International Journal of Antimicrobial Agents

Molecular docking studies of bioactive constituents of Long pepper, Ginger, Clove and
Black pepper to target the Human Cathepsin-L protease: as a natural therapeutic
strategy against SARS-Cov-2
--Manuscript Draft--

Manuscript Number:

Article Type: VSI: COVID-19: Therapeutic and Prevention

Keywords: Molecular docking; SARS-CoV-2; Human cathepsin L protease; natural bioactive

components

Corresponding Author: Kaushal Kishor Sharma, Ph.D.

Jiwaji University
bahorapur, Madhya Pradesh INDIA

First Author: Brijendra Singh, M.Sc.

Order of Authors: Brijendra Singh, M.Sc.

D D Agarwal, PhD

Kaushal Kishor Sharma, Ph.D.

Manuscript Region of Origin: INDIA

Abstract: Human cathepsin-L protease present in the endosomal compartment of the target host
cell make a cleavage of S protein of SARS-Cov2 virus and activate the membrane
fusion which mediate the entry of virus in to the host cell. Thus, it suggests the
cathepsin-L protease is critical for entry of SARS-Cov2. Currently, chemically
synthesized cathepsin-L inhibitor are present, but, the consumption of chemical
synthesized drugs are also an alarming stage due to its side effects, illness and age
reduction. Therefore, in this study, natural bioactive constituents of Long pepper,
Ginger, clove and black pepper that has been widely known for antiviral effect and
other medicinal properties were used for molecular docking against the Human
cathepsin-L receptor (PBD ID 2XU1). Molecular docking (using a software, Autodock
4.2) were performed on Bioactive constituents of Long pepper, Ginger, Clove and
Black pepper were reported in NCBI-Pubchem database are docked against the
human cathepsin-L protease and elucidate the binding energies, visualization and
analysis of interacting residue and non-interacting residue (using Discovery studio) at
the docking site of cathepsin-L protease and compared the docking analysis of these
bioactive constiuents with pre-clinical cathepsin-L inhibitor (Chemicalname- N -[(1,1-
Dimethylethoxy)carbonyl]-L-tryptophan-2-[[[2-[(2 ethylphenyl)amino]-2-
oxoethyl]thio]carbonyl]hydrazide; Pub Chem CID: 16725315). The pharmacokinetic
properties and toxicity evaluation were calculated by Datawarrior software and Osiris
Molecular Property explorer software respectively. Results displayed that Piperolactam
A (CID 3081016) and Kaempferol (CID 5280863) were found to be a more acceptable
natural therapeutic compounds among other selected bioactive constituents with
appropriate binding energy, pharmacokinetic property with no toxicity.

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Cover Letter

Cover letter

Dear Editor

Please consider our manuscript entitled “Molecular docking studies of bioactive constituents
of Long pepper, Ginger, Clove and Black pepper to target the Human Cathepsin L protease:
as a natural therapeutic strategy against SARS-Cov-2 by (Brijendra Singh, D.D Agarwal
Kaushal K. Sharma) submitted for special issue VSI: COVID-19: Therapeutic and
Prevention. This manuscript was not earlier submitted for publication to other journals. It is
not either under peer review in another journal. In current scenario world is facing the
pandemic of COVID 19. The pandemic is affected many countries and even the total loss in
economy and human life still not in fixed figures. This is the responsibility of scientific
community to save world from the pandemic and find the suitable cure. The vaccine and other
organic compound are under trail and they will take much time to formulate as drug. The
plants has great therapeutic value especially when it comes to spices they have significant
therapeutic value. Here we find out how the bioactive constituents can be used to treat Covid
19. I hope that the paper confirms to the rigorous standards of your journal, and I look
forward to hearing your positive comments and decision concerning the publication of our
paper.

Best regards
Corresponding author
Dr. Kaushal Kishor Sharma
Reserach Associate
Bioinformatics Infrastructure Facility (DBT)
A.P.J.Abdul Kalam Central Instrumentation Facility
Jiwaji University , Gwalior M.P , India
E.Mail : send2kaushal@gmail.com
Author contribution

CONFLICT OF INTEREST

The authors do not have any actual or potential conflict of interests including
financial, personal or academic.
Author contribution

Credit author statement

Brijendra Singh: study design, conduct the molecular docking and visualization, data
interpretation, D.D Agarwal Conceptualization, Data interpretation ,Kaushal K. Sharma:
Review and edit the manuscript

Corresponding Author:

Dr Kaushal K. Sharma
Research Associate
Bioinformatics Infrastructure Facility
DBT (Dr. APJ Abdul Kalam Central
Instrumentation Facility), Jiwaji
University Gwalior (M.P) 474001, India
Email id:send2kaushal@gmail.com
Reviewer suggestions

Reviewer Suggestion

Reviewer can be assigned by the concern of editor

Graphical Abstract (for review)
Highlights

 World health organization (WHO) has declared the Covid-19 outbreak, a global epidemic

that led to high morbidity and mortality across the world.

 Human cathepsin-L and S protein of SARS-Cov2 virus mediate the entry of virus in to the

host cell.

 Natural antiviral bioactive constituents of Long pepper, Ginger, clove and black pepper

that has been used for molecular docking against the Human cathepsin-L receptor.

 Long pepper, Ginger, clove and black pepper bioactive constituents is a promising

inhibitor of the human cathepsin L protease in the lysosome and restrict the entry of the

SARS-Cov-2 virus.
Manuscript Click here to view linked References

1 Title
2
3 Molecular docking studies of bioactive constituents of Long pepper, Ginger, Clove and
4 Black pepper to target the Human Cathepsin-L protease: A Natural Therapeutic Strategy
5 against SARS-Cov-2
6 Brijendra Singha, D. D Agarwalb, Kaushal K. Sharmac*
7 a
Jiwaji University, Gwalior – 474 011, India
b
8 A.P.J Abdulkalam Central Instrumentation Facility
9 School Of Studies in Chemistry, Jiwaji University Gwalior (M.P) 474001, India
c
10 Bioinformatics Infrastructure Facility DBT (Dr. APJ Abdul Kalam Central Instrumentation
11 Facility), Jiwaji University Gwalior (M.P) 474001, India
12
13 Corresponding Author:
14 Dr Kaushal K. Sharma
15 Research Associate
16 Bioinformatics Infrastructure Facility
17 DBT (Dr. APJ Abdul Kalam Central
18 Instrumentation Facility), Jiwaji
19 University Gwalior (M.P) 474001, India
20 Email id:send2kaushal@gmail.com
21

22

23

24

25

26

27

28

29

1
30 Molecular docking studies of bioactive constituents of Long pepper, Ginger,
31 Clove and Black pepper to target the Human Cathepsin-L protease: as a
32 natural therapeutic strategy against SARS-Cov-2

33

34 Abstract

35 Human cathepsin-L protease present in the endosomal compartment of the target host cell make
36 a cleavage of S protein of SARS-Cov2 virus and activate the membrane fusion which mediate
37 the entry of virus in to the host cell. Thus, it suggests the cathepsin-L protease is critical for entry
38 of SARS-Cov2. Currently, chemically synthesized cathepsin-L inhibitor are present, but, the
39 consumption of chemical synthesized drugs are also an alarming stage due to its side effects,
40 illness and age reduction. Therefore, in this study, natural bioactive constituents of Long pepper,
41 Ginger, clove and black pepper that has been widely known for antiviral effect and other
42 medicinal properties were used for molecular docking against the Human cathepsin-L receptor
43 (PBD ID 2XU1). Molecular docking (using a software, Autodock 4.2) were performed on
44 Bioactive constituents of Long pepper, Ginger, Clove and Black pepper were reported in NCBI-
45 Pubchem database are docked against the human cathepsin-L protease and elucidate the binding
46 energies , visualization and analysis of interacting residue and non-interacting residue (using
47 Discovery studio) at the docking site of cathepsin-L protease and compared the docking analysis
48 of these bioactive constiuents with pre-clinical cathepsin-L inhibitor (Chemicalname-N-[(1,1-
49 Dimethylethoxy)carbonyl]-L-tryptophan-2-[[[2-[(2 ethylphenyl)amino]-2-
50 oxoethyl]thio]carbonyl]hydrazide; Pub Chem CID: 16725315). The pharmacokinetic properties
51 and toxicity evaluation were calculated by Datawarrior software and Osiris Molecular Property
52 explorer software respectively. Results displayed that Piperolactam A (CID 3081016) and
53 Kaempferol (CID 5280863) were found to be a more acceptable natural therapeutic compounds
54 among other selected bioactive constituents with appropriate binding energy, pharmacokinetic
55 property with no toxicity.

56 Keywords: Molecular docking, SARS-Cov-2, Human cathepsin L protease, natural bioactive

57 components
58

2
59

60

61

62

63 Introduction

64 World health organization (WHO) has declared the Covid-19 outbreak, a global epidemic that
65 led to high morbidity and mortality across the world. Illness, common cold, pneumonia, and
66 lower respiratory tract infection, cough, shortening of breath and dyspnea are belongs to the
67 symptoms of Covid-19 infected patients. Although, in more severe cases, this infection can also
6, 35
68 cause multiple organ failure and even death . According to World Health Organization
69 (WHO), in November 2002, there was an outbreak in the Guagelong province of china caused by
70 the pathogenic virus known as SARS-Cov virus, affected 5 continents and 29 countries resulted
71 in 800 Cases and 774 death by 23 September 2003 and also affected the global economy around
72 $30 to $180 billion. SARS-Cov infected patients have also developed symptoms like severe
73 acute respiratory illness, including fever, cough, and shortness of breath. Again in 2012, around
74 2494 diagnosed cases and 858 deaths was reported majority in Saudi Arabia due to the MERS-
75 COV (The middle East Respiratory Syndrome Coronavirus) infection (Eurosurveillance Editorial
10, 24
76 . Recently, Covid-19, a disease caused by SARS-Cov2/2019-nCov has been outbreak from
77 the city of Wuhan China to other Asian countries and other continents like Africa, America, and
78 Europe are also affected (World Health Organization, 2020). According to the European Centre
79 for Disease Prevention and Control, the total number of 3,186,400 cases of Covid-19 and
80 225,466 deaths has been reported from 31st Dec 2019 to 30th April 2020. In the current situation,
81 no specific vaccine or medications are not available for the treatment of Coronavirus infected
82 people.

83 Based on the phylogenetic clustering, Coronaviruses are envelope virus consist of the positive
84 sense RNA genome and found to be the largest group of viruses belonging to the
85 Coronaviridaefamily and placed under the Nidovirales order. Coronaviridaefamily is consists of
86 one of the Coronavirinae subfamilies and further divided into 4 classes known as alpha, beta,

3
 11
87 gamma, and delta . Pathogenic viruses (SARS-Cov, MERS-Cov and SARS-Cov2/2019) are
21
88 belonging to the delta class of Coronavirus . Similar to SARS-Cov and MERS-Cov, SARS-
89 Cov2 attack the lower respiratory tract and cause viral pneumonia 19,39. Before human to human
13
90 transmission, Chinese horse shoe bat is believed to be a host of SARS-Cov2 . Cryo-EM
91 structure analysis revealed the four structural components of coronaviruses: Spike (S)
38
92 glycoprotein, Envelope (E) protein, Membrane (M) protein and Nucleocapsid (N) protein .
93 Recent studies revealed the binding affinity of S protein of SARS-Cov2 has 10-20 times higher
34
94 affinity than the S protein of SARS-Cov . Spike glycoprotein consists of extracellular S1
95 protein mediate the angiotensin-converting enzyme 2ACE-2 receptor binding present on the
96 surface of the host cell and anchored S2 protein domain facilitates the fusion of SARS-Cov2-
26
97 ACE2 complex with the target cell membrane . ACE-2 receptor binding to the S protein
98 induces the changes in the conformation of the S protein and led to formation of the ACE-2-
25
99 SARS-Cov2 complex .Further, SARS-Cov2 utilize the endosomal acid protease (cathepsin L)
100 for fusion active state and translocation of ACE2-SARS-Cov2 complex to endosome studies
101 have also suggested that the critical role cathepsin L in priming of S protein of SARS-Cov2 in
102 the lysosome, protease activation of S protein and play an important role in the entry of ACE-
25, 26
103 2SARS-Cov2 complex through endocytosis . Therefore, we hypothesized that cathepsin L
104 protease activity inhibitor may play a promising role in restricting the SARS-Cov2 virus entry.

105 Antiviral drugs designing and synthesis are time-consuming and financial challenges that cannot
106 be affordable by a large number of sections of society. Also, the consumption of
107 drugs/antibiotics is also an alarming stage due to its side effects, illness and age reduction.
108 Recently, the highest mortality rate in SARS-Cov-2 infected patients underlying cardiovascular
109 diseases, diabetes, blood pressure and kidney diseases has been reported 36 . Patients having such
110 comorbidities are more vulnerable to the side effect of chemically synthesized drugs resulted in a
111 high mortality rate. Therefore, a natural therapeutic bioactive component needs to be studied to
112 combat the SARS-CoV-2. In our study, natural bioactive components having an antiviral activity
113 present in natural origin spices (Long pepper, Ginger, Clove and black pepper) were screened
114 and insilico analysis was performed for the characterization of human cathepsin L inhibitor.

115 Material and Methods

116 Target Protein:

4
117 The Human cathepsin L protease receptor (PBD ID 2XU1) having a resolution of 1.45 Å and
118 four chains A, B, C and D used for molecular docking. The three-dimensional crystal structure of
119 the receptor were retrieved from the Protein Data Bank (https://www.rcsb.org/) as shown in (Fig.
120 1A).
121 Receptor preparation:
122 The crystal structure of the receptor file was saved as Protein.pdb. Autodock 4.2 was used for
123 removal of the water molecule and any non-protein atoms from the 3-D structure of the receptor.
124 Subsequently, the addition of polar hydrogen atoms and Kollman charges were added, followed
125 by management of its conformer and the minimization process. Further, the Accelrys Discovery
126 Studio 2.0 software package was used for the CHARMM27 force field.
127 Ligand preparation:
128 Bioactive constituents of Long pepper, Ginger, Clove and Black pepper were reported in NCBI-
129 Pubchem database as listed in (table 1) are docked against the human cathepsin L receptor,
130 Additionally, preclinical Cathepsin L inhibitor (Fig.1B) namely (Chemical name-N-[(1,1-
131 Dimethylethoxy)carbonyl]-L-tryptophan-2-[[[2-[(2-ethylphenyl)amino]-2-
132 oxoethyl]thio]carbonyl]hydrazide; Pub Chem CID: 16725315) (Ou et al., 2020) were also
133 docked against the human cathepsin L receptor and to compare with the docking interactions of
134 bioactive constitiuents of Long pepper, Ginger, Clove and Black pepper against Cathepsin L
135 receptor

136 All naturally occurring bioactive constituents downloaded from Pubchem in the SDF file.
137 Further, Discovery studio 2.0 software was used to convert the SDF file into the PDB file format.
138 Subsequently, Charges and torsion were added to the ligands, and pdbqt file was generated for
139 docking studies.

140 Molecular Docking

141 MGLTools 1.5.6 suite by using AutoDock tools version (4.2) were used for generating the input
142 files in the pdbqt format by adding hydrogen bonds and gasteiger charges. Subsequently, to
143 define the binding site, grid box were generated along the x (126), y (92) and z (84) respectively
144 with spacing 0.972 Å and 29.85, -10.33 and -19.18 as x-center, y-center and z-center across the
145 protein by using AutoGrid software. All these parameters were saved as a grid parameter file

5
146 and further used by the Autodock software to defining the active binding sites of ligands present
147 in the receptor.

148 Analyzing and Output Visualisation using Discovery Studio

149 Log and ligan_out.pdbqt file obtained from AutoDock 4.2 was used to predict the binding
150 affinity and docking interactions of one ligand to the receptor. Binding energy and Docking site
151 (interacting and non-interacting molecules) of bioactive constituents of Long pepper, Ginger,
152 Clove and Black pepper against human cathepsin L receptor were compared with the binding
153 energy and docking site of cathepsin L Inhibitor (Pub Chem CID: 16725315)+ cathepsin L
154 receptor complex by using Discovery Studio Software package.
155 Pharmacokinetic Profiling and Toxicity Studies

156 Selected bioactive constituents of Long pepper, Ginger, Clove and Black pepper were
157 further evaluated for their pharmacokinetic properties and presence of any toxicity effects like
158 mutagenicity, tumorigenicity, irritant effect and reproductive effects by using Datawarrior
22
159 software and Osiris Molecular Property explorer software respectively . The pharmacokinetic
160 properties like calculated partition coefficient (cLogP), molecular weight, hydrogen bond donor
161 and acceptor sites were predicted based on Lipinsky’s rule of five and Veber.

162 Results

163 Binding energy and docking pose of human cathepsin L inhibitor (Pub Chem CID:
164 16725315)

165 The binding energy of inhibitor (Pub Chem CID: 16725315) obtained by molecular docking
166 simulation against the human cathepsin L is 9.2 kcal/mol (Table 1) and the docking ligand
167 interacting with the amino acid residue i.e., PRO D:15; LEU D:45; PRO C:15; ARG D:44; SER
168 C:47; LEU C:45 and GLY D:43 present at the active site of the receptor (Cathepsin L) by alkyl,
169 pi-alkyl, unfavorable donor-donor and conventional hydrogen bond (Fig. 2C). Whereas, non-
170 interacting amino acid residue i.e., THR D:14; VAL D:13; GLY C:11; PHE C:39; ILE D:46;
171 GLY C:43; PHE D: 39; ARG C:44; LYS D:10; GLU D:9; LYS C:17 and VAL C:13 were also
172 found to be present at the surface of the docking ligand.

173 Binding energy and docking pose of active constituents of Long pepper (Piper longum)

6
174 Among the constituents of long pepper, PiperolactanA (CID: 30810116) was found to be the
175 highest binding energy (-9.4 Kcal/mol) as shown in (Table 1), which is greater than the binding
176 energy of human cathepsin L inhibitor (Pub Chem CID: 16725315). Moreover, amino acid
177 residue i.e., ARG C:44; LEU C:45; PHE D:39; PHE C:39; LEU D:45; LYS D:10; GLY C:11;
178 GLY C:43, GLY D:43 and ARG D:44 present at the surface of docking PiperolactanA (Fig.3C)
179 are similar to the amino acid residue present at docking pose of cathepsin L inhibitor and
180 cathepsin L receptor complex. Also, LEU C:45 and LEU D:45 are the common interacting amino
181 acid residue present at the docking pose of both Piperlactan A+ cathepsin L and Inhibitor (Pub
182 Chem CID: 16725315+ cathepsin L receptor complex. The similarity in the interacting and non-
183 interacting amino acid residue indicating that Piperlactan A, a constituent of long pepper can also
184 act as an inhibitor of human cathepsin L.
185 Bisdemethoxycurcumin (CID 5315472) were found to be docked with the binding energy
186 (-8.5Kcal/mol) at the active site of cathepsin L. Amino acid residue i.e., ARG D:8; PRO D:15;
187 PRO C:15 with pi alkyl and alkyl bond; PHE D:39 with pi-pi T shaped bond; ARG C:44 with an
188 unfavorable bond; GLY C:11 and GLY D:43 with conventional hydrogen bond making an
189 interaction with the active site of human cathepsin L receptor. PRO D:15 and PRO C:15 are the
190 interacting amino residue and THR D:14; LEU C:45; VAL D:13 are the non-interacting amino
191 acid found at the surface of docking pose in both the Bisdemethoxycurcumin + cathepsin L and
192 Inhibitor (Pub Chem CID: 16725315)+ cathepsin L receptor complex.
193 Caumaperine (CID 10131321) exhibits the binding energy (-7.8 Kcal/mol) with the
194 amino acid residue i.e., PRO C:15; PRO D:15; PHE D:39 and ARG C:44 with the alkyl or pi-
195 alkyl bond interaction present at the active site of human cathepsin L receptor. Amino acid
196 residue ie., PRO D:15; THR C:14; LEU C:45; GLY C:43; LEU D:45; THR D:14; VAL C:13;
197 PHE C:39; GLY C:11, GLY D:43 and VAL D:13 found at the surface at the docking pose of
198 Caumaperine is similar to the docking pose of Inhibitor (Pub Chem CID: 16725315)+ cathepsin
199 L receptor complex. Whereas, (PRO D:15) is the common interacting amino acid present at both
200 the receptor complexes.
201 Other constituents of Long pepper such as Demethoxycurcumin (CID 5469424) exhibit a
202 binding energy (-6.4 Kcal/mol), Isopiperine (CID 1548913) exhibit a binding energy (4.2
203 Kcal/mol), Piperidin4ol (CID 79341) exhibit a binding energy (-4.2 Kcal/mol), Piperidine, 1-(5-
204 (1,3- benzodioxol-5-yl)-1-oxo-2,4-pentadienyl) (CID 4840) exhibit a binding energy (-6.5

7
205 Kcal/mol) and docked at the surface of human cathepsin L receptor poses no similarity with the
206 docking pose of Inhibitor (Pub Chem CID: 16725315)+ cathepsin L receptor complex.
207 Binding energy and docking pose of active constituents of Ginger -
208 Zingiberene (CID 92776) were found to be docked at the surface of cathepsin L receptor with the
209 binding energy (-7.2Kcal/mol) and the amino acid residue i.e., PRO D:15; PHE D:39; LEU C:45;
210 ARG C:44;LEU D:45; PHE C:39 intereact via alkyl and pialkyl bond respectively with the
211 docking ligand. Amino acid residue i.e., PRO D:15; LEU C:45; LEU D:45 are the interacting
212 amino residue and PHE D:39; ARG C:44; PHE C:39; THR D:14; VAL C:13; GLY C:11 and
213 GLY D:43 are the non-interacting amino acid residue were found at the surface of docking pose
214 of Zingiberene is similar to amino acid residue found at the docked pose of Inhibitor (Pub Chem
215 CID: 16725315)+ cathepsin L receptor complex indicating the similar docking site of both the
216 Zingiberene and inhibitor (Pub Chem CID: 16725315) ligand.
217 6Gingerdiol (CID 11369949) bind with the amino acid residue i.e., GLY D:196; GLU D:191;
218 TYR D:198; VAL D:16; VAL D:13; TYR C:198; GLU C:191 with pi-alkyl, pi-pi t shaped and
219 conventional hydrogen bond with the binding energy (-7.1Kcal/mol). Amino acid residue i.e,
220 VAL D:13; THR D:14; THR C:14; PRO D:15 were found at the surface of 6Gingerdiol-
221 cathepsin L is similar to Inhibitor (Pub Chem CID: 16725315)- cathepsin L receptor complex.
222 6Gingerol (CID 442793) were found to be docked at the surface of cathepsin L receptor with the
223 binding energy (-7.0 Kcal/mol) and the amino acid residue i.e., PRO C:15; VAL D:13; PRO
224 D:15; GLU C:191; ARG D:8 are interacting via alkyl, pialkyl, carbon-hydrogen bond,
225 unfavorable donor-donor and conventional hydrogen bond with the docking ligand. Amino acid
226 residue i.e., PRO C:15 and PRO D:15 are the interacting amino residue and GLY C:11; THR
227 C:14; THR D:14; LEU D:45; LEU C:45 is the non-interacting amino acid residue were found at
228 the surface of docking pose of 6Gingerol is similar to amino acid residue found at the docked
229 pose of Inhibitor (Pub Chem CID: 16725315)+ cathepsin L receptor complex indicating the
230 6Gingerol molecule also poses a similar docking site at the surface of human cathepsin L
231 receptor comparison with docking site of inhibitor (Pub Chem CID: 16725315) ligand.
232 8Gingerdione (CID 14440537) bind with the amino acid residue i.e., TYR D:12; LEU D:45;
233 PHE C:39, GLY D:43 and ARG C:44 with pi-alkyl, pi-pi t shaped and conventional hydrogen
234 bond with the binding energy (-6.3 Kcal/mol). The interacting amino acid residue (LEU D:45)
235 and non-interacting amino acid residue LYS D:10; PHE D:39; ILE C:46; LEU C:45 and GLY

8
236 C:11; ARG D:44 and GLY C:43 are the non-interacting amino acid were found at the surface of
237 8Gingerdione-cathepsin L receptor complex is similar to Inhibitor (Pub Chem CID: 16725315)-
238 cathepsin L receptor complex.
239 10Gingerdione (CID 14440539) were found to be docked at the surface of cathepsin L receptor
240 with the binding energy (-6.4 Kcal/mol) and the amino acid residue i.e., PHE D:39; LEU D:45;
241 PHE C:39, ILE C:46 and GLY D:43 are interacting via alkyl, pialkyl, pi-pi T shaped bond and
242 conventional hydrogen bond with the docking ligand. LEU D:45 is the interacting amino acid
243 residue and GLY C:43; ARG D:44; GLY C:11; LEU C:45; LYS D:10 and GLU C:87are the
244 non-interacting amino acid residue were found at the surface of docking pose of 6Gingerol is
245 similar to amino acid residue found at the docked pose of Inhibitor (Pub Chem CID:
246 16725315)+ cathepsin L receptor complex.
247 10 Paradol (CID 51352076) dock at the surface of human cathepsin L receptor the binding
248 energy (-7.1Kcal/mol) and interact with GLU D:9 amino acid residue via., unfavorable acceptor
249 –acceptor bond with. Non-interacting amino acid residue i.e, LYS C:17; GLU C:86; PRO C:15;
250 SER C: 47; THR C:14; LYS D:10 and GLU C:87 were found at the surface of 10 Paradol-
251 cathepsin L is similar to Inhibitor (Pub Chem CID: 16725315)- cathepsin L receptor complex.
252 10 Shagaol (CID 6442612) exhibits the binding energy (-5.4 Kcal/mol) with the amino
253 acid residue i.e., LEU C:45; PHE D:39 and PHE C:39 with conventional hydrogen bond
254 interaction present at the active site of human cathepsin L receptor. Interacting amino acid
255 residue ie., LEU C:45 and non-interacting amino acid residue i.e., ARG C:44; GLY C:11; ARG
256 D:44; ILED:46; LEU D:45, GLY D:43 and GLY C:43 found at the surface at the docking pose
257 of 10 Shagaol –cathepsin L receptor complex is similar to the docking pose of Inhibitor (Pub
258 Chem CID: 16725315)+ cathepsin L receptor complex.
259 6Paradol (CID 94378) interacts with the amino acid residue i.e., TYR C:91 and GLU D:9
260 with pi-alkyl and conventional hydrogen bond present at the active site of human cathepsin L
261 receptor and exhibits the binding energy (-5.6 Kcal/mol). Amino acid residue i.e., GLU D:9;
262 LYS C:17; GLU C:86; GLU C:87; PRO C:15; THR C:14 and SER C:47 found at the surface at
263 the docking pose of 6Paradol–cathepsin L receptor complex is similar to the docking pose of
264 Inhibitor (Pub Chem CID: 16725315)+ cathepsin L receptor complex.
265 Remaining constituents of ginger such as 6 Shagol (CID 5281794) exhibit binding energy
266 (-6.20 Kcal/mol), 8 Gingerol (CID 168114) exhibit binding energy (-4.7 Kcal/mol), 8 Paradol

9
267 (CID 213821) exhibit a binding energy (-6.0 Kcal/mol), 8 Shagaol (CID 6442560) exhibit a
268 binding energy (-6.3 Kcal/mol), 10 Gingerol (CID 168115) exhibit a binding energy (-5.7
269 Kcal/mol), 12 Gingerol (CID 168115) exhibit a binding energy (-6.0 Kcal/mol), 12 Shagaol (CID
270 9975813) exhibit a binding energy (-6.0 Kcal/mol) and Zingerone (CID 31211) exhibit a binding
271 energy (5.7 Kcal/mol) and docked at the surface of cathepsin L receptor without any similarity
272 with the docking pose of Inhibitor (Pub Chem CID: 16725315)+ cathepsin L receptor complex.
273 Binding energy and docking pose of active constituents of Clove –
274 Kaempferol (3,4’5,7- tetrahydroxyflavone) (CID 5280863), a type of natural flavonoid were
275 found to be docked at the surface of cathepsin L receptor with the binding energy (-9.3
276 Kcal/mol)as shown in (Table.1) and the amino acid residue i.e., ARG C:8; PRO D:15; VAL
277 C:16; ARG D:8; GLU C:191; and GLU C:195 present at the docking site of cathepsin L receptor
278 interact via Van der Waals, conventional hydrogen bond, pi-cation and pi alkyl bond respectively
279 with the docking ligand (Fig.4C). Amino acid residue i.e., PRO D:15 is the interacting amino
280 residue and THR D:14; VAL D:13; GLY C:11 and VAL C:13 are the non-interacting amino acid
281 residue were found at the surface of docking pose of Kaempferol is similar to amino acid residue
282 found at the docked pose of Inhibitor (Pub Chem CID: 16725315)+ cathepsin L receptor
283 complex indicating the similar docking site of both the Kaempferol and inhibitor (Pub Chem
284 CID: 16725315) ligand.
285 Rhammetin (CID 5281691) were found to be docked at the surface of cathepsin L receptor with
286 the binding energy (-8.9 Kcal/mol) and the amino acid residue i.e., LEU D:45; GLY D:43and
287 ARG C:44 present at the docking site of cathepsin L receptor interact via Van der Waals, alkyl
288 and pi alkyl bond respectively with the docking ligand. Amino acid residue i.e., LEU D:45 and
289 GLY D:43 is the interacting amino residue and ARG C:44, PHE C:39, GLY C:11, ARG D:44,
290 GLY C:43, LEU C:45, GLU D:9 and LYS D:10 are the non-interacting amino acid residue were
291 found at the surface of docking pose of Rhammetin is similar to amino acid residue found at the
292 docked pose of Inhibitor (Pub Chem CID: 16725315)+ cathepsin L receptor complex.
293 βCadinene (CID 10657) exhibit the binding energy (8.0 Kcal/mol) docked at the surface of
294 cathepsin L receptor with the amino acid residue i.e., VAL C:16; VAL D:16; ARG D:8; VAL
295 D:13 and PRO C:15 with Van der Waal bond and alkyl bond. Interacting amino acid residue ie.,
296 PRO C:15 and non-interacting amino acid residue i.e., VAL D:13; PRO D:14; VAL C:13; THR
297 and C:14 found at the surface at the docking pose of βCadinene –cathepsin L receptor complex is

10
298 similar to the docking pose of Inhibitor (Pub Chem CID: 16725315)+ cathepsin L receptor
299 complex.
300 Αlpha-cedrene (CID 6431015) were found to be docked at the surface of cathepsin L receptor
301 with the binding energy (-8.0Kcal/mol) and the amino acid residue i.e., VAL C:16; TYR C:198;
302 PRO D:15; ARG C:8; VAL D:13; ARG D:8 and PRO C:15 present at the docking site of
303 cathepsin L receptor interact via Van der Waals, alkyl and pi alkyl bond respectively with the
304 docking ligand. Amino acid residue i.e., PRO D:15 and PRO C:15 is the interacting amino
305 residue and VAL D:13 and VAL C:13 are the non-interacting amino acid residue were found at
306 the surface of docking pose of Αlpha-cedrene is similar to amino acid residue found at the
307 docked pose of Inhibitor (Pub Chem CID: 16725315)+ cathepsin L receptor complex.
308 Αlpha-muurolene (CID 12306047) were found to be docked at the surface of cathepsin L
309 receptor with the binding energy (-7.6 Kcal/mol) and the amino acid residue i.e., TYR D:198;
310 VAL C:16; ARG C:8; VAL C:13; PRO C:15; ARG D:8 and VAL D:13 present at the docking
311 site of cathepsin L receptor interact via Van der Waals, alkyl and pi alkyl bond respectively with
312 the docking ligand. Amino acid residue i.e., PRO C:15 is the interacting amino residue and VAL
313 C:13; THR D:14; PRO D:15; THR C:14 is the non-interacting amino acid residue were found at
314 the surface of docking pose of Αlpha-muurolene is similar to amino acid residue found at the
315 docked pose of Inhibitor (Pub Chem CID: 16725315)+ cathepsin L receptor complex.
316 Eugenitin (CID 3083581) exhibits the binding energy (-7.5 Kcal/mol) docked at the surface of
317 cathepsin L receptor with the amino acid residue i.e., PHE D:39; LEU D:45; ARG C:44 and
318 LEU C:45 with pi-pi T-shaped, alkyl and pi-alkyl bond. LEU D:45 and LEU C:45 are the
319 interacting amino acid residue and VAL D:13; PRO D:14; VAL C:13; THR, GLY D:43 and
320 C:14 are non-interacting amino acid residue found at the surface of βCadinene –cathepsin L
321 receptor complex is similar to the docking pose of Inhibitor (Pub Chem CID: 16725315)+
322 cathepsin L receptor complex.
323 Jasmone (CID 1549018) were found to be docked at the surface of cathepsin L receptor with the
324 binding energy (-6.0 Kcal/mol) and the amino acid residue i.e., PRO C:15; TYR C:91; PHE C:28
325 and LYS C:17 present at the docking site of cathepsin L receptor interact via Van der Waals,
326 alkyl and pi alkyl bond respectively with the docking ligand. Amino acid residue i.e., PRO C:15
327 is the interacting amino residue and LYS C:17; THR C:14; GLU D:9; GLU C:86; SER C:47 is
328 the non-interacting amino acid residue was found at the surface of docking pose of Jasmone is

11
329 similar to amino acid residue found at the docking pose of Inhibitor (Pub Chem CID:
330 16725315)+ cathepsin L receptor complex.
331 Eugenol (CID 3314) exhibits the binding energy (-5.3 Kcal/mol) docked at the surface of
332 cathepsin L receptor with the amino acid residue i.e., PHE D:39; LEU D:45 and ARG C:44 with
333 pi-pi T-shaped, alkyl and pi-alkyl bond. LEU D:45 is the interacting amino acid residue and PHE
334 D:39; ARG C:44; PHE C:39; GLY C:43, GLY D:43 and LEU C:45 are non-interacting amino
335 acid residue found at the surface of Eugenol –cathepsin L receptor complex is similar to the
336 docking pose of Inhibitor (Pub Chem CID: 16725315)+ cathepsin L receptor complex.
337 However, remaining active constituents of clove such as 4 Allylanisole (CID 8815) exhibit a
338 binding energy (-4.9 Kcal/mol), 4 Allylphenole (CID 68148) exhibit a binding energy (-5.5
339 kcal/mol), alpha Pinene (CID 6654) exhibit a binding energy (-5.1 Kcal/mol), Anethol (CID
340 637563) exhibit a binding energy (-4.8 Kcal/mol), beta Selinene (CID 10856614) exhibit a
341 binding energy (-6.8 Kcal/mol), beta Caryophyllene (CID 5281515), beta Pinene (CID 14896)
342 exhibit a binding energy (-5.2 Kcal/mol), Campesterol (CID 173183) exhibit a binding energy (-
343 8.1 Kcal/mol), Caryophyllene oxide (CID 1742210) exhibit a binding energy (-6.5 Kcal/mol),
344 Copaene (CID 19725) exhibit a binding energy (-6.9 Kcal/mol), Eucalyptol (CID 2758) exhibit a
345 binding energy (-6.1 Kcal/mol), Eugenyl acetate (CID 7136) exhibit a binding energy (-6.0
346 Kcal/mol), Gallic acid (CID 370) exhibit a binding energy (-6.5Kcal/mol), Globulol (CID
347 12304985) exhibit a binding energy (-6.7 Kcal/mol), Jasmone (CID 1549018) exhibit a binding
348 energy (-6.0 Kcal/mol), Ledol (CID 92812) exhibit a binding energy (-6.4 Kcal/mol), Maslinic
349 acid (CID 73659) exhibit a binding energy (-8.3 Kcal/mol), Methyl salicylate (CID 4132)
350 exhibit a binding energy (-5.0 Kcal/mol), Oleanolic acid (CID 2758) exhibit a binding energy (-
351 8.0 Kcal/mol) and Stigmasterol (CID 5280794) exhibit a binding energy (-7.9 Kcal/mol), docked
352 at the surface of human cathepsin L receptor poses no similarity with the docking pose of
353 Inhibitor (Pub Chem CID: 16725315)+ cathepsin L receptor complex.
354 Binding energy and docking pose of active constituents of Black pepper–
355 (2E,4E)-2-Methyl-1-[(S)-2-(pyrrolidine-1-carbonyl)-pyrrolidin-1-yl]-octa-2,4-dien-1-one (CID
356 44292388) were found to be docked at the surface of cathepsin L receptor with the binding
357 energy (-6.7Kcal/mol) and the amino acid residue i.e., PRO C:15; PRO C:90; and ASN C:18
358 interact via Van der Waals, conventional hydrogen bond, Carbon hydrogen bond and alkyl bond
359 respectively with the docking ligand. Amino acid residue i.e., PRO C:15 is the interacting amino

12
360 residue and THR C:14; GLU D:9; SER C:47 and GLU C:86 are the non-interacting amino acid
361 residue were found at the surface of docking pose of 2E4E2Methyl1-Cathepsin L is similar to
362 amino acid residue found at the docked pose of Inhibitor (Pub Chem CID: 16725315)- cathepsin
363 L receptor complex.
364 Piperolein B (CID 21580213) exhibit the binding energy (-6.8 Kcal/mol), docked at the surface
365 of cathepsin L receptor with the amino acid residue i.e., LEU C:45; PHE D:39; LEU D:45; ILE
366 C:46; VAL C:106 and SER C:85 with alkyl, pi-alkyl bond and pi-pi Donar hydrogen bond. LEU
367 C:45 and LEU D:45 are the interacting amino acid residue and PHE D:39; ILE C:46; PHE C:39;
368 ARG C:44; ARG D:44; GLU C:87 and LYS D:10 are non-interacting amino acid residue found
369 at the surface of Piperolin B–cathepsin L receptor complex is similar to the docking pose of
370 Inhibitor (Pub Chem CID: 16725315)+ cathepsin L receptor complex.
371 Sabinene (CID 10887971) exhibits the binding energy (-6.1 Kcal/mol), docked at the surface of
372 cathepsin L receptor with the amino acid residue i.e., LEU C:45; PHE C:39; LEU D:45; ARG
373 C:44 and PHE D:39 with alkyl and pi-alkyl bond. LEU C:45 and LEU D:45 are the interacting
374 amino acid residue and PHE D:39; PHE C:39; ARG C:44, GLY D:43 and ARG D:44 are non-
375 interacting amino acid residue found at the surface of Sabinene –cathepsin L receptor complex is
376 similar to the docking pose of Inhibitor (Pub Chem CID: 16725315)+ cathepsin L receptor
377 complex.
378 In addition, other active constituents of black pepper such as 2E octadecanoyl pyrrolidine (CID
379 5746599) exhibit a binding energy (-3.8 Kcal/mol), 6 Cadinol (CID 6428423) exhibit a binding
380 energy (-6.6 Kcal/mol), beta Sitestero (CID 222284) exhibit a binding energy (-6.6 Kcal/mol),
381 beta Caryophyllene (CID 20831623) exhibit a binding energy (-6.5), beta Guaiene (CID
382 15560252) exhibit a binding energy (-7.4 Kcal/mol), EZ Farnesol (CID 1549109), exhibit a
383 binding energy (-4.9 Kcal/mol), hexadecanoyl pyrrolidine (CID 247220) exhibit a binding
384 energy (-6.4 Kcal/mol), Limonene (CID 22311) exhibit a binding energy (-6.1 Kcal/mol),
385 Linalool (CID 6549) exhibit a binding energy (-4.9 Kcal/mol), Pellitorine (CID 5318516) exhibit
386 a binding energy (-5.1 Kcal/mol), Piperanine (CID 5320618), exhibit a binding energy (-7.9
387 Kcal/mol), Piperettine (CID341140014) exhibit a binding energy (-6.6 Kcal/mol), Piperine (CID
388 638024) exhibit a binding energy (-6.8 Kcal/mol), Piperolein (CID 11141599) exhibit a binding
389 energy (-7.6 Kcal/mol), Piperolin B (CID 21580213) exhibit a binding energy (-6.8 Kcal/mol),
390 Piperylene (CID 62204) exhibit a binding energy (-3.7 Kcal/mol) docked at the surface of

13
391 human cathepsin L receptor poses no similarity with the docking pose of Inhibitor (Pub Chem
392 CID: 16725315)+ cathepsin L receptor complex.
393 Pharmacokinetic and toxicity evaluation
394 Active constituents of Long pepper, Ginger, Clove and Black pepper having similar docking site
395 interactions and acceptable range of binding energy were further evaluated their drug-likeness,
396 pharmacokinetic properties and toxicity effects based on Lipinski’s-Rule- of- five. As shown in
397 (Table 2), active constituents of Long pepper, Ginger, Clove and Black pepper were found to be
398 the suitable range of pharmacokinetic properties such as acceptable molecular weight (acceptable
399 range <500g/mol), acceptable hydrogen bond acceptor (acceptable range <10), acceptable
400 hydrogen bond donor (acceptable range <5), acceptable TPSA (Two-dimensional polar surface
401 area, acceptable range <140Å), acceptable lipophilicity property ( acceptable range -0.4 to 5.6)
402 and these active constituents also having good and moderate solubility in water, except 10
403 Gingerdione (CID 14440539), 10 paradol (CID 51352076), 10 shagaol (CID 6442612) having
404 poor water solubility. Also, no mutagenic, tumorogenic, irritant and reproductive effect revealed
405 by toxicity profiling of active constituents of Long pepper, Ginger, Clove and Black as shown in
406 (Table.3)
407 Discussion-
408 Similar to SARS-Cov virus, the entry stage of SARS-Cov2 virus are also having the three-step
409 process i.e., 1- S protein-ACE2 receptor binding, 2- changes in the conformation of S protein, 3-
410 involvement of endosomal Cathepsin L protease enzyme activates the S protein fusion activity
8, 14
411 with the endosome . Previously, it was believed that the fusion activity with the endosomal
412 membrane requires an absolute acidic environment or low pH. Another study has provided
413 evidence that viral S protein can make membrane fusion with the help of Cathepsin L protease
32
414 enzyme can also make a membrane fusion activity at neutral pH . In vitro study was
415 demonstrated the Cathepsin L inhibitors such as E63c, E64d and MDL28170 restrict the SARS-
416 Cov virus entry or SARS-Pseudovirus 15, 33. Therefore, the current insilico study has targeted the
417 human cathepsin L protease enzyme to block the initial entry stage of SARS-Cov-2 with the
418 Bioactive constituents of Long pepper, Ginger, Clove and Black pepper. Also, in our current
419 study, we hypothesized these active constituents as a herbal medicine in the treatment of viral
420 infection and also having multiple benefits with a lower risk of adverse side effects. Recently,
421 WHO and few clinical studies have reported the most of the SARS-Cov-2 infected patients was

14
422 die due to comorbidities conditions. Therefore, there should be an effective therapeutic in such a
423 way that it may not aggravate the patient’s condition.

424 Long pepper is recognized for its medicinal properties and its active constituents are commonly
425 used for the treatment of various diseases such as viral hepatitis, respiratory infections, chronic
426 bronchitis, asthma, cough, Chronic malaria, constipation, diarrhea, cholera, Gonorrhea and
12, 20
427 tumors . Also, Long pepper is known for its remarkable improvement in inflammatory
428 diseases, depression, diabetes and cancer patients, reported the inhibitory activity of active
12
429 constituents of Long pepper on the Hepatitis B virus infection . In-vitro antiviral activity and
430 anticancer activity of methanol and chloroform extraction of Long pepper against the vesicular
431 stomatitis Indian virus and human parainfluenza virus have also demonstrated by Priya and
432 Kumar (2017). Therefore, in the present study, we have performed the molecular docking of
433 bioactive constituents of Long pepper and analyze its binding energy and docking ligand
434 interactions with the human cathepsin L protease enzyme and compare with the binding energy
435 and docking pose interactions of human cathepsin L inhibitor (Pub Chem CID: 16725315). The
436 finding from this insilico study suggests that all the active constituents as shown in (Table 1)
437 having acceptable binding energy and docking interactions with the active site of human
438 cathepsin L protease. Based on the comparison with the binding energy (-9.3kcal/mol) and
439 docking interactions of Inhibitor (SID 26681509)+ cathepsin L receptor complex, we selected
440 out the Piperolactam A (CID: 30810116), Bisdemethoxycurcumin (CID 5315472) and
441 Caumaperine (CID 10131321) are the most acceptable active constituents on the various
442 parameters i.e., binding energy, interacting and non-interacting amino residue present at the
443 docking site, pharmacokinetic and toxicity evaluation profile. Furthermore, thebinding energy of
444 Piperolactam A (-9.4 Kcal/mol) is larger than the binding energy of human cathepsin L inhibitor
445 (-9.3Kcal/mol). Also, amino acid residue ARG C:44; LEU C:45; PHE D:39; PHE C:39; LEU
446 D:45; LYS D:10; GLY C:11; GLY C:43 and ARG D:44 out of them LEU C:45 and LEU D:45
447 forming a stable alkyl and pi-alkyl bond at the docking site of Piperolactam A is similar with the
448 Inhibitor (SID 26681509)+ cathepsin L receptor complex revealing the potency of Piperolactam
449 A to act as inhibitory action on the human cathepsin L protease. Similarly, the docking pose of
450 Bisdemethoxycurcumin (CID 5315472) and Caumaperine (CID 10131321) was also found in a
451 similar docking pose with the (SID 26681509)+ cathepsin L receptor complex. Although, the
452 binding energy of Bisdemethoxycurcumin (CID 5315472) (-8.5 Kcal/mol) and Caumaperine
15
453 (CID 10131321) (-7.8 Kcal/mol) was lower than the human cathepsin L inhibitor (-9.3Kcal/mol),
454 in an acceptable range that can also be considered a promising inhibitor of Cathepsin L to restrict
455 the early entry of SARS-Cov2 virus into the cell.

456 Ginger also known as Zingiber officinale Roscoe, belong to the Zingiberaceae has been
457 commonly used as a spice in food. Several studies have demonstrated its herbal medicinal
458 properties in various biological activities i.e., antimicrobial, antioxidant, anti-inflammatory,
459 anticancerous and also having potential in the treatment of respiratory diseases, cardiovascular
27, 29
460 diseases, obesity and neurodegenerative diseases . Moreover, preclinical evaluation has
16
461 revealed the antiviral activity of the ginger extract against the Hepatitis C virus (HCV) . San
462 Chang et al., 2013 has reported the antiviral activity of fresh ginger by blocking the viral
463 attachment and internalization process resulted in a reduction in plaque formation in the
464 respiratory tract induced by Human Syncytial Virus (HRSV). Therefore, in our present study
465 active constituents of ginger were screened through molecular docking process and analyze the
466 binding affinity against the human cathepsin L protease and measure the potentiality of active
467 constituents of ginger to block the internalization process of SARS-Cov-2 virus. Based on our
468 result, we screened out the selected active constituents of ginger i.e., Zingiberene (B.E.-
469 7.2Kcal/mol), 6Gingerdiol (B.E.-7.1Kcal/mol), 6Gingerol (B.E.-7.0Kcal/mol), 8Gingerdione
470 (B.E.-6.3Kcal/mol), 10Gingerdione (B.E.-6.4Kcal/mol), 10 Paradol (B.E.-5.7Kcal/mol), 10
471 Shagaol (-5.4Kcal/mol) and 6Paradol (B.E.-5.6Kcal/mol) showed acceptable binding energy and
472 similar docking pose with the comparison with the docking pose of Inhibitor (SID 26681509)+
473 cathepsin L receptor complex. Thus the finding suggests the significant interactions of active
474 constituents of ginger against the cathepsin L protease indicating the potential of selected active
475 constituents of ginger to act as an inhibitor.

476 Clove (Syzygium aromaticum) is one of the traditional spice use in food, preservative and
477 medicinal herbs. In the last few decades, several studies have confirmed the antiviral,
478 antibacterial, antioxidant and anti-cancerous properties of clove 7. A Phenolic compound
479 extracted from cloves such as Eugenol, eugenol acetate and gallic acid are commonly used in
480 pharmaceutical industries for their biological applications 9. Invitro study has also demonstrated
481 the antiviral activity of aqueous extract of both clove and ginger against the feline calicivirus 1.
482 The inhibitory action against the Herpes simplex virus type-1 (HSV-1) has shown by the

16
483 eugenol, β-caryophyllene, β-caryophyllene, an active constituent of clove 2. Consequently, in our
484 present study, essential active constituents of the clove are screened and targeted against the
485 active site of human cathepsin L protease. We screened out the active constituents of clove i.e.,
486 Kaempferol (B.E. -9.3Kcal/mol), βCadinene(B.E. -8.0Kcal/mol), Αlpha-cedrene(B.E. -
487 8.0Kcal/mol), Αlpha-muurolene (B.E. -7.6Kcal/mol), Eugenitin (B.E. -7.5 Kcal/mol), Jasmone
488 (B.E. -6.0Kcal/mol) and Eugenol (B.E. -5.3Kcal/mol) based on the acceptable range of binding
489 energy and similar docking site with the docking site of Inhibitor (SID 26681509)+ cathepsin L
490 receptor complex. Several studies have also demonstrated the antiviral activity of Kaempferol
491 and its derivative such as Kaempferol 3-O-α-L-rhamno pyranoside and Kaempferol-7-o-
492 glucoside against the viruses ie., Human cytomegalovirus (HCV), Japanese encephalitis virus
493 (JEV) and influenza virus-like H1N1 and H9N2 5, 40
.

494 Black pepper (Piper Nigrum L.) is one of the spices used in food but also included under the
495 medicinal herbs due to its antimicrobial, antioxidant, antiproliferative and gastroprotective
30
496 module properties . Mair et al., 2016 has also observed the antiviral and anti-proliferative
497 properties in in-vitro study of active constituents of black pepper. Consequently, in our insilico
498 study, we found the acceptable range of binding energy of following active constituents of black
499 pepper against the human cathepsin L receptor i.e(2E,4E)-2-Methyl-1-[(S)-2-(pyrrolidine-1-
500 carbonyl)-pyrrolidin-1-yl]-octa-2,4-dien-1-one (B.E. -6.7Kcal/mol), Piperolein B (B.E. -
501 6.8Kcal/mol) and Sabinene B (B.E. -6.8Kcal/mol). Also, these active constituents show a
502 similarity in the docking pose with the (SID 26681509)+ cathepsin L receptor complex
503 indicating the potentiality of these active constituents to restrict the activity of cathepsin L
504 protease.

505 According to the Lipinski rule of five predictors, for adequate absorption and permeation is more
506 likely when there are Hydrogen bond donors are ≥ 5, Hydrogen bond acceptors is ≥ 10 to avoid,
507 Molecular weight not more than 500g/mol, Topological polar surface is (TSPA) for the
508 measurement of polar surface area should not be greater than 140Å and Lipophilicity (Clog P)
3, 17
509 not greater than 5, so that bioactive molecules can easily penetrate the cell membrane .
510 Consequently, pharmacokinetic properties of selected active constituents of Long pepper,
511 Ginger, Clove and Black pepper are found suitable in various parameters of Lipinski rule of five
512 (Table 2)such as Molecular weight, Hydrogen bond donor, Hydrogen bond acceptor, Topological

17
513 polar surface area (TPSA), water solubility and Lipophilicity for better bioavailability to the cell.
514 Thus, our insilico study, Overall, we found the Piperolactam A (CID 3081016) and Kaempferol
515 (CID 5280863) was found to be a more acceptable natural therapeutic compounds among other
516 selected bioactive constituents which posses to act as an inhibitor to target the human cathepsin
517 L protease as they are having equal or higher binding energy as shown in (Table 1) and similar
518 docking pose comparatively with the Inhibitor (SID 26681509)+ cathepsin L receptor complex.
519 So, it can be concluded that these bioactive constituents not only act a promising inhibitor of the
520 human cathepsin L protease in the lysosome and restrict the entry of the SARS-Cov-2 virus, but
521 also having possibilities to overcome the comorbidities conditions and multiple organ injuries in
522 the treatment of SARS-Cov-2 infected patients. However, still, there is a need for the in-vivo
523 antiviral activity should be further tested in animal models to put forward therapeutic
524 repositioning of bioactive constituents of Long pepper, Ginger, Clove and Black pepper.

525 CONFLICT OF INTEREST

526 The authors do not have any actual or potential conflict of interests including financial,
527 personal or academic.

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602 Z., 2020. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its
603 immune cross-reactivity with SARS-CoV. Nature communications, 11, 1, pp.1-12.
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605 treatment of gastrointestinal cancer. Gastroenterology research and practice, 2015.
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614 (Zingiberofficinale) has anti-viral activity against human respiratory syncytial virus in
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617 2005. Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus
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622 Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell.
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624 multi-organ injuries in the treatment of COVID-19. The Lancet, 395, 10228, p.e52.
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628 report, 72.
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20
635 40. Zakaryan, H., Arabyan, E., Oo, A., Zandi, K., 2017. Flavonoids: promising natural
636 compounds against viral infections. Archives of virology, 162, 9, pp.2539-2551.

637 Table legends:

638 Table 1. Binding energy of bioactive constituents of Long pepper, Ginger, Clove and Black
639 pepper
640 Table.2 “Lipinski’s rule of five” for the bioactive constituents of Ginger, clove, long pepper
641 and black pepper targeting human cathepsin L protease
642
643 Table.3 Toxicity evaluation of bioactive constituents of ginger, clove, long pepper and black
644 pepper
645

646

647 Figure Legends:

648 Figure:1 (A) Crystal structure of Human cathepsin L protease (PDB ID: 2XU1) having a
649 resolution of 1.45Å and four chains A, B ,C and D; (B) Cathepsin L inhibitor namely
650 (Chemicalname-N-[(1,1-Dimethylethoxy)carbonyl]-L-tryptophan-2-[[[2[(2ethylphenyl)amino]-2
651 oxoethyl]thio]carbonyl]hydrazide; Pub Chem CID: 16725315)
652
653 Figure:2 (A) The structure of Human cathepsin L protease- Cathepsin L inhibitor complex. The
654 red box indicating the docking pocket of Cathepsin L inhibitor (Gray color). (B) Zoom-in view
655 of the docking site of Cathepsin L inhibitor. (C) The two-dimensional binding interactions of
656 Cathepsin L inhibitor shows the main interactions with amino acid residue i.e., PRO D:15; LEU
657 D:45; PRO C:15; ARG D:44; SER C:47; LEU C:45 and GLY D:43 present at the active site of
658 receptor (Human Cathepsin L protease ) by alkyl, pi-alkyl, unfavorable donor-donor and
659 conventional hydrogen bond.
660
661 Figure:3 (A) The structure of Human cathepsin L protease- PiperolactanA complex. The red box
662 indicating the docking pocket of PiperolactanA (Gray color). (B) Zoom-in view of the docking
663 site of PiperolactanA. (C) The two-dimensional binding interactions of PiperolactanA shows the
664 main interactions with amino acid residue i.e., ARG C:44; LEU C:45; PHE D:39; PHE C:39 and
665 LEU D:45 present at the active site of receptor (Human Cathepsin L protease) by Van der Waals,
666 p-Donor hydrogen bond,pi-alkyl, pi-sigma, and pi-pi T-shaped bond.
667
668
669 Figure:4 (A) The structure of Human cathepsin L protease Kaempferol complex. The red box
670 indicating the docking pocket of Kaempferol (Gray color). (B) Zoom-in view of the docking site
671 of Kaempferol. (C) The two-dimensional binding interactions of Kaempferol shows the main
672 interactions with amino acid residue i.e., ARG C:8; PRO D:15; VAL C:16; ARG D:8; GLU
673 C:191 and GLU C:195 present at the active site of receptor (Human Cathepsin L protease) by
674 Van der Waals, conventional hydrogen bond, pi-cation and pi alkyl bond.

21
675

676

677

678

679

680

22
Table

Table 1. Binding energy of bioactive constituents of Long pepper, Ginger, Clove and Black pepper

Chemical name Chemical structure Binding

energy
-9.2
N-[(1,1-Dimethylethoxy)carbonyl]-
L-tryptophan-2-[[[2-[(2-
ethylphenyl)amino]-2-
oxoethyl]thio]carbonyl]hydrazide
(CID 16725315)

Ginger
6 gingerdiol (CID 11369949) -7.1

6 gingerol (CID 442793) -7.0

(6 paradol CID 94378) -5.3

6 shagaol (CID 5281794) -6.2

-
8 gingerdione (CID 14440537) -6.3

8 gingerol (CID 168114) -4.7

8 paradol (CID 213821) -6.0

8 shogaol (CID 6442560) -6.3

10 Gingerdione( CID 14440539) -6.4

10 Gingerol (CID 168115) -5.7

10 paradol (CID 51352076) -5.7

10 shagaol (CID 6442612) -5.4

12 gingerol (CID 168115) -5.9

12 shagaol (CID 9975813) -6.0

6 PARADOL (94378) -5.6

Zingerone (CID 31211) -5.7

Zingiberene (CID 92776) -7.2

Clove
4 Allylanisole (CID 8815) -4.9
4 Allyphenol (CID 68148) -5.5

Alpha Cedrene (CID 6431015) -8.0

Alpha Muurolene (CID 12306047) -7.6

Alpha Pinene (CID 10657) -5.1

Anethol (CID 637563) -4.8

B Cadinene (CID 10657) -8.0

Beta_Caryophyllene (CID -5.2

5281515)

Beta Caryophyllene (CID 3314) -5.7

Beta Caryophyllene (CID 5281515) -6.2

Beta Copaene (CID 87529) -6.8

Beta Pinene (CID 14896) -5.2

beta Silinene (CID 10856614) -6.8

Campesterol (CID 173183) -8.1

Cryophylleneoxide (CID 1742210) -6.5

Copaene (CID 19725) -6.9

Eucalyptol (CID 2758) -6.1

Eugenitin (CID 3083581) -7.5

Eugenol (CID 3314) -5.3

BETA SELININE (CID 442393) -6.9

Cryophylleneoxide (CID 1742210) -6.5

Copaene (CID 19725) -6.9

Eucalyptol (CID 2758) -6.1

Eugenylacetate (CID 7136) -6.0

Gallic acid ( CID 370) -6.5

Globulol (CID 12304985) -6.7

Jasmone (CID 1549018) -6.0

Kaempferol (CID 5280863) -9.3

Ledol (CID 92812) -6.4

Maslinic acid (73659) -8.3

Methyl Salicylate (CID 4133) -5.0

Oleonolic acid (CID 10494) -8.0

Rhamnetin (CID 5281691) -8.9

Stigmasterol (CID 5280794) -7.9

Black pepper
(2E,4E)-2-Methyl-1-[(S)-2- -6.7
(pyrrolidine-1-carbonyl)-pyrrolidin-
1-yl]-octa-2,4-dien-1-one (CID
44292388)

(2E)-octadecanoyl] pyrrolidine -3.8

(CID 574659)

Hexadecanoylpyrrolidine -6.4
(CID 247220)

Pellitorine (CID 5318516) -5.1

Piperettine (CID 341140014) -6.6

Piperanine (CID 5320618) -7.9

Piperolein B (CID 21580213) -6.8

Piperolein A (CID 11141599) -7.6

6-cadinol (CID 6428423) -6.6

(E,Z)-Farnesol (CID 1549109) -4.9

beta-Guaiene (CID 15560252) -7.4

Linalool CID 6549) -4.9

Limonene (CID 22311) -6.1

Sabinene (CID 10887971) -6.1

beta-Pinene (CID 14896_ -5.8

b-sitostero (CID 222284) -6.6

Stigmasterol (CID 5280794) -7.9

Piperine (CID 638024) -6.8

PIPRIDINE (CID 8082) -3.9

Laung pepper
Bisdemethoxycurcumin (CID -8.5
5315472)

Coumaperine (CID 10131321) -7.8

demethoxycurcumin (CID 5469424) -6.4

Isopiperine (CID 1548913) -8.0

-4.2
Piperidin-4-ol (CID 79341)

Piperidine, 1-(5-(1,3- benzodioxol- -6.5

5-yl)-1-oxo-2,4-pentadienyl) (CID
4840)
Piperolactam A (CID 3081016) -9.4
Table
Table.2 “Lipinski’s rule of five” for the bioactive constituents of Ginger, clove, long pepper
and black pepper targeting human cathepsin L protease

S.no. Compound Molecular weight HBD HBA TPSA Water Lipophilicity Druglikeness
solubility

Ginger
1. 6 gingerdiol 296.40g/mol 3 4 69.92Å soluble 3.13 0.55
(CID 11369949)
2 6 gingerol 294.39g/mol 2 4 66.76 Moderate 3.13 0.55
(CID 442793) Å soluble
3 8gingerdione 320.42g/mol 1 4 63.60 Moderate 3.81 0.55
(CID 14440537) Å soluble
4 10 gingerdione 348.48g/mol 1 4 63.60 Poor 4.58 0.55
(CID 14440539) Å soluble
5 10 Paradol 334.49g/mol 1 3 46.53 Poor 5.39 0.55
(CID 51352076) Å soluble
6 10 shagaol 332.48/mol 1 3 46.53 Poor 5.24 0.55
(CID 6442612) Å soluble
7 6 Paradol 278.39/mol 1 3 46.53 Moderate 3.96 0.55
(CID 94378) Å soluble
8 Zingiberene 204.35/mol 0 0 0Å soluble 4.47 0.55
(CID 92776)
Clove
9 Alpha Cedrene 204.35/mol 0 0 0Å soluble 4.36 0.55
(CID 6431015)
10 Alpha muurolene 204.35g/mol 0 0 0Å soluble 4.08 0.55
(CID 12306047)
11 Beta 204.35g/mol 0 0 0Å soluble 4.06 0.55
cadinene(CID
10657)
12 Eugenitin (CID 220.22g/mol 1 4 59.67 soluble 2.12 0.55
3083581) Å
13 Eugenol (CID 164.20g/mol 1 2 29.46 soluble 2.25 0.55
3314) Å
14 Jasmone (CID 164.24g/mol 0 1 17.07 soluble 2.74 0.55
1549018) Å
15 Kaempferol (CID 286.24g/mol 4 6 111.13 soluble 1.58 0.55
5280863) Å
16 Rhammetin (CID 316.26g/mol 4 7 120.36 soluble 1.63 0.55
5281691) Å
Long pepper
17 Bisdemethoxycur 308.3g/mol 2 4 74.60 soluble 2.83 0.55
cumin (CID Å
5315472)
18 Caumaperine 257.33g/mol 1 2 40.54 soluble 2.74 0.55
(CID 10131321) Å
19 PiperolactanA 265.26g/mol 2 3 62.32Å soluble 2.85 0.55
(CID: 30810116)
Black pepper
21 Piperolein B 343.46 g/mol 0 3 38.77 Modera 4.47 0.55
(CID Å te
21580213) soluble
22 Sabinene (CID 136.23 g/mol 0 0 0.00 Soluble 3.25 0.55
10887971) Å
20 (2E,4E)-2- 304.43g/mol 0 2 40.62Å Soluble 2.79 0.55
Methyl-1-[(S)-2-
(pyrrolidine-1-
carbonyl)-
pyrrolidin-1-yl]-
octa-2,4-dien-1-
one (CID
44292388)
Table

Table.3 Toxicity evaluation of bioactive constituents of ginger, clove, long pepper and
black pepper

Compounds Mutagenic Tumorigenic Irritant Reproductive Effect

Ginger

6 gingerdiol (CID 11369949) None None None None

6 gingerol (CID 442793) None None None None

8gingerdione (CID 14440537) None None None None
10gingerdione(CID14440539) None None None None

10 Paradol (CID 51352076) None None None None

10 shagaol(CID 6442612) None None None None
6 Paradol (CID 94378) None None None None

Zingiberene (CID 92776) None None None None

Clove
AlphaCedrene (CID 6431015) None None None None

Alpha muurolene (CID None None None None

12306047)
Beta cadinene (CID 10657) None None None None
Eugenitin (CID 3083581) None None None None
Eugenol (CID 3314) None None None None
Jasmone (CID 1549018) None None None None
Kaempferol (CID 5280863) None None None None
Rhammetin (CID 5281691) None None None None
Long pepper
Bisdemethoxycurcumin (CID None None None None
5315472)
Caumaperine (CID 10131321) None None None None
PiperolactanA(CID30810116) None None None None

Black pepper

Piperolein B (CID 21580213) None None None None

Sabinene (CID 10887971) None None None None

(2E,4E)-2-Methyl-1-[(S)-2- None None None None
(pyrrolidine-1-carbonyl)-
pyrrolidin-1-yl]-octa-2,4-dien-1-
one (CID 44292388)
Figure Click here to access/download;Figure;Fig.1.tif
Figure Click here to access/download;Figure;Fig.2.tif
Figure Click here to access/download;Figure;Fig.3.tif
Figure Click here to access/download;Figure;Fig.4.tif