||Cholest-5-en-3-ol, 6-nitro-, acetate (ester), (3.beta.)-||C20H42O2S||346.64||2.98|
Table 5.9: Molecular structure of phytoconstituents identified in hexane leaves extract of Hypericum mysorense
|S.No.||Compound name||Molecular structure|
|3||9,12,15-Octadecatrienoic acid, (Z,Z,Z)-|
|9||(1-Ethyl-3,7-dimethylocta-2,6 dienylthio) benzene|
|11||2-Benzofurancarboxylic acid,2,4,5,6,7,7a-hexahydro-4,4,7a-trimethyl-, methyl ester, cis-|
|16||Cholest-5-en-3-ol, 6-nitro-, acetate (ester), (3.beta.)-|
5.3.3 GC-MS analysis of ethanol stem extract from Hypericum mysorense
The GC-MS chromatogram of ethanol extract of stem of Hypericum mysorense was shown in the Fig 5.9. The GC-MS spectral studies revealed the presence of fourteen compounds namely 2-Formylhistamine; Phenol, 2-(1-methylethoxy)-, methylcarbamate; 1,6;3,4-Dianhydro- 2-o-acetyl-. beta.-d-galacto pyranose; 1,2,3,4-Cyclopentanetetrol, (1.alpha.,2.beta.,3.beta., 4.alpha.)-; 2-[1,2-Dihydroxyethyl]-9-[.beta.-d-ribofuranosyl]hypoxanthine; 3,7,11,15-Tetramethyl-2-hexadecen-1-ol; Eicosanoic acid; Bicyclo [4.1.0]heptane, 7-pentylb-; Propylure; Octasiloxane, 1,1,3,3,5,5,7,7, 9,9,11,11,13,13,15,15-hexadecamethyl; 3.Beta.-acetoxy- bisnor-5-cholenamide; 3-Butoxy-1,1,1,5,5,5- hexamethyl-3-(trimethyl siloxy) trisiloxane; 3-isopropoxy- 1,1,1,5,5,5-hexamethyl- 3-(trimethylsiloxy) trisiloxane; 1,2-Bis (trimethylsilyl) benzene. Compounds with their retention time (RT), molecular formula (MF), molecular weight (MW), peak area (%) and molecular structure were presented in the Table 5.10 & 5.11.
Fig 5.9: GC-MS chromatogram of ethanol stem extract of Hypericum mysorense
Table 5.10: Identified phytoconstituents of ethanol stem extract of Hypericum mysorense
|Molecular formula||Molecular weight||Peak area (%)|
Table 5.11: Molecular structure of phytoconstituents identified in ethanol stem extract of Hypericum mysorense
|S.No.||Compound name||Molecular structure|
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