DETERMINING THE PHARMACOLOGICAL THERAPEUTIC PROPERTIES OF A NOVEL PEPTIDE FROM ANDROGRAPHIS PANICULATA EP1-LIKE GLYCOPROTEIN 2 AGAINST ROS GENERATED BY ETHANOL IN ZEBRAFISH LARVAL MODEL

Abstract

Research into peptide-based therapeutics is gaining increasing attention due to their specificity for targets and minimal adverse effects or toxicity. However, the bioactivity of peptides still requires further exploration of their physicochemical properties, amino acid sequences, and structural configurations. Peptides can be derived from a range of sources, including plants, animals, microbes, and chemical synthesis. This study focuses on identifying a novel bioactive peptide derived from the EPI-Iike glycoprotein 2 of Andrographis paniculata. The peptide sequence was predicted using the computational tool HeliQuest, and molecular docking studies were conducted with the web-based algorithm HPEPDOCK. The peptide identified, named AN 16 with Lhe sequence "APLSLLLLFLTLQSAN," consists of 16 amino acids and was assessed for its antioxidant potential using an ethanol-mediated oxidative stress model in zebrafish larvae. Toxicity analysis in the zebrafish larval model, including heart rate, survival rate, and morphological assessments, demonstrated that the AN 16 peptide is non-toxic. The peptide's efficacy was further supported by reductions in reactive oxygen species (ROS) levels and regulation of antioxidant enzyme levels. Molecular docking results confirmed that AN 16 interacts with proteins involved in antioxidant proteins. Overall, the data highlight the potential therapeutic applications of the AN 16 peptide in reducing damage associated with oxidative stress.

Specification

PREAMBLE TO THE DESCRIPTION

THE FIELD OF INVENTION (Pharmaceuticals)
This innovation is in alignment with advancements in biotechnology and pharmaceuticals, especially
in the field of peptidomic therapeutics, which uses peptides obtained from naturally occurring sources
such as plants. It highlights the control of reactive oxygen species (ROS) levels and the functions of
glutathione peroxidase (GPX), catalase (CAT), and superoxide dismutase (SOD), showing
encouraging possibilities in pharmaceutical development and functional supplements.
BACKGROUND OF THE INVENTION
The demand for novel therapeutic agents with low side effects is highlighted by challenges related to
the physicochemical qualities, targeted delivery, bioavailability, and efficacy of therapeutic drugs.
Peptides derived from natural sources are renowned for their high bioactivity, notable specificity, and
no adverse side effects. Research was done on the plant Andrographis paniculata, which is wellknown
for its medicinal properties. Specifically, the peptide sequence AN 16 that is produced from
its EPI-like glycoprotein 2 was studied. Using a model of zebrafish larvae exposed to ethanolinduced
reactive oxygen species (ROS), the antioxidant activity of AN16 was evaluated. To
demonstrate the potential biological and therapeutic implications of AN 16, this work attempts to
clarify the active mechanism behind its antioxidant properties.
SUMMARY OF THE INVENTION
The AN16 peptide, which is produced from Andrographis paniculata's EPl-like glycoprotein 2, was
investigated for its antioxidant potential. The investigation employed bioinformatics tools like
HPEPDOCK (peptide-protein docking server), Peptide Ranker, HeliQuest, and to investigate the
antioxidant potency of ethanol-mediated reactive oxygen species (ROS) in the zebra fish model. The
study evaluated the levels of reactive oxygen species (ROS) in zebra fish larvae as well as the activity
of the antioxidant enzymes glutathione peroxidase (GPX), catalase (CAT), and superoxide dismutase
(SOD). The heart rate and survival rates of the larval zebra fish were assessed to perform the toxicity
analysis. Based on its peptide ranking score and binding affinity with proteins implicated in
neurotransmitter processes, the AN 16 peptide was chosen. Results demonstrated that the AN 16
peptide greatly lowers ROS levels and modifies enzyme function, demonstrating that it might possess
therapeutic and protective impacts.


COMPLETE SPECIFICATION
Specifications
• The NCB I database provided the protein sequence of Demographics paniculata EP 1-like
glycoprotein 2 (accession ID XP 051152319.1). The peptide sequence and its helical structure
were determined by machine learning predictions with HeliQuest.
• PeptideRanker and the peptide calculator were used to assess the predicted peptide sequences'
physicochemical characteristics. These investigations led to the selection of the peptide AN 16,
which has the sequence "APLSLLLLFLTLQSAN." Its three-dimensional structure was verified,
and a molecular docking study was performed.
• The AN 16 peptide demonstrated remarkable interactions and binding scores with antioxidant
proteins, according to the results of molecular docking. Research on the developmental toxicity
of the AN 16 peptide in zebra fish larvae showed that exposure to it up to 96 hours after fertilisation
(hpf) did not result in any negative effects. Its therapeutic potential is further demonstrated by the
decrease in ethanol-mediated oxidative stress in zebrafish larvae.
• By regulating reactive oxygen species (ROS), this study significantly increases the antioxidant
potency of the AN 16 peptide. Its extensive effectiveness in treating conditions related to oxidative
stress.
• Furthermore, our results indicated that the AN 16 peptide's antioxidant capacity mitigated the
production of intercellular ROS in zebrafish larvae.




DESCRIPTION
The NCB! database provided the protein sequence of Andrographis paniculata EP 1-like glycoprotein
2 (accession ID XP_051152319.1). Through HeliQuest, machine learning algorithms were used to
predict the peptide sequence and its helical structure. To assess the predicted peptide sequences'
physicochemical characteristics, PeptideRanker and the peptide calculator were utilised. The
sequence "APLSLLLLFL TLQSAN" found in peptide AN 16 was chosen as a result of this
investigation. Molecular docking investigations were carried out and the 3D structure of AN 16 was
validated. The AN 16 peptide demonstrated notable interactions and binding scores with antioxidant
proteins, according to the molecular docking data. Studies on the developmental toxicity of AN 16 in
zebrafish larvae showed no negative effects from exposure to the compound up to 96 hours after
fertilisation (hpf). Additionally, the therapeutic potential of the peptide was highlighted by its ability
to reduce ethanol-mediated oxidative stress in zebrafish larvae. The study highlights the AN 16
peptide's increased antioxidant capacity via regulating reactive oxygen species (ROS), demonstrating
its broad applicability in treating oxidative stress-related conditions.



CLAIM
We Claim
I.· Claim: A strategy to manufacture the AN 16. peptide, which is generated from Demographis
paniculata's EP 1-like glycoprotein 2, and then employ mole~ular docking techniques to investigate its
antioxidant efficacy. ·
2. Claim: The AN 16 peptide from Andrographis paniculata EP 1-like glycoprotein 2 targets ethanolmediated
ROS problems in zebrafish larvae and is intended to provide antioxidant protection,
potentially finding use in other organisms.
3. Claim: An ANI6 peptide formulation that can be employed in biomedical research, derived from
Andrographis paniculata EP 1-like glycoprotein 2, exhibits significant antioxidant activity by
protecting cell damage caused by ethanol-mediated oxidative stress.
4. Claim: Pharmacological composition comprising the 16 amino acid AN 16 peptide, which is
derived from A nrlrographis paniculata EP 1-like glycoprotein 2, exhibits strong antioxidant activity
in zebra fish larvae by lowering ethanol-mediated intracellular ROS modulating essential antioxidant
enzymes such as glutathione peroxidase (GPX), catalase (CAT), and superoxide dismutase (SOD).
5. Claim: An environmentally safe and sustainable AN 16 peptide that functions as an antioxidant and
provides a biodegradable solution for managing oxidative stress and environmental conservation
initiatives. It is produced from Andrographis paniculata EPl-like glycoprotein 2.

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