IN SILICO SCREENING OF BIOACTIVE COMPONENTS OF BASIL (Ocimum basili-

ucum) AS INHIBITOR TO FUSION PROTEIN OF Rotaviruses

A High School Thesis Presented to the Faculty of

HONORATO C. PEREZ SENIOR MEMORIAL SCIENCE HIGH SCHOOL
Mabini Extension, Cabanatuan City

In Partial Fulfilment
Of The Requirements For The Subject
SCIENCE RESEARCH II

DAOS, YOHAN 3RDY C.

ESCOREL, JOHN ALDRINE C.
MAGNO, HYUNG JIN KYLE A.

MARCH, 2020

i
 APPROVAL SHEET

We the members of the Thesis Examining Committee have found this study entitled: “IN SILICO
SCREENING OF BIOACTIVE COMPONENTS OF BASIL (Ocimum basiliucum) AS IN-
HIBITOR TO FUSION PROTEIN OF Rotaviruses
” conducted by the following researchers: DAOS, YOHAN 3RDY C., ESCOREL JOHN ALDRINE C., MAG-
NO, HYUNG JIN KYLE A., Acceptable and substantial: thereby passing the Mid Oral Defense.

__________________ _________________________ ____________________________

DENNIS S. BUNAG GLADYS JOY B. CAOILE R.N MARL CLESTER A. RUFINO Ph.D.
Member Member Member

__________ __________ __________

Date Signed Date Signed Date Signed

______________________ __________________ ____________________________

Ma. MENISA A. CABUAL ISMAEL A. DE LARA FAYE MARION C. SEBASTIAN R.Ch.
Member Member Member

__________ __________ __________

Date Signed Date Signed Date Signed

__________________________________
RAQUEL MOANA M ABOLENCIA Ed.D.
Research Adviser

__________
Date Signed

_________________________
LEONARA C. DE JESUS Ph.D.
Principal II

__________
Date Signed

ii
ACKNOWLEDGMENT

iii
 TABLE OF CONTENTS

CONTENTS PAGE

TITLE PAGE i

APPROVAL SHEET ii

ACKNOWLEDGMENT iii

TABLE OF CONTENTS iv
Table of Figures v
Table of Appendices vi
Table of Tables vii

ABSTRACT viii

BACKGROUND OF THE STUDY 1

Statement of the Problem 2

Hypothesis 3
Significance of the Study 3
Scope and Delimitation 3

METHODOLOGY 3

Methods of Research Used 3

Materials 3
General Procedure 3
Research Paradigm 3
Research Design 4
Statistical Analysis 5

RESULTS AND DISCUSSION 5

CONCLUSION 6

RECOMMENDATION 6

BIBLIOGRAPHY 6

APPENDICES ix

CURRICULUM VITAE xv

iv
IN SILICO SCREENING OF BIOACTIVE COMPONENTS OF BASIL (Ocimum basili-
ucum) AS INHIBITOR TO FUSION PROTEIN OF Rotaviruses

Basil, a herb belonging to the mint family and have an anti-viral property, will be used to know the possi-
bility if it could stop the reproduction of Rotavirus. And turns out, Basil seems more effective than a commercially
available drug in inhibiting Rotavirus.

The study began on finding the bioactive components of Basil which are mainly Eugenol, Geranial, Gera-
niol, Linalool, and Methyl Chavicol which then used to inhibit the binding sites of Rotavirus through software called
Arguslab which will give the researchers the numerical value if a component is effective or less effective in inhibi-
tion. The bioactive components of Basil did a good job but unfortunately 2 other drugs (Diphenoxylate, and
Loperamide) outdone them.

Results show that in inhibiting Rotavirus, only one out of three commercially available drugs is less effec-
tive than Basil, which is Pepto-Bismol.

In the inhibition of rotavirus, Methyl Chavicol was proven to be the best ligand in slowing down Rotavirus.
Although there are better drugs to be used, Basil will serve as another option in curing diarrhea.

viii
 IN SILICO SCREENING OF BIOACTIVE COMPONENTS OF BASIL
(Ocimum basiliucum) AS INHIBITOR TO FUSION PROTEIN OF Rota-
viruses
H.J.K MAGNO, Y. 3.C DAOS, J.A.C. ESCOREL
GRADE: X-BELARDO, X-DEL MUNDO
CATEGORY: BIOLOGY
SUB CATEGORY: ELECTRONICS
SCHOOL: HONORATO C. PEREZ SR. MEMORIAL SCIENCE HIGH SCHOOL
SCHOOL ADDRESS: MABINI EXTENSION, CABANATUAN CITY
AUTHORS’ EMAIL: magnohyungjinkyle0962@gmail.com, escorelaldrine@gmail.com, daosyohan@yahoo.com
INTRODUCTION
Basil, also known as Saint Joseph's Wort, is
an herb belonging to the mint family. It is used in
cooking and may have some health benefits.
Basil's proposed benefits include reducing inflamma-
tion, and it is said to have anti-aging and antibacterial
properties. (Nordqvist, J. Updated last January 3,
2018)
Ocimum basilicum is an annual plant found
in the wild tropical, subtropical and temperate regions
of the world. It is a member of the family labiate (la-
miaceae). It contains a wide variety of constituents of
medicinal importance. Ocimum basilicum is a com-
mon herb, grown in many households with a broad
range of therapeutic properties. It would be a blessing
in disguise if this herb becomes a medicine for the
common man. Various plant parts such as leaves,
seeds, and roots are recommended for the common
people as folk medicines. Ocimum basilicum has re-
puted medicinal uses as antioxidant, antibacterial,
antimicrobial, antifungal, antiviral, cytoprotective,
anticonvulsant, hypoglycaemic, hypolipidemic, hepa-
toprotective, renoprotective, neuroprotective, spermi-
cidal, dermatologic and insecticidal.
A plant of the tropics, it can also be grown in
the subtropics and as an annual in the warm temperate
zone. In the tropics, it succeeds up to an elevation of
1,000 meters. It grows best in areas where annual day-
time temperatures are within the range 18 - 27°c but
can tolerate 7 - 36°c. It is intolerant of frosts. It prefers
a mean annual rainfall in the range 1,000 - 1,600mm
but tolerates 600 - 4,300mm. Prefers a sunny, sheltered
position. Prefers a rich light well-drained to dry soil.
Prefers a pH in the range 6 - 7, tolerating. There are
reports that the plant can become a weed in cultivated
fields. The plant takes 70 - 110 days from germination
to first harvest, and can thereafter be cut up to three
more times at 60-day intervals, the harvesting taking
place just before the crop starts flowering.
The average yield is 1.2 - 1.5 tonnes per hec-
tare of dry, cleaned drug. Several different constituents
make up the essential oil in basil, and the proportions
of these vary considerably between plants growing in
different regions of the world. From this variety, many
named varieties with different flavor characteristics
have been developed. (Tropical Plants Database, Ken
Fern. tropical.theferns.info. 2019-09-26. <tropi-
cal.theferns.info/viewtropical.php?id=Ocimum+basilic
um>)
Rotavirus is a genus of double-stranded RNA
viruses in the family Reoviridae. Rotaviruses are the
most common cause of diarrhoeal disease among in-
fants and young children. Nearly every child in the
world is infected with rotavirus at least once by the age
of five. Immunity develops with each infection, so
subsequent infections are less severe; adults are rarely
affected. There are nine species of the genus, referred
to as A, B, C, D, E, F, G, H and I. Rotavirus A, the
most common species, causes more than 90% of rota-
virus infections in humans.
The virus is transmitted by the fecal-oral
route. It infects and damages the cells that line the
small intestine and causes gastroenteritis (which is
often called "stomach flu" despite having no relation to
influenza). Although Rotavirus was discovered in 1973
by Ruth Bishop and her colleagues by electron micro-
graph images and accounts for approximately one-
third of hospitalizations for severe diarrhea in infants
and children its importance has historically been un-
derestimated within the public health community, par-
ticularly in developing countries. In addition to its im-
pact on human health, rotavirus also infects animals
and is a pathogen of livestock
1
 Rotaviral enteritis is usually an easily man-
aged disease of childhood, but in 2013, rotaviruses
caused 37 percent of deaths of children from diarrhea
and 215,000 deaths worldwide, and almost two million
more became severely ill. Most of these deaths oc-
curred in developing countries. In the United States,
before initiation of the rotavirus vaccination program
in the 2000s, rotavirus caused about 2.7 million cases
of severe gastroenteritis in children, almost 60,000
hospitalizations, and around 37 deaths each year. Fol-
lowing the rotavirus vaccine introduction in the United
States, hospitalization rates have fallen significantly.
Public health campaigns to combat rotavirus focus on
providing oral rehydration therapy for infected chil-
dren and vaccination to prevent the disease. The inci-
dence and severity of rotavirus infections have de-
clined significantly in countries that have added a ro-
tavirus vaccine to their routine childhood immuniza-
tion policies.
Viral diarrheas are the cause of high mortality
among children and animals, including many mamma-
lian and avian species. But despite considerable re-
search over several decades, the mechanisms underly-
ing rotaviral diarrheal disease remain unclear com-
pared with those of bacterial secretory enterotoxins,
such as cholera toxin and the Escherichia coli heat-
labile and heat-stable toxins.
Rotavirus infection has always been consid-
ered to be confined to the upper two-thirds of the villi
of the small intestine, but recent reports suggest that
extra-intestinal manifestations may occur. Diarrhea can
occur with no visible tissue damage and, conversely,
the histological lesions can be asymptomatic. The se-
verity of intestinal histological lesions is clearly de-
pendent on both host and viral factors. However, even
when slight erosion of the epithelial surface was found
to exist in various animal models, there was no evi-
dence of any significant enterocyte loss or flattening of
the mucosa. Thus, the idea is gaining ground that diar-
rhea is not necessarily a consequence of any physical
lesion but can precede it, as if cell dysfunction were
the cause, not the consequence, of the histological
damage .
Rotavirus diarrhea was first considered to be
malabsorptive. Since 1996, the prevailing idea in the
rotavirus field is that the nonstructural NSP4 protein
might play a crucial role in fluid and electrolyte secre-
tion, and hence might represent a novel viral secretory
enterotoxin. Being absent from the mature, infective
virion particle, NSP4 needs to be synthesized in virus-
infected villus enterocytes and the NSP4 cleavage
product NSP4-(112–175) has been reported to be se-
creted into the extracellular medium and/or the intesti-
nal lumen. Like the full-length NSP4 and NSP4-(114–
135) peptide, the NSP4-(112–175) peptide induced
diarrhea in neonatal mice in the absence of any histo-
logical damage to the intestinal mucosa. It has been
hypothesized that the NSP4-(112–175) secreted pep-
tide would be available to bind a yet-unidentified api-
cal membrane receptor in villus and perhaps crypt en-
terocytes and enteroendocrine cells, to trigger a series
of events that would lead to secretory diarrhea. Recent
studies also indicate that NSP4 is released from the
basal side of infected enterocytes, but its role in rota-
virus disease remains to be defined. (Virol J. 4-31-
2007. How do the rotavirus NSP4 and bacterial enter-
otoxins lead differently to diarrhea?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC18390
81/)
Based on the study conducted by the World
Health Organization (WHO) in 2016, the death rate
from diarrhoeal diseases decreased by almost 1 million
between 2000 and 2016, but still caused 1.4 million
deaths which put diarrhoeal diseases the top 9 cause of
death worldwide. Still based on the study, it is stated
that lower-income countries have higher death per-
centage of diarrhoeal diseases compared to those of
higher-income countries. (WHO. May 24, 2018. The
top ten causes of death.)
The rotavirus nonstructural protein NSP4 was
the first viral enterotoxin discovered. It induces diar-
rhea and causes Ca2+-dependent transepithelial secre-
tion.
A transmembrane glycoprotein, NSP4 is or-
ganized into three main domains: a three-helical TM
domain in the N-terminus (also a viroporin domain), a
central cytoplasmic coiled-coil domain for multimeri-
zation, and a C-terminal flexible region. It can also be
secreted out of the cell. As of 2019, only structures of
the central domain, which is responsible for diarrhea,
has been solved. It oligomerizes into dimeric, tetram-
eric, pentameric, and even higher-order forms.
Due to the Anti-viral Property of Basil (Oci-
mum basilicum), the researchers have decided to con-
duct a research about the properties of its Bioactive
components to inhibit fusion proteins of Rotaviruses
and create a base study for medicinal companies to
create another alternative source of medicine for diar-
rhoeal diseases.
Statement of the Problem
The main focus of this study is to determine
and evaluate the bioactive components Basil (Ocimum
basilicum) as ligands to inhibit 3MIW (NSP4).
The researcher aims to find answers to the
following questions:
2
1. How may the enzyme-ligand interaction of
3MIW and bioactive components of Basil (Ocimum
basilicum) is described in terms of Binding affinity?
2. Is there any significant difference between the
binding affinity of the bioactive components of Basil
(Ocimum basilicum) and Diphenoxylate, Loperamide,
Pepto-Bismol as control?
3. In terms of inhibition, what ligand was de-
termined to be the best in terms of binding affinity
Hypotheses:
The following hypotheses are stated in the
null form.
1. There is no significant difference in the inhib-
iting efficiency of the bioactive components of Basil
(Ocimum basilicum)
2. There is no significant difference between the
inhibiting capacity of bioactive components of Basil
(Ocimum basilicum) and control against 3MIW.
3. There is no determined ligand to be the best
in inhibiting the Rotavirus
Significance of the Study
For the Researchers. This study will serve
as background for further in vitro and in vivo studies
in discovering drugs against 3MIW.
For the Community. This study helps in
search of anti-respiratory tract infection and anti-
respiratory inflammation from medicinal plants for the
demand for plant-based medicines that are considered
to be safer, non-toxic and less harmful than synthetic.
For the Pharmaceutical Industry. This study
will provide information about bioactive components
that could be isolated in Basil (Ocimum basilicum) as
potential drugs against the fusion protein of Rotavirus.
For the School. To help the students in con-
ducting new studies in research using in silico screen-
ing by technology and software.
Scope and Delimitation
This study is limited only in assessing the
inhibiting capacity of bioactive components of Basil
(Ocimum basilicum)) and to compare with rotavirus as
controls against 3MIW and active sites.
The main focus of this study is to identify the
bioactive components of Basil (Ocimum basilicum)
that will be bind to inhibit the 3MIW. It will be com-
pared to rotavirus as controls. ArgusLab Software will
be used to determine its effectiveness. A total of three
trials were conducted each of the bioactive constitu-
ents and the results gathered were analyzed using T-
test and Analysis of Variance.
METHODOLOGY
To provide the general approach and tools
they will use in the study, it will discuss the methods
of research used including measurement and controls
in any changes in the variables.
Methods of Research Used
The type of research that the researchers are
going to apply is Experimental Research.
The researcher used books, the Internet and
other references in conducting this study. Not all data
came from the researchers themselves.
Materials
 Laptop
 Arguslab Software
General Procedures
Gathering of Data
Materials and Tools
Searching For
Bioactive Components
Docking of the
Molecules
Interpretation
Figure 1. Shows the Procedures for the in silico
screening of bioactive components of Basil (Ocimum
basilicum) as inhibitor to fusion protein of rotaviruses.
3
 a) In phase 1, the researchers have
gathered all the data, materials Table 2 Binding Affinity of the bioactive components
and tools needed for the study of Basil (Ocimum basilicum): Eugenol, Geranial, Ge-
from multiple reliable sources. raniol, Linalool, Methyl Chavicol (Estragole) com-
pared to Loperamide in inhibiting sites of 3MIW.
b) In phase 2, the researchers Re- Ligand Mean t t Inter-
searched for the bioactive com- kcal/mol calc crit preta-
ponents and their molecular tion
structure of Basil (Ocimum basil- Bind- Eugenol
ing Loperami
icum) to be used for dockings. Site de
Bind- Geranial
c) For phase 3, the researchers have ing Loperami
done the dockings of the ligands Site de
from Basil (Ocimum basilicum) Bind- Geraniol
ing Loperami
and the controls to the residues of
Site de
rotavirus using Arguslab. Bind- Linalool
ing Loperami
d) For phase 4, the researchers have Site de
interpreted the data from dock- Bind- Estragole
ing Loperami
ings made from phase 2. Which
Site de
shows who is better in inhibiting
Rotavirus. Table 3 Binding Affinity of the bioactive components
of Basil (Ocimum basilicum): Eugenol, Geranial, Ge-
Research Design raniol, Linalool, Methyl Chavicol (Estragole) com-
pared to Pepto-Bismol in inhibiting sites of 3MIW.
Table 1 Binding Affinity of the bioactive components
of Basil (Ocimum basilicum): Eugenol, Geranial, Ge- Ligand Mean t t Inter-
raniol, Linalool, Methyl Chavicol (Estragole) com- kcal/mol calc crit preta-
pared to Diphenoxylate in inhibiting sites of 3MIW. tion
Bind- Eugenol
Ligand Mean t t Inter- ing Pepto-
kcal/mol calc crit preta- Site Bismol
tion Bind- Geranial
Bind- Eugenol ing Pepto-
ing Diphe- Site Bismol
Site noxylate Bind- Geraniol
Bind- Geranial ing Pepto-
ing Diphe- Site Bismol
Site noxylate Bind- Linalool
Bind- Geraniol ing Pepto-
ing Diphe- Site Bismol
Site noxylate Bind- Estragole
Bind- Linalool ing Pepto-
ing Diphe- Site Bismol
Site noxylate
Bind- Estragole
ing Diphe-
Site noxylate

4
Statistical Analysis Ligand Mean t calc t In-
kcal/mol crit ter-
The statistical analysis used was T-test pre-
ta-
Results and Discussion tion
Bind- Eugenol -6.16 3.181E 2 ≠
Table 4 Binding Affinity of the bioactive components ing Loperami -7.58 -09
of Basil (Ocimum basilicum): Eugenol, Geranial, Ge- Site de
raniol, Linalool, Methyl Chavicol (Estragole) com- Bind- Geranial ≠
-6.18 2.07E- 2
pared to Diphenoxylate in inhibiting sites of 3MIW. ing Loperami -7.58 08
Site de
Ligand Mean t t Inter- Bind- Geraniol ≠
-6.21 1.895E 2
kcal/mol calc crit preta- ing Loperami -7.58
tion -08
Site de
Bind- Eugenol -6.16 0.07 2 ≠ Bind- Linalool ≠
ing
-6.17 8.558E 2
Diphe- -7.07 ing Loperami -7.58
Site -09
noxylate Site de
Bind- Geranial -6.18 0.08 2 ≠ Bind- Estragole -6.29 4.369E 2 ≠
ing Diphe- -7.07 ing Loperami -7.58
Site
-08
noxylate Site de
Bind- Geraniol -6.21 0.09 2 ≠ Legend: ≠ - There is no significant difference.
ing Diphe- -7.07
Site noxylate The table above shows the comparison of the
Bind- Linalool -6.17 0.08 2 ≠ control (Loperamide) and the bioactive components of
ing Diphe- -7.07 basil. (The more negative the better) This table shows
Site noxylate that Loperamide is better than the bioactive compo-
Bind- Estragole -6.29 0.1 2 ≠ nents. And that there is no significant difference be-
ing Diphe- -7.07 tween them
Site noxylate
Legend: ≠ - There is no significant difference. Table 3 Binding Affinity of the bioactive components
of Basil (Ocimum basilicum): Eugenol, Geranial, Ge-
The table above shows the comparison of the raniol, Linalool, Methyl Chavicol (Estragole) com-
control (Diphenoxylate) and the bioactive components pared to Pepto-Bismol in inhibiting sites of 3MIW.
of basil. (The more negative the better) This table
shows that Diphenoxylate is better than the bioactive Ligand Mean t t Inter-
components. And that there is no significant difference kcal/mol calc crit preta-
between them tion
Bind- Eugenol -6.16 0.02 2 ≠
Table 5 Binding Affinity of the bioactive components ing Pepto- -5.97
of Basil (Ocimum basilicum): Eugenol, Geranial, Ge- Site Bismol
raniol, Linalool, Methyl Chavicol (Estragole) com- Bind- Geranial -6.18 0.04 2 ≠
pared to Loperamide in inhibiting sites of 3MIW. ing Pepto- -5.97
Site Bismol
Bind- Geraniol -6.21 0.02 2 ≠
ing Pepto- -5.97
Site Bismol
Bind- Linalool -6.17 0.04 2 ≠
ing Pepto- -5.97
Site Bismol
Bind- Estragole -6.29 0.01 2 ≠
ing Pepto- -5.97
Site Bismol

5
Legend: ≠ - There is no significant difference.
The table above shows the comparison of the
control (Pepto-Bismol) and the bioactive components
of basil. (The more negative the better) This table
shows that Pepto-Bismol is worse than the bioactive
components.
Conclusions
The bioactive components of Basil (Ocimum
basilicum) were docked to the active sites of 3miw
(NSP4) to inhibit the attachment of the virus to the
host among the bioactive components of Basil (Oci-
mum basilicum). Methyl Chavicol (Estrgole) is deter-
mined to be the best ligand to inhibit. Statistical analy-
sis shows that Methyl Chavicol (Estrgole) is more ef-
fective than a commercially available drug (Pepto-
Bismol) in inhibiting 3miw.
Although there are better drugs to be used,
Basil will serve as another option in curing diarrhea
Bibliography
Mesde, L. A. C. Tambalque, E. J. A.
December 14, 2016. IN SILICO SCREENING
OF BIOACTIVE COMPONENTS OF TAWA-
TAWA (Euphorbia hinta Linn.) AS INHIBI
TOR TO FUSION PROTEIN OF Paramyxo
viridae viruses. https://prezi.com/bmofv7-
6w9lk/in-silico-screening-of-bioactive-
components-of-tawa-tawa-e/
Nordqvist, J. January 3, 2018
Why everyone should eat basil.
https://www.medicalnewstoday.com/articles/2
66425.php
Fern K. Tropical Plants Database,
Tropical.theferns.info. 2019-09-26. <tropi
cal.theferns.info/viewtropical.php?id=Ocimu
m+basilicum>

Recommendation World Health Organization. May 24, 2018.

The top ten causes of death.
This study only focuses on the fusion of the https://www.who.int/news-room/fact-
host to the viruses that cause diarrhoeal diseases. It is sheets/detail/the-top-10-causes-of-death
highly recommended for future researchers to:
Virol J. 4-31-2007. How do the rotavirus NSP4 and
1. Isolate Eugenol, Geranial, Geraniol, Lin- bacterial enterotoxins lead differently to diar
alool, Methyl Chavicol from Basil (Oci- rhea?
https://www.ncbi.nlm.nih.gov/pmc/articles/P
mum basilicum) MC1839081/

2. Do a vitro and in vivo analysis for further

validation of the inhibiting capacities.

3. To conduct a new study focusing on the

prevention of the attachment of the viral
pathogens to the host and the future re-
searchers can conduct an enhanced study
with another set of medicinal plants
available in our country

6
Appendices A Tables

ix
Appendices B Formula
Appendices C Pictures