Claims:
Claims:
1. A herbal formulation for preventing the lung cancer and coronavirus, comprises:
Cordyceps militaris powder;
Ganoderma lucidum powder;
Shiitake mushroom powder;
Spirullina (Arthrospira platensis) powder; and
Ginger powder.
2. The herbal formulation as claimed in claim 1, wherein the Cordyceps militaris powder is present in an amount of 50%.
3. The herbal formulation as claimed in claim 1, wherein the Ganoderma lucidum powder is present in an amount of 15%.
4. The herbal formulation as claimed in claim 1, wherein the Shiitake mushroom powder is present in an amount of 15%.
5. The herbal formulation as claimed in claim 1, wherein the Spirullina (Arthrospira platensis) powder is present in an amount of 10%.
6. The herbal formulation as claimed in claim 1, wherein the ginger powder is present in an amount of 10%.
, Description:FORM 2
The Patents Act 1970
(39 of 1970)
&
The Patent Rules 2003
COMPLETE SPECIFICATION
(See Section 10 and rule 13)

TITLE OF THE INVENTION:
CORDYCEPS MILITARIS BASED HERBAL FORMULATION FOR PREVENTING THE LUNG CANCER AND SARS-COV-2

APPLICANT:
Name: AKS UNIVERSITY
Nationality: Indian
Applicant: DEPARTMENT OF BIOTECHNOLOGY, AKS UNIVERSITY, SATNA, Madhya Pradesh-485001, INDIA

PREAMBLE OF THE DESCRIPTION:
THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED

A) TECHNICAL FIELD OF INVENTION
[001] The present invention generally relates to herbal composition and particularly to herbal composition for treatment of lung cancer and coronavirus. The present invention more particularly relates to a herbal composition comprising species of fungi and essential herbal additives that are effective against lung cancer and coronavirus.

B) BACKGROUND OF INVENTION
[002] Cordyceps extract or cordyceps militaris extract has been used as a traditional medicine for many years in Asian countries. Recently, many studies have tried to elucidate the pharmacological mechanism of Cordyceps, which include immune activation, anti-inflammatory, anticancer, and anti-viral effects. Cordyceps is composed of mainly four purified components which are cordycepin, cyclosporin, acetoxyscirpenol, and ergosterolperoxide. The most bountiful amino acids of C. militaris are lysine, glutamic, proline and threonine which are present in the fruiting body, while proline and lysine are present in the corpus.
[003] The complex of bioactive compounds derived from Ganoderma lucidum extract, presumably a mix of polyphenols and carbohydrate-flavonoid complexes, can be beneficially exploited in anticancer and anti-inflammatory therapies against melanoma and breast cancer.
[004] Lentinula edodes, known as shiitake, has been used as food and in medication because its mycelium and fruiting body have indicated a few remedial properties. Lentinula edodes, have antiviral action against poliovirus type 1 (PV-1) and cow-like herpes virus type 1 (BoHV-1). Lentinula edodes has lentinan and KS-2 as antiviral compounds.
[005] Spirulina (Arthrospira platensis) ingredient of Spirulina, ß-carotene, has been reported to have antioxidant and anti-inflammatory. Spirulina was once situated specifically on its workable as a source of protein, vitamins, specifically nutrition B12 and provitamin A (ß-carotene), and fundamental greasy acids like ? - linolenic corrosive. As of late more consideration has been given to study its restorative impacts, which incorporate diminishment of cholesterol and nephrotoxicity by using overwhelming metals, anticancer properties, protection towards radiation, and upgrade of the immune framework (Belay et al., 1994). Spirulina too possesses other herbal capacities such as antiviral, antibacterial, antifungal, and antiparasite exercises.
[006] Investigation shows that Ginger, Zingiber officinale, has a typical flavour and furthermore a generally utilized therapeutic plant in antiquated time. Ginger is a fixing to Ge-Gen-Tang. GGT has been demonstrated to have antiviral properties against human respiratory syncytial virus (HRSV). However, it is obscure whether ginger is powerful against human respiratory syncytial virus (HRSV).
[007] There have been identified secondary treatment drugs for coronavirus but these medicines come with major side effects. Hence there is a need to come up with a herbal composition which does not have any side effects and is very much effective against coronavirus as well as lung cancer.
[008] The value additions and above-mentioned shortcomings, disadvantages and problems are addressed herein, as detailed below.

C) OBJECT OF INVENTION
[009] The primary object of the present invention is to provide a herbal formulation for the treatment of lung cancer and coronavirus diseases.
[0010] Another object of the present invention is to provide a herbal formulation comprising Cordyceps militaris (mushroom), Ganodurma lucidum (mushroom) and Lentinula edodes (Shiitake mushroom), Arthrospira platensis (spirulina) and Zingiber officinale (ginger).
[0011] Yet another object of the present invention is to provide a herbal formulation that comprises species of fungi and are effective against coronavirus.
[0012] Yet another object of the present invention is to provide a herbal formulation comprising cordycepin which is a good therapeutic drug compound against viral proteins of SARS-CoV-2.
[0013] Yet another object of the present invention is to identify effective herbal treatment without any side effects for deadly coronavirus.
[0014] These and other objects and advantages of the embodiments herein will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings.

D) SUMMARY OF INVENTION
[0015] The various embodiments of the present invention provide a herbal formulation for preventing the lung cancer and coronavirus, comprises:
[0016] a novel herbal formulation for prevention of lung cancer and diseases of corona virus. According to an embodiment of the present invention, the Cordyceps militaris powder is present in an amount of 50%.
[0017] According to an embodiment of the present invention, the Ganoderma lucidum powder is present in an amount of 15%.
[0018] According to an embodiment of the present invention, the Shiitake mushroom powder is present in an amount of 15%.
[0019] According to an embodiment of the present invention, the Spirullina (Arthrospira platensis) powder is present in an amount of 10%.
[0020] According to an embodiment of the present invention, the ginger powder is present in an amount of 10%.
[0021] These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.

E) BRIEF DESCRIPTION OF DRAWINGS
[0022] The other objects, features and advantages will occur to those skilled in the art from the following description of the preferred embodiment and the accompanying drawings in which:
[0023] FIG. 1A shows TLC profiles of test solutions of Cordyceps militaris at 366nm, named as plate 1.1, wherein F1-F9 are test solutions, F10 is unknown sample and F11-F12 are standard (std) at 2µl and std (6µl), according to an embodiment of the present invention.
[0024] FIG. 1B shows TLC profiles of test solutions of Cordyceps militaris at 366 nm after derivatization with 5% methanolic H2SO4, named as plate 1.2, according to an embodiment of the present invention.
[0025] FIG. 1C shows TLC profiles test solutions of Cordyceps militaris named as plate 1.1, at day light after derivatization with 5% methanolic H2SO4, according to an embodiment of the present invention.
[0026] FIG. 2A and FIG. 2B show the HPLC analysis of the samples, according to an embodiment of the present invention.
[0027] FIG. 3 shows the graphical representation of cell viability assay with different fraction of cordyceps formulation extract, according to an embodiment herein.
[0028] FIG. 4 shows the graphical representation of cell viability assay with different concentration of DMSO, according to an embodiment of the present invention.
[0029] FIG. 5 shows the cell viability with DMSO, according to an embodiment of the present invention.
[0030] FIG. 6 shows the images of all six targeted protein structures, according to an embodiment of the present invention.
[0031] FIG. 7 showing Molecular Docking of Cordycepin over targeted proteins of SARS-CoV-2, according to an embodiment of the present invention.
[0032] FIG. 8 showing Molecular Docking of Remdesivir over targeted proteins of SARS-CoV-2, according to an embodiment of the present invention.

F) DETAILED DESCRIPTION OF DRAWINGS
[0033] In the following detailed description, a reference is made to the accompanying drawings that form a part hereof, and in which the specific embodiments that may be practiced is shown by way of illustration. The embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that the logical, mechanical, electronic and other changes may be made without departing from the scope of the embodiments. The following detailed description is therefore not to be taken in a limiting sense.
[0034] The various embodiments of the present provide a herbal formulation for prevention of coronavirus disease, SARS-COV-2 and also lung cancer comprising Cordyceps militaris (mushroom), Ganodurma lucidum (mushroom) and Lentinula edodes (Shiitake mushroom), Arthrospira platensis (spirulina) and Zingiber officinale (ginger).
[0035] According to an embodiment of the present invention, the Cordyceps militaris powder is present in an amount of 50%.
[0036] According to an embodiment of the present invention, the Ganoderma lucidum powder is present in an amount of 15%.
[0037] According to an embodiment of the present invention, the Shiitake mushroom powder is present in an amount of 15%.
[0038] According to an embodiment of the present invention, the Spirullina (Arthrospira platensis) powder is present in an amount of 10%.
[0039] According to an embodiment of the present invention, the ginger powder is present in an amount of 10%.
EXPERIMENTAL DETAILS
[0040] The Cordycep based formulation contains Cordyceps militaris (mushroom), Ganodurma lucidum (mushroom) and Lentinula edodes (Shiitake mushroom), Arthrospira platensis (spirulina) and Zingiber officinale (ginger). The formulation of the present study has significant antioxidant and anti-cancerous properties. The present study will justify the ability of Cordycepin as major compound having capability to restrict the viability of human lung cancer cell line cancerous cell line A549 (hLCCL).
[0041] Material and Method: The major study was conducted at Department of Biotechnology of AKS University, Satna in association with AIIMS Bhopal (M.P.) India.
[0042] Sample collection: The Cordyceps militaris, Ganodurma lucidum and Lentinula edodes samples were collected from (CEBRT) laboratory Department of Biotechnology AKS University Satna and other samples of Ginger and Spirulina were collected from local market Satna (M.P).
[0043] Processing of ingredients: All the ingredients were dried at 40°C for 24h. After that each ingredient was grinded and made into a fine powder. All the ingredients were mixed together in proper ratio and a final formulation was made. The prepared powder was sealed in translucent or polythene bag and used for further analysis of anticancer activity. Table 1 shows the composition of the herbal formulation according to the present invention.
Table 1: various ingredients in Cordyceps Herbal Formulation with the sample in closed vessel
S. No Ingredient Name Amount in
1. Cordyceps militaris powder 50%
2. Ganoderma lucidum powder 15%
3. Shiitake mushroom powder 15%
4. Spirullina (Arthrospira platensis) powder 10%
5. Ginger powder 10%

[0044] Column chromatography: Column chromatography was performed with the packing of glass columns. A glass column was taken and filled with a small amount of cotton with the help of a wood applicator stick. After that silica gel was prepared with 50 ml of hexane and poured into the column. Then, 1 g of silica gel and 5 g of sample were mixed and placed on top of the silica gel packed column. After sample loading, different solvents were used for fraction collections as mentioned in table 2.
Table 2: solvents used for column chromatography
S. No Fraction Solvent
1. F1 Hexane
2. F2 Toluene
3. F3 Dichloromethane
4. F4 Di ethyl ether
5. F5 Ethyl acetate
6. F6 Acetone
7. F7 Acetic acid
8. F8 Ethanol
9. F9 Methanol
10. F10 Water

[0045] HPTLC analysis: HPTLC analysis of fractions collected from column chromatography of C. militaris was performed using 10 cm size activated silica gel HPTLC plates (Merck, Darmstadt, Germany). For this purpose, fraction samples were loaded on activated silica gel 60 HPTLC plate for linear ascending development of the bands on plates to a distance of 80 mm. Methanol was used as optimized mobile phase in a TLC chamber. Fractions were run for 20 min at 25°C for under mobile phase to separate and observe the different compounds as bands. For this, the dried plates were photo-documented and scanned at 254 nm and 366 nm wavelengths under UV light using deuterium light source with the slit dimensions of 4.000.30 mm.
[0046] HPLC analysis: The cordycepin concentration was also determined by HPLC which consists of a UV detector and a reverse phase column (C-18 RP, 4.6 mm×150 mm, 5µm). Standard calibration curve was drawn by dissolving different concentrations of Cordycepin (10-50 µg/ml) in doubled distilled water. Samples were filtered through 0.45 µm membrane filter in order to remove suspended particles before performing assay. HPLC conditions for cordycepin detection were as follows: injection volume, 20 µl; mobile phase, water: methanol (70:30); flow rate, 0.5 ml min-1 with isocratic elution and detection wavelength 260 nm.
[0047] MTT Cell viability assay: MTT Cell viability assay on lung cancer cell in different concentration of cordyceps militaris formulation is shown in Table 3.
Table 3: MTT cell viability assay of cordyceps militaris formulation
Software Version 2.05.5

Experiment File Path: C:\Users\acer\Desktop\ritu\mush..mtt 08.05.2020.xpt
Protocol File Path: D:\Dr Neha Arya\Jaya\Protocols\MTT Assay_570_650.prt
Plate Number Plate 2
Date 08/05/2020
Time 8:23:48 PM
Reader Type: Eon
Reader Serial Number: 14021011
Reading Type Reader

Procedure Details
Plate Type 96 WELL PLATE
Shake Linear: 0:03 (MM:SS)
Frequency: 567 cpm (3 mm)
Read Absorbance Endpoint
Full Plate
Wavelengths: 570
Pathlength Correction: 570 / 650
Absorbance at 1 cm: 0.18
Read Speed: Normal, Delay: 100 msec, Measurements/Data Point: 8

[0048] Extraction method: For determining the anticancer activity of different fractions (cordyceps militaris formulation), MTT assay was used. For this extract was completely dried and were dissolved in DMSO. A549 (lung cancer cell line) were maintained in DMEM with10% FBS, 1% L-glutamine and1% penstrep. 5000 Cells were seeded in each well of 96 well plates, at 60-70% confluency cells were treated with different fraction at various concentrations. After 48 hrs of incubation, media was removed, washed with 1x PBS and 100ul of MTT (0.5 mg/ml) media was added in each well, after 4 hrs of incubation MTT was removed and formazon crystals were dissolved in 200 ul DMSO. Further plate was read by ELISA reader at 570nm for absorbance density value to determine cell viability.
[0049] Computational Determination of Inhibitory Compound Against SARS-CoV-2: Cordycepin (3'deoxyadenosine) is a purine nucleoside antimetabolite secluded from the organism Cordyceps militaris having various pharmacological and remedial implications including antineoplastic, cancer prevention agent, and mitigating exercises (Gu et al. 2007; Yue et al. 2013; Tuli et al. 2014) with a non-mutagenic and non-harmful properties (Aramwit et al. 2015). The current atomic docking study, endeavors to recognize possible restorative focuses for COVID-19 that are related with numerous trademarks, which can encourage normal disclosure of compelling treatments for COVID-19. Our sub-atomic docking examination of cordycepin ligand on certain pathogenicity related proteins of the SARS-COV-2 was evaluated to discover inhibitory activities on the collaborations between viral proteins and the host cell receptors consequently popular passage might be obstructed to have cell. The momentum study, endeavors to look through the tertiary structure of target proteins of SARS-CoV-2 like protease, glycoprotein, nucleocapsid phosphoprotein and non-underlying protein 16-10 (nsp10) complex along with examination of physicochemical properties, pharmacokinetic properties and ADME profiling for cordycepin and remdesivir to comprehend the remedial properties against focused receptor proteins of SARS-CoV-2 and host cell. Last end is drawn dependent on the forecast of different energies and affirmation-based hydrogen bonds associated with authoritative of ligand of cordycepin and remdesivir with focused receptor proteins of SARS-CoV-2.
[0050] Identification of targeted protein models of SARS-CoV-2: RCSB PDB (https://www.rcsb.org/) database was used to identify the 3D structures of target proteins of SARS-CoV-2 from the database. We have taken main protease (M pro) (PDB ID: 6LU7), membrane glycoprotein or RBD-Ace2 complex (PDB ID: 6M17), nucleocapsid phosphoprotein (PDB ID: 6VYO) anon-structural protein 16-10 complex or RNA methyltransferase or MTase (PDB ID: 6W4H), RDRp (PDB ID: 6NUR) and exonuclease Nsp10/14 complex (5NFY). Their detailed description is presented in result section and targeted proteins were further considered for molecular docking.
[0051] Molecular docking of cordycepin and remdesivir over target protein models of SARS-CoV-2: The ligand cordycepin and remdesivir (DB14761) pdb files obtained from Drug Bank database was pre-processed by using Discovery Studio visualizer version 3.5 (Dassault Systèmes BIOVIA) and AutoDock version 4.2 (Morris et al. 2009) to predict the Ligand- Protein interaction for structure based molecular docking. The 3D structure for docking analysis of all proteins was prepared by removing the water molecules, metals and ligands. Charges and hydrogen bonds were added to the molecule. Lamarckian Genetic Algorithm (GA) was set for 10 independent runs for each docking studies. Molecular Docking were performed by using Auto Dock 4.2 (Morris et al., 2009) and parameters were set for image construction as Temperature (T) 298.15 K and the docked ligand protein complexes were visualized on Pymol version 2.1.0. The grid generation and docking were subsequently executed through AutoGrid 4.2 and AutoDock 4.2, respectively. The binding energies and affinities for ligands were obtained from the log files of the docks generated by AutoDock. Drug targets were screened to determine the binding affinity of ligands with selected proteins from estimated free energy of binding at 298.15 K. The image construction was further visualized by employing PyMol visualizing tool. The images were constructed by taking ligands into green/red stick confirmations showing interaction with targeted proteins. Separate image was constructed where viral target proteins of SARS-CoV- 2 were present in surface confirmation in which docked ligand (cordycepin and remdesivir) was visualized in the protein pockets.
[0052] Molecular Dynamics Simulation: The MD simulation was performed to obtain the free energy of the binding of cordycepin with targeted proteins of SARS-CoV-2. Due to its modular design and because it does not need calculations on a training set. They have been successfully used to replicate and rationalize experimental findings and to optimize virtual screening and docking outcomes (Genheden & Ryde, 2015). Molecular Dynamics AMBER 20 force field package (Salomon-Ferrer et al. 2012) simulation was run to estimate the free energy of the docked molecule. Minimization and equalization steps in MM/PBSA were performed at 10ns for 1,50,000 steps for all six docked molecules at Temperature 300K and total energy (Etot) was estimated. Further, on the basis of best total energy, NAMD simulation was performed to estimate the rmsd (root mean square deviation) values at 10ns for 1,000000 runs on 1000 minimization steps on temperature of 310K. The system configuration for programming MD simulation was of Intel(R) Core (TM) i7-8700 CPU @ 3.20GHz of 64 bits with 16GB RAM.
RESULT AND DISCUSSION
[0053] Column chromatography analysis results:
[0054] HPTLC Fingerprinting: FIG. 1A shows TLC profiles of test solutions of Cordyceps militaris at 366nm, named as plate 1.1, wherein F1-F9 are test solutions, F10 is unknown sample and F11-F12 are standard (std) at 2µl and std (6µl), according to an embodiment of the present invention.
[0055] FIG. 1B shows TLC profiles of test solutions of Cordyceps militaris at 366 nm after derivatization with 5% methanolic H2SO4, named as plate 1.2, according to an embodiment of the present invention.
[0056] FIG. 1C shows TLC profiles test solutions of Cordyceps militaris named as plate 1.1, at day light after derivatization with 5% methanolic H2SO4, according to an embodiment of the present invention.
[0057] HPLC Analysis: FIG. 2A The HPLC chromatograms of standard cordycepin present in fig 2A
[0058] and FIG. 2B show the HPLC chromatograms of cordycepin present in Cordyceps militaris fraction compared with the standard, according to an embodiment of the present invention
[0059] Table 4 below shows the validation report for obtaining Cordycepin from Cordyceps militaris. The result was obtained from National Botanical Research Institute, Lucknow, India.
Table 4: Showing the validation report for obtaining Cordycepin from Cordyceps militaris
S. No. Name of the sample Condycepin content by HPLC
1. Extract of Cordyceps militaris 1.44 µg/mg

[0060] Viable cells reduce the tetrazolium salt to formazon resulting in blue color whereas dead cells will not. Fraction of viable cells were calculated using formula [% Viability = (Abs test / abs control) x 100]. Anticancer property of different components of mushroom was determined by MTT assay. For this, cultured A549 were treated with different concentration of each fraction. Table 4.1 shows the last 5 fraction concentration and effects.
Table 4.1 showing the results for fraction 5, 6, 7, 8, 9 and 10
Sample Control 0.25 ul 0.5 ul 1ul 2ul
fraction 5 1.391 0.0685 0.079 0.067 0.0705
fraction 6 1.391 0.0775 0.0645 0.068 0.068
fraction 7 1.391 1.141 0.4105 0.0725 0.063
fraction 8 1.391 1.072 1.0075 0.2665 0.069
fraction 9 1.391 0.566 0.521 0.1195 0.0685
fraction 10 1.391 0.071 0.0745 0.07 0.0685

[0061] As shown in figure ...fraction 5, 6, 7, 8, 9 and 10 were able to kill cells indicating anticancer activity further FIG. 3 shows the graphical representation of cell viability assay with different fraction of cordyceps formulation extract, according to an embodiment herein. With respect to FIG. 3, it was seen that when concentration is increased cell viability is decreased.
[0062] FIG. 4 shows the graphical representation of cell viability assay with different concentration of DMSO, according to an embodiment of the present invention. Table 5 shows the different concentration and their effects with DMSO treatment.

Table 5: different concentration and their effects with DMSO treatment.
control 0.25 ul 0.5 ul 1ul 2 ul 3ul 4ul
DMSO 1.391 1.119 1.31 1.435 0.831 0.2925 0.205
[0063] 5000 Cells were seeded in each well of 96 well plates, at 60-70% confluence cells were treated with different fraction at various concentrations. After 48 hrs of incubation, media was removed, washed with 1x PBS and 100ul of MTT (0.5 mg/ml) media was added in each well. after 4 hrs of incubation MTT was removed and formazon crystals were dissolved in 200 ul DMSO. Further plate was read by ELISA reader at 570nm for absorbance density value to determine cell viability. Viable cells reduce the tetrazolium salt to formazon resulting in blue colour (Control) whereas dead cancerous cells showing colourless appearance.
[0064] The extract was completely dried and were dissolved in DMSO. A549 (lung cancer cell line) were maintained in DMEM with10%FBS, 1% L-glutamine and1% penstrep. 5000 Cells were seeded in each well of 96 well plates; at 60-70% confluency cells were treated with different fraction at various concentrations. Further plate was read by ELISA reader at 570nm for absorbance density value to determine cell viability. FIG. 5 shows the cell viability with DMSO, according to an embodiment of the present invention. Viable cells reduce the tetrazolium salt to formazon resulting in blue colour whereas dead cells will not. Fraction of viable cells was calculated using formula.
[0065] And next study of formulation in computational approaches provides molecular interactions of viral proteins of SARS- CoV-2 with cordycepin as an inhibitor. The computational approach is drawn to understand the binding of Cordycepin at specific sites to restrict SARS-CoV-2 by molecular docking and molecular dynamics studies, which reflects that Cordycepin an nucleoside analogue can also respond as antiviral.
[0066] Identification of targeted protein of SARS-CoV-2 from RCSB PDB: Three dimensional structures of six target proteins of SARS-CoV-2 were retrieved from protein databank RCSB PDB (https://www.rcsb.org/) for molecular docking analysis. Targeted proteins were chosen for this study as they play crucial role in viral assembly and entry into host cell. FIG. 6 shows the images of all six targeted protein structures, according to an embodiment of the present invention. X ray diffraction and electron microscopy-based structures are being considered for identifying the atomic resolution of target proteins of SARS-CoV-2 as they are highly refined and accurate. The identified proteins viz. 6LU7, 6M17, 6VYO, 6W4H, 6NUR and 5NFYexhibited excel entre solution with refined method of structure formation. The resolution defines the quality of data obtained by observing the scattering and diffraction pattern of X-ray of proteins.
[0067] Prediction of physicochemical descriptors of Cordycepin: Swiss ADME tool was used to predict the physicochemical descriptors. The physicochemical descriptors of cordycepin include physicochemical properties, pharmacokinetic properties, drug like score and ADME profiling. Predicted results are presented in Table 6.
[0068] It was found that cordycepin do not inhibit cytochrome 450 – 1A2, 2C19, 2C9, 2D6, 3A4. The predicted physicochemical descriptors provided information to categorize cordycepin as suitable therapeutic candidate. The ideal drug like molecule should have logarithms of partition coefficient (LogP) between -0.4 and 5.6 and the molar refractivity should be in the range of 40-130. This is based on relationship between the volume and molecular weight of the molecule. The predicted results favour the suitability of cordycepin as drug. The higher rate of absorption of cordycepin in GI (Gastro intestine) makes it a better option for oral administration (Table 6). Oral tract and GI are considered as the most favourable site for the routing of drug as per norms of drug development in medicinal chemistry. The solubility and bioavailability of cordycepin is as high as logS -1.25 and 0.55, respectively and the drug likeness score is 0.49 (Table 6). Overall, the predicted results favour the suitability of cordycepin as drug.
Table 6: Experimental data taken from RCSB PDB database of target proteins of SARS-CoV-2
PDB ID Protein Name Resolution Method Total Structure Weight (kDa) Reference
6LU7 Main Protease 2.16 Å X ray diffraction 34.51 Liu et al. 2020
6M17 Membrane Glycoprotein (RBD/ACE2 complex) 2.90 Å Electron microscopy 394.46 Yan et al. 2020
6VYO Nucleocapsid Phosphoprotein 1.70 Å X ray diffraction 57.67 Chang et al.2020

6W4H Nsp16-10 complex (RNA Methyltransferase or MTase) 1.80 Å X ray diffraction 49.79
Rosas-Lemus et al. 2020
6NUR RNA Dependent RNA polymerase (RDRp) 3.10 Å Electron microscopy 162.52 Kirchdoerferand Ward 2019
5NFY Exonuclease Nsp10/14 Complex 3.38 Å X ray diffraction 304.66 Ferron et al. 2018

[0069] The potential targets of proteins are Main protease, membrane glycoprotein (RBD-ACE2 complex), nucleocapsid phosphoprotein, nsp16-10 complex (RNA methyltransferase or MTase), RDRp and exonuclease nsp10/14 complex. As compare to remdesivir, cordycepin had shown effective docking on AutoDock4.0 at 10 ns. Further from MD Simulation we were able to recognize 6VYO and 6M17 as potentially inhibited protein of SARS-CoV-2 by Cordycepin as it was validated by using NAMD-VMD simulation procedure where the RMSD values obtained for time 10ns at 1,000,000 steps confirming the effectiveness of cordycepin against Exonuclease Nsp10/14 complex and Membrane glycoprotein (RBD/ACE2 complex). Overall, cordycepin can stand as a good therapeutic drug compound against viral proteins of SARS-CoV-2. The computational approach presented here is robust and powerful, which can be applied to generate potential leads for other therapeutic areas, as well.
[0070] FIG. 6 shows the Target Protein Structures of SARS-CoV-2 from RCSB PDB database, according to an embodiment of the present invention. With respect to FIG. 6, Main Protease (PDB ID: 6LU7) having resolution of 2.16Å, the crystal structure was formed by X ray diffraction having total structural weight of 34.51 kDa (a), Membrane Glycoprotein (PDB ID: 6M17) having resolution of 2.90Å, the crystal structure was formed by electron microscopy having total structural weight of 394.46 kDa (b), Nucleocapsid Phosphoprotein (PDB ID: 6VYO) having resolution of 1.70Å, the crystal structure was formed by X ray diffraction having total structural weight of 57.67 kDa (c), Non-structural protein 16-10 complex (PDB ID: 6W4H) having resolution of 1.80Å, the crystal structure was formed by X ray diffraction having total structural weight of 49.79 kDa (d), RNA Dependent RNA polymerase (RDRp) having resolution of 3.10Å, the crystal structure was formed by X ray diffraction having total structural weight of 162.52 kDa(e), Exonuclease Nsp10/14 complex having resolution of 3.38Å, the crystal structure was formed by X ray diffraction having total structural weight of 304.66 kDa (f).
[0071] Ligand-Protein interaction by molecular docking using AutoDock 4.2: Docking over targeted proteins (main protease, membrane glycoprotein, nucleocapsid protein and nsp16-10 complex) of cordycepin and remdesivir against SARS-CoV-2 was made with the help of AutoDock 4.2 (Morris et al. 2009). Various modes of ligand protein interactions were generated with binding and total internal energy after successful docking of legends over targeted proteins. It was evaluated from each confirmation. The least binding affinity, total internal energy, contacting residues in docked position and RMSD values were evaluated for both the ligands showing interaction with targeted proteins of SARS-CoV-2. All the docked structures were visualized by using Pymol visualization tool. On visualizing the docked results, it was observed that least energy confirmations of both ligand molecules were docked with targeted protein. The ligands are shown as green and red stick models whereas the protein was shown as lines and surface. The interactions were shown by green dash lines referring to the hydrogen bonding interactions between ligands and proteins. The binding pocket of proteins with the ligands was shown in red and green sticks in order to achieve the visibility.
[0072] The visualization of docked cordycepin with targeted proteins of SARS-CoV-2 is presented. Results presented in further suggest considering cordycepin as potential candidate against SARS-CoV-2 because of being traditional medicine with natural and effective properties. Cordycepin is showing effective binding -3.5 kcal/mol with MPro (main protease) of SARS-COV-2 forming two hydrogen bonds at position Leu287 and Thr199 while showing less interaction with remdesivir of -0.52 kcal/mol forming 1 hydrogen bond at position Tyr154. Similarly, for membrane glycoprotein (RBD/ACE2 complex), the cordycepin bind at position Leu675 and Asn 674 with 2 hydrogen bonds but remdesivir was capable to bind at Ser709 position with 1 hydrogen bond/ binding energy -1.92 kcal/mol. The nucleocapsid phosphoprotein 6VYO displayed binding energy -5.06 kcal/mol with cordycepin; the highest among all with 3 hydrogen bond and interacted at position Phe66, Gly69 and Ala134 (Table 7).
Table 7: Physicochemical descriptors, lipophilicity, solubility, pharmacokinetics and drug likeness estimation for Cordycepin by SWISS ADME tool
Physicochemical Descriptors of Cordycepin
Molecular Weight 251.24 gm/mol
Formula C10H13N5O3
Number of Heavy Atoms 18
Number of Hydrogen Bond Acceptors 6
Number of Hydrogen Bond Donor 3
Number of Rotatable Bonds 2
Molar Refractivity 61.51
Lipophilicity
Log Po/w (Octanol/Water Partition Coefficient) 1.30
Water Solubility
Log S -1.25
Class Very Soluble
Pharmacokinetics
GI Absorption (Gastro-Intestinal) High
Blood Brain Barrier permeant No
P-gp Substrate (Permeability Glycoprotein) No
CYP1A2 Inhibitor (Cytochrome P450 1A2) No
CYP2C19 Inhibitor (Cytochrome P450 2C19) No
CYP2C9 Inhibitor (Cytochrome P450 2C9) No
CYP2D6 Inhibitor (Cytochrome P450 2D6) No
CYP3A4 Inhibitor (Cytochrome P450 3A4) No
Log Kp (Skin permeation) -8.27 cm/s
Drug Likeness
Lipinski Yes, 0 Violations
Veber Yes
Bioavailability Score 0.55
Drug Like Score 0.49
[0073] In contrast, remdesivir interacted 6VYO at Thr166 with 1 hydrogen bond/binding energy -2.09 kcal/mol. Nsp 16-10 complex RNA methyltransferase or MTase of SARS-CoV-2 displayed to interact with cordycepin with 2 hydrogen bonds at position Asn4367 and Lys4281. The binding energy for 6W4H was -3.77 kcal/mol when interacted with cordycepin. On the other hand, it was -3.48 kcal/mol with 1 hydrogen bond when interacted with remdesivir on position Ile4308. The binding energy while interacting with cordycepin for RDRp 6NUR was -3.33 kcal/mol having 2 hydrogen bonds at position Asp390 and Ser397. Conversely, 6NUR when interacted with remdesivir displayed -1.15 kcal/mol of binding energy beside 1 hydrogen bond at Leu41. The binding energy was -2.88 kcal/mol beside 2 hydrogen bonds at Thr39 and Asn40 for 5NFY exonuclease Nsp10/14 complex when interacted with cordycepin. On the contrary, remdesivir displayed -2.25 kcal/mol binding energy at Gln22, Met49 and Asn129 (Table 8). The results corroborate significantly beside the visualization data of docked cordycepin and remdesivir, respectively which is in close proximity to the targeted proteins of SARS-CoV-2.
Table 8: Molecular docking of Cordycepin on target proteins of SARS-CoV-2 to estimate Binding energy, Internal energy, Contacting Residues in Docked Position and Number of Hydrogen Bond formation from AutoDock 4.2.
Molecular docking of Cordycepin on target proteins of SARS-CoV-2
Target Proteins PDB ID Ligand Binding Energy (kcal/mol) Total Internal Energy (kcal/mol) Contacting Residues in Docked Position No. of H- bond forms RMSD
Main Protease 6LU7 Cordycepin -3.5 -1.91 Leu287
Thr199 2 60.24
Membrane Glycoprotein (RBD/ACE2 complex) 6M17 Cordycepin -2.95 -2.09 Leu675 Asn674 2 307.83
Nucleocapsid Phosphoprotein 6VYO Cordycepin -5.06 -2.0 Phe66 Gly69 Ala134 3 23.26
Nsp16-10 complex ((RNA Methyltransferase or MTase)) 6W4H Cordycepin -3.77 -1.43 Asn4367 Lys4281 2 60.57
RNA Dependent RNA polymerase (RDRp) 6NUR Cordycepin -3.33 -2.02 Asp390 Ser397 2 277.88
Exonuclease Nsp10/14 Complex 5NFY Cordycepin -2.88 -2.12 Thr39 Asn 40 2 139.76

Table 9: Molecular docking of Remdesivir on target proteins of SARS-CoV-2 to estimate Binding energy, Internal energy, Contacting Residues in Docked Position and Number of Hydrogen Bond formation from AutoDock 4.2.
Molecular docking of Remdesivir on target proteins of SARS-CoV-2
Protein Models PDB ID Ligand Binding Energy (kcal/mol) Total Internal Energy (kcal/mol) Contacting Residues in Docked Position No. of H- bond forms RMSD
Main Protease 6LU7 Remdesivir -0.52 -4.05 Tyr154 1 59.34
Membrane Glycoprotein (RBD/ACE2 complex) 6M17 Remdesivir -1.92 -2.27 Ser709 1 271.45
Nucleocapsid Phosphoprotein 6VYO Remdesivir -2.09 -2.73 Thr166 1 41.07
Nsp16-10 Complex (RNA methyltransferase or MTase) 6W4H Remdesivir -3.48 -3.04 Ile4308 1 66.85
RNA Dependent RNA polymerase (RDRp) 6NUR Remdesivir -1.15 -3.29 Leu41 1 259.62
Exonuclease Nsp10/14 Complex 5NFY Remdesivir -2.25 -2.8 Gln22 Met49 Asn129 3 148.52

[0074] FIG. 7 showing Molecular Docking of Cordycepin over targeted proteins of SARS-CoV-2, according to an embodiment of the present invention. With respect to FIG. 7, ligand cordycepin (green sticks) interacting with amino acid residues of main protease (PDB ID: 6LU7) and docked cordycepin (green sticks) visualized in pocket of (multicolour) surface structure of main protease (a), ligand cordycepin (green sticks) interacting with amino acid residues of membrane glycoprotein (PDB ID: 6M17) and docked cordycepin (green sticks) visualized in pocket of (multicolour) surface structure of membrane glycoprotein (b), ligand cordycepin (green sticks) interacting with amino acid residues of nucleocapsid phosphoprotein (PDB ID: 6VYO) and docked cordycepin (green sticks) visualized in pocket of (multicolour) surface structure of nucleocapsid phosphoprotein (c), ligand cordycepin (green sticks) interacting with amino acid residues of nsp16-10 complex (PDB ID: 6W4H) and docked cordycepin (green sticks) visualized in pocket of (multicolour) surface structure of nsp16-10 complex (d), ligand cordycepin (green sticks) interacting with amino acid residues of RNA Dependent RNA polymerase (PDB ID: 6NUR) and docked cordycepin (green sticks) visualized in pocket of (multicolour) surface structure of RNA Dependent RNA polymerase (e), ligand cordycepin (green sticks) interacting with amino acid residues of exonuclease nsp10/14 complex (PDB ID: 5NFY) and docked cordycepin (green sticks) visualized in pocket of (multicolour) surface structure of exonuclease nsp10/14 complex (f).
[0075] FIG. 8 showing Molecular Docking of Remdesivir over targeted proteins of SARS-CoV-2, according to an embodiment of the present invention. With respect to FIG. 8, ligand remdesivir (green sticks) interacting with amino acid residues of main protease (PDB ID: 6LU7) and docked remdesivir (green sticks) visualized in pocket of (multicolour) surface structure of main protease (a), ligand remdesivir (green sticks) interacting with amino acid residues of membrane glycoprotein (PDB ID: 6M17) and docked remdesivir (red sticks) visualized in pocket of (multicolour) surface structure of membrane glycoprotein (b), ligand remdesivir (green sticks) interacting with amino acid residues of nucleocapsid phosphoprotein (PDB ID: 6VYO) and docked remdesivir (red sticks) visualized in pocket of (multicolour) surface structure of nucleocapsid phosphoprotein (c), ligand remdesivir (green sticks) interacting with amino acid residues of nsp16-10 complex (PDB ID: 6W4H) and docked remdesivir (red sticks) visualized in pocket of (multicolour) surface structure of nsp16-10 complex (d), ligand remdesivir (green sticks) interacting with amino acid residues of RNA Dependent RNA polymerase (PDB ID: 6NUR) and docked remdesivir (green sticks) visualized in pocket of (multicolour) surface structure of RNA Dependent RNA polymerase (e), ligand remdesivir (green sticks) interacting with amino acid residues of exonuclease nsp10/14 complex (PDB ID: 5NFY) and docked remdesivir (green sticks) visualized in pocket of (multicolour) surface structure of exonuclease nsp10/14 complex (f).
[0076] Molecular Dynamics Simulation: MD simulation study was used to obtain free energy from the binding of cordycepin to targeted SARS-CoV-2 proteins and the results obtained in the form of average total energy (Etot) from the resulting file (prod3.out) at 10ns for 1,50,000 steps at temperature 300K. The estimated total energy for all six docked molecules were performed present in Table 9. but, in the study, 6nur was not able to executed due to failure in MD simulation processing after equalization, the output file was failed to generate the RMSD data. Further, RMSD values for 6VYO_Cordycepin and 6M17_Cordycepin were obtained by the graph plotted for RMSD against Time at 10ns for 1,000,000 steps in which it was found that the RMSD values for6VYO_Cordycepin was found to be in range from 0.086Å to 1.69Å and standard deviation was obtained as 0.261. Further, with similar conditions of nanoscale molecular dynamics, RMSD values for 6M17_Cordycepin was found to be in range from 0.025Å to 2.705Å and standard deviation was obtained as 0.524.
Discussion
[0077] Lung cancer remains the main source of malignant growth mortality in people in India. Furthermore, around the world. About 90% of lung malignant growth cases are brought about by smoking and the utilization of tobacco items. Be that as it may, different factors, for example, radon gas, asbestos, air contamination, and persistent diseases can add to lung carcinogenesis. What is more, different acquired constantly systems of weakness to lung malignant growth has been proposed. Lung malignant growth is separated into two wide histological classes, which develop and spread in an unexpected way: small cell lung carcinomas (SCLC) and non-small cell lung carcinomas (NSCLC). Therapy choices for lung malignancy incorporate a medical procedure, radiation treatment, chemotherapy, and therapeutic treatment. Helpful modalities suggestions rely upon a few variables, including the sort and phase of disease. In spite of the enhancements in conclusion and treatment made during the previous 25 years, the guess for patients with lung malignancy is as yet unsuitable. The reactions to current standard treatments are poor aside from the most confined malignancies. Be that as it may, a superior comprehension of the biology appropriate to these difficult malignancies may prompt the improvement of more effective and maybe more explicit medications. Numerous studies have shown that Cordycepin as one valuable compound can inhibit many malignant tumors through different pathways. Since Cordycepin-induced death of cancer cells are performed via multi-target pathways, it is difficult to some extent for cancer cells to develop drug resistance. Moreover, another advantage of Cordycepin is that the small side effect is shown when inhibiting the growth and progression of cancer cells. Therefore, Cordycepin may be considered as one wonderful drug candidate for lung cancer treatment. During this study, we assessed the anticancer property of C. militaris by MTT assay to check the cancer cell line viability. As mentioned in results of MTT assay fractions of C. militaris having cordycepin as bioactive compounds were capable to restrict the cancer cell line indicating anticancer activity.
[0078] Synthetic medicines (allopathic) after the Second World War completely overshadow the application of herbal folk medicine but during the last 35 years herbal legacy has revived as a paramount contender of synthetic products which are used in different formulations. Medicinal mushroom Cordyceps sp. has been described to have a number of pharmacological effects including immuno-modulating, hepatic, renal, cardiovascular, respiratory, nervous, sexual, immunological systems, anti-microbial activities besides several lifestyle ailments. The chemical synthesis of cordycepin is completed mainly through the replacement of the OH group at the 3'-position in the ribofuranosyl moiety with H, generating a deoxy analogue of adenosine (Huang et al. 2018). The computational approaches provide molecular interactions of viral proteins of SARS-CoV-2 with cordycepin as an inhibitor. COVID-19 is a global threat with no acclaimed drug against it. Comparative studies on mode of mechanism of action of cordycepin with remdesivir were made through computational approaches and found that cordycepin is a better substitute to remdesivir being a natural product with broad spectrum in bioavailability, absorption and solubility capacity in hydrated form.
[0079] Main protease, nucleocapsid glycoprotein, membrane glycoprotein, and nsp 16-10 complex protein are key proteins that play significant role in the pathogenesis of virus during entry and mechanism into the host cells, thus this makes it a potential target for ligand interaction. The binding affinity of ligand cordycepin was more effective compared to remdesivir as inhibitor against viral proteins of SARS-CoV-2 in our study.
[0080] Clinical trial data of remdesivir, a synthetic potential anti-viral drug, revealed it an adenosine triphosphate analogue as a possible treatment for Ebola (Warren et al. 2016). Sheahan et al. (2017) subsequently reported its action against the coronavirus family of viruses. Remdesivir is also being researched as a possible action to SARS-CoV-2, the coronavirus responsible for COVID-19 (de Wit et al., 2020). Thus, comparative investigations gave us an opportunity to detect the binding potentiality of the two legend and established the fact that cordycepin had much better ability than remdesivir to bind on various viral proteins of SARS-CoV-2. Our studies suggested that the main protease (6LU7) at Leu287 and Thr199 displayed interaction with cordycepin with 2 H bonds making it a therapeutic ligand to protect host’s antibody response. Membrane glycoprotein of novel coronavirus participate actively in its life cycle from assembly to pathogenesis. The interaction with cordycepin at Leu675, Asn674 position provides 2 H bond with a possibility to restrict its activity in host cells. Our results further suggest that ligand interact at Phe66, Gly69 and Ala134of nucleocapsid phosphoprotein with 3 H bonds. The nsp16-10 complex (RNA methyltransferase or MTase) is yet another target exhibiting binding over Asn4367 and Lys4281. RDRp had shown interaction at Asp390 and Ser397 and Exonuclease nsp10/14 complex of SARS-CoV at Thr39 and Asn40; this makes it a unique therapeutic candidate displaying interactions with SARS-CoV-2 viral proteins. On the other hand, docked remdesivir against the identified targeted proteins was less effective. Clinical study reported that remdesivir acts as a viral protein inhibitor (Holshue et al. 2020 and Wang et al. 2020). Our docked results on remdesivir suggested that main protease of SARS-COV-2 had interacted at Tyr154, at Thr166 with membrane protein, at Ser709 with membrane glycoprotein, at Thr166 with nucleocapsid phosphoprotein at Thr166 with nsp16-10 complex had interacted with remdesivir at Ile4308 with 1 Hydrogen bond. Docking results suggested that ligand cordycepin got docked effectively onto the viral proteins (Main protease, membrane glycoprotein, nucleocapsid phosphoprotein and nsp16-10 complex). Based on the lowest docked energy and hydrogen bond formation, cordycepin can stand as a good therapeutic drug compound against viral proteins of SARS-CoV-2. Molecular dynamics (MD) simulations, using empirical force fields such as the "CHARMM force field" that play an important role in the theoretical study of biomolecules, compute the atomic trajectories by solving the motion equations numerically. By this process, the time dependent behaviour of a molecular system is determined. We used certain software such as MM/PBSA and NAMD-VMD to perform a molecular dynamics simulation (Sasumana et al. 2018). Through this, we solve biomolecules, simulate molecules to calculate the total energy for which we minimize energy, and after the simulation, we evaluate the trajectory, RMSD values, and molecule energies. Total energy estimate (Etot) in Kcal/mol obtained as performed at 10ns for 1,50,000 steps at 300 K temperature. The average total energy (Etot) was determined to be higher than other docked molecules for 6VYO_Cordycepin and 6M17_Cordycepin and on these grounds he graph plotted for RMSD against Time at 10ns showed that the RMSD values for 6VYO_Cordycepin ranged overall from 0.06Å to 1.69Å and the standard deviation was obtained as 0.261. Furthermore, RMSD values for 6M17_Cordycepin were found to be in the range of 0.025Å to 2.705Å with similar nanoscale molecular dynamic conditions and standard deviation was obtained as 0.524. Thus, total energy and RMSD values were in favour to prove that cordycepin has potential to bind effectively with viral proteins. Exonuclease Nsp10/14 complex and Membrane Glycoprotein (RBD/ACE2 complex) of SARS-CoV-2 were showing highest binding affinity with cordycepin as per the validation by simulation studies. Exonuclease Nsp10/14 complex is known for viral RNA proofreading and the most abundant protein in the viral envelope is the membrane glycoprotein. Despite the relatively poor sequence homology of amino acids just under 30%, the overall structure of coronavirus glycoprotein is highly conserved and these functional annotations of both viral proteins becomes remarkable bench point to explain that cordycepin is effectively binding with both proteins and positioning of amino acids involve in binding with cordycepin at Leu675 Asn674 of 6M17 and Phe66 Gly69 Ala134 of 6VYOwith perfect binding energy and RMSD values were estimated in this paper.
[0081] Conclusion
[0082] Cordyceps based formulation in the present study, the effects of cordycepin on the human lung cancer cell line were investigated via MTT assay. The results showed that Cordyceps based formulation could inhibit the cell proliferation. These results implied that the inhibitory role of Cordyceps based formulation on A549 may be exerted through down-regulation of the phosphorylation level. It was firmly believed that cordycepin exerts its anticancer activity via multiple mechanisms. Though this study just revealed the tip of the iceberg, the inhibition effect of cordycepin on cancer could provide useful information to modify the chemical structure of cordycepin to seek more potent anticancer agents.
[0083] Cordycepin is an important pharmacologically active non-toxic, non-mutagenic bioactive molecule produced by medicinal mushroom Cordyceps sp. The current study has attempted to predict the binding interactions of ligand cordycepin and remdesivir with target proteins of SARS-CoV-2. The identification of protein(s) in this study are based on relative importance of their targeted site for ligand interaction in establishing pathogenesis in the host cells during entry and multiplication for further docking analysis and ligand interaction. The potential targets of proteins are Main protease, membrane glycoprotein (RBD-ACE2 complex), nucleocapsid phosphoprotein, nsp16-10 complex (RNA methyltransferase or MTase), RDRp and exonuclease nsp10/14 complex. The overall total energy estimated from MM/PBSA was able to recognize 6VYO and 6M17 as potentially inhibited protein of SARS-CoV-2 by Cordycepin and it was validated by using NAMD-VMD simulation procedure where the RMSD values obtained for time 10ns at 1,000,000 steps confirming the effectiveness of cordycepin against Exonuclease Nsp10/14 complex and Membrane glycoprotein (RBD/ACE2 complex). But, in future more confined and detailed study can make this a hope. Overall, cordycepin can stand as a good therapeutic drug compound against viral proteins of SARS-CoV-2 when compared with remdesivir. The computational approach presented here is robust and powerful, which can be applied to generate potential leads for other therapeutic areas, as well.

G) ADVANTAGES OF INVENTION
[0084] The present invention provides a novel herbal formulation for prevention of lung cancer and diseases of corona virus. The formulation was examined in wet and dry laboratory procedures where it was found to possess anti-viable property for human lung cancer cell line and against SARS-CoV-2 viral proteins. The herbal component cordycepin is found to possess the ability to restrict the spread of viral proteins of SARS-CoV-2.
[0085] It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the claims presented in the complete specification or non-provisional application.