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Discoveries in Agriculture and Food Sciences - Vol. 12, No. 4
Publication Date: August 25, 2024
DOI:10.14738/dafs.124.16859.
Kaewsrichan, J., Wongwitwichot, P., Tipmanee, V., & Pengnoo, A. (2024). Productive Growing System of Andrographis Paniculata
and Inhibitions of Reverse Transcriptase and 3clpro Enzymes by the Isolated Andrographolide. Discoveries in Agriculture and Food
Sciences, 12(4). 21-34.
Services for Science and Education – United Kingdom
Productive Growing System of Andrographis Paniculata and
Inhibitions of Reverse Transcriptase and 3clpro Enzymes by the
Isolated Andrographolide
Jasadee Kaewsrichan
Department of Pharmaceutical Chemistry and Drug Delivery System Excellence
Center, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai,
Songkhla, 90112, Thailand
Paweena Wongwitwichot
Department of Pharmaceutical Chemistry and Drug Delivery System Excellence
Center, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai,
Songkhla, 90112, Thailand
Varomyalin Tipmanee
Department of Biomedical Sciences and Bioengineering, Faculty of Medicine,
Prince of Songkla University, Hat-Yai, Songkhla, 90112, Thailand
Ashara Pengnoo
Department of Earth Science, Faculty of Natural Resources,
Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand
ABSTRACT
Molecular interactions between andrographolide (ADP) and proteins/enzymes of
different pathogenic viruses were computationally predicted by using docking
method. Binding energy corresponded to a protein-ADP pairing was theoretically
accounted in comparison with other current antiviral drugs as positive ligands. In
silico, interactions between ADP and PLpro (PDB, 6WX4), ADP and 3CLpro (PDB,
7LMD), ADP and ACE-2 receptor-spike protein complex (PDB, 6M0J), as well as ADP
and M2 protein (PDB, 6BKK) were stronger than those of between rimantadine,
remdesivir, lopinavir, or ritonavir and the described proteins. The inhibitions of
ADP against reverse transcriptase and 3CLpro activities were greater than those of
lopinavir, ritonavir, and oseltamivir, according to in vitro assays. ADP was isolated
as a major active compound from Andrographis paniculata grown outdoor in newly
established, cost-effective, hydroponic system. An average fresh mass per plant was
as high as 170 g from a 35x35 cm2 spacing hole. Simple and productive procedures
were recently suggested for ADP extraction. These acquired data would be applied
for pharmaceutical manufacturing.
Keywords Andrographis paniculate, Andrographolide, Molecular docking, Reverse
transcription, 3C rhinoviral protease, Outdoor hydroponic system
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Discoveries in Agriculture and Food Sciences (DAFS) Vol 12, Issue 4, August- 2024
Services for Science and Education – United Kingdom
INTRODUCTION
Viruses are biological entities capable of replication in living organisms only. Distinct
mechanisms have been used by a virus to affect an organism. This is why a virus that causes
illness in cats may not infect humans. There are serious and persistent threats from viruses to
humankind at present, due to the emergence, re-emergence, or evolution of drug-resistant
strains. Consequently, continuous search of new and potent antiviral drugs is important. Viral
life cycle is termed for a group of stages used by a virus to infect cells. These include (i),
attachment to host cell membrane; (ii), entry into host cells; (iii), genome uncoating, replication,
transcription, and translation; (iv), assembly; and (v), budding and release. Each stage is
completed by particular enzymes, which indicated to be dependent on the genome of the
infected virus. For instance, replication of most DNA viruses arises in the host cell nucleus and
is carried out by the host enzymes. Another large DNA virus, called Vaccinia, is an exception in
that replication and transcription occur in the host cell cytoplasm using enzymes encoded by
the infected virus. Genetic information of RNA viruses is stored in the form of RNA molecules.
Their replication can be achieved by means of RNA-dependent RNA polymerase (RdRp) or
RNA-dependent DNA polymerase (so-called reverse transcriptase) for RNA or cDNA synthesis,
respectively. Indeed, the viruses must encode reverse transcriptase on their own because of not
available from cells of humans. Then, integration between the cDNA and the host genome can
be happened, leaving the viral genome persistent in the infected host cells. Consequently, it is
helpful to hinder viral infection, replication, and spreading by inactivation of these associating
enzymes [1], which have been drug targets for searching new lead compounds by
computational methodology.
Andrographolide (ADP) is a major secondary metabolite of Andrographis paniculata with
diterpenoids structure. The herb has been used in traditional Eastern medicine for treatments
such as common cold, sore throat, liver disease, jaundice, diabetes, dysentery, chronic malaria,
and snake bite [2,3], and be included in Thai Herbal Pharmacopoeia since 2017. Few years ago,
the potentials of ADP and the A. paniculata extracts against SARS-CoV-2 were reported [4]. Hard
capsules containing dried powder or extracts of the herb were dispensed to COVID-19 patients
with mild symptoms in Thailand of which the treatment outcomes were desirable. Later,
different interactions between ADP and structural or non-structural proteins of SARS-CoV-2
such as papain-like protease, coronavirus main proteinase, and the spike protein, were
demonstrated by many in silico researches [5]. By network pharmacological analysis, immune
modulatory effects of ADP on p53 signaling and chemokines/natural killer cell mediating
cytotoxicity have been apparent, including its inhibitions towards infectious viral strains like
herpes simplex virus 1, human immunodeficiency virus, influenza A virus, hepatitis C virus, as
well as dengue virus. So far, broad inhibitory spectra of ADP have been indicated [6]. However,
molecular mechanisms that underline the described therapeutic actions of ADP are scant at
present, leading to the discoveries important.
In this research, molecular docking method was carried out to predict what proteins/enzymes
of human infectious strains of viruses are potential as ADP targets. By comparing with other
commonly used antiviral drugs, possible/attractive interactions based on binding energy
(kcal/mole) could be suggested. Two enzymatic assays [7] were performed in vitro for insisting
the computationally simulated results. The recently used ADP was purified from A. paniculata
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Kaewsrichan, J., Wongwitwichot, P., Tipmanee, V., & Pengnoo, A. (2024). Productive Growing System of Andrographis Paniculata and Inhibitions of
Reverse Transcriptase and 3clpro Enzymes by the Isolated Andrographolide. Discoveries in Agriculture and Food Sciences, 12(4). 21-34.
URL: http://dx.doi.org/10.14738/dafs.124.16859
grown outdoor in our modified hydroponic garden. Purity and content of the ADP were
demonstrated by using HPLC technique [8]. Using our developed procedures for plating A.
paniculata and extracting ADP, it seemed to increase yield and quality of the extracted ADP
(closures on planting of A. paniculata and purification of ADP by two petty patents). The
obtained data might be applicable in producing a pharmaceutical grade of ADP for
manufacturing scale.
METHODS
Chemicals and Reagents
Chemicals and solvents of analytical/chromatographic grades were used. Methanol and
absolute ethanol were bought from Merck (Darmstadt, Germany). ADP working standard
(98.15% purity) was obtained from Department of Medical Sciences Reference Standards,
Ministry of Public Health, Thailand. Abacavir sulfate, lopinavir, ritonavir, and oseltamivir
phosphate were purchased from Sigma-Aldrich. Mouse muscle cell line, C2C12, was acquired
from ATCC. Chemicals/reagents for cell culture technique such as Dulbecco’s Modified Eagle
medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, fungizone, RIPA buffer,
etc., were purchased from Gibco (Life Technologies, NY, USA). Oligonucleotide primers were
ordered from Macrogen HQ (Seoul, Republic of Korea). The HRV 3C protease activity assay kit
(Colorimetric) was bought from BioVision (MA, USA).
Molecular Docking Study
Three-dimensional structures of ADP (Pubchem ID: 5318517) and oseltamivir (Pubchem ID:
65028) were downloaded from PubChem database, while those of lopinavir (ChemSpider ID:
83706) and ritonavir (ChemSpider ID: 347980) were from ChemSpider server. Translation to
Protein Data Bank (PDB) format files by using Online SMILES Translator and Structure File
Generator (https://cactus.nci.nih.gov/translate/) was then performed. Crystal structures of
enzymes were acquired from RCSB protein database (http://www.rcsb.org). These included
influenza A neuraminidases: H1N1 (PDB ID: 6HP0), H3N2 (PDB ID: 4GZT), H3N8 (PDB ID:
4WA4), N4 (PDB ID: 2HTW); influenza A M2 protein (PDB ID: 6BKK); hepatitis C virus
nonstructural protein 5B (NS5B) isolate 1 (PDB ID: 4TLR) and subtype 1b (PDB ID: 5CZB);
hepatitis B virus mutant capsid proteins: (PDB ID: 5T2P) and (PDB ID: 6J10); SARS CoV-2
proteases: 3-chymotrypsin-like protease (PDB ID:7LMD) and papain-like protease (PDB ID:
6WX4); and ACE-2 receptor-spike protein complex (PDB ID: 6M0J). Any attached molecules
including water were dissected from the crystal structures. But polar hydrogen atoms were
added. These were operated by using AutoDock4. Files of target proteins and test ligands were
saved as PDBQT format for molecular docking. Again, AutoDock4 was used for selection of
active site residues and molecular docking Grid, and docking protocols of the active site
predictions. Grid site was set spacing at 0.375 Å. The x-y-z dimensions were set at 120-120-120
Å3. The rigid entity of protein structures was used. But the ligand was set as a flexible molecule.
Docking study was rendered by using the Lamarckian genetic algorithm (GA) and implemented
by AutoDock4. The number of GA runs was 50 with a popular size of 200. Binding energy
(ΔGbind) was analyzed by using ADT. Interactions were visualized by using BIOVIA Discovery
Studio (BIOVIA, 2020). Protein-ligand structure at the binding site was visualized by using
Visual Molecular Dynamics (VMD) package.