EVALUATION OF THE THERAPEUTIC POTENTIALS OF CYPRINUS CARPIO-DERIVED KK14 PEPTIDE ON INTERCELLULAR RE

Abstract

This study explores the antioxidant properties of peptide derived from zebrafish using a zebrafish larvae model subjected to oxidative stress. The KK 14 peptide sequence was retrieved from the the liver-expressed antimicrobial peptide-28 (LEAP 28) of Cyprinus carpio L., and 14-amino acid peptides with an alpha-helical structure were generated using the HeliQuest tool. The peptide structures were screened with PeptideRanker, and KK 14 (KIRMSPL WRIMGFK) was selected based on its predicted bioactive potential. Concentrations of the peptide ranging from 5 to 45 f!M were tested in vitro and in vivo using a zebrafish model. Using DCFDA and acridine orange staining, the effects of KK14 on intracellular reactive oxygen species (ROS) levels were assessed. Zebrafish larvae were exposed to oxidative stress through hydrogen peroxide (H202) and subsequently treated with the KK14 peptide. Results showed that KK 14 significantly reclured intracellular ROS levels and stress-induced apoptosis in zebrafish larvae, indicating a strong antioxidant effect. These finding suggest that the KK14 peptide has therapeutic potential for oxidative stress-related disorders. This study highlights KK14 peptide as a promising antioxidant candidate for biomedical research and drug development.

Specification

PREAMBLE TO THE DESCRPTION
THE FIELD OF INVENTION (Pharmaceuticals)
The innovation domain centers on advancements in biotechnology and 'p harmaceuticals, particularly
in antioxidant therapy and the development of novel peptides from natural sources. The aim is to
leverage these innovations to manage oxidative stress and enhance antioxidant functions, with
potential applications in pharmaceutical formulations, nutraceuticals, and functional foods.
BACKGROUND OF THE INVENTION
Oxidative stress, resulting from an imbalance between reactive oxygen species (ROS) production and
the antioxidant defense system, contributes to numerous diseases and disorders. Antioxidants are
essential for neutralizing ROS and reducing oxidative damage, presenting potential therapeutic
strategies for managing oxidative stress-related conditions. Fish-derived antimicrobial peptides
(AMPs) have demonstrated antioxidant potential due to their diverse biological activities. The
common carp (Cyprinus carpio L.), a key dietary fish in China and other regions, is a promising
source of these peptides. Research indicates that AMPs may serve as effective therapeutic agents for
various diseases. Notably, two hydrophobic peptides derived from P-defensin genes, skin mucus, and
eDNA sequences of hepcidin and LEAP-2A and LEAP 28 have been identified in common carp.
The LEAP 28 eDNA encodes a 91-amino acid sequence. This study aims to explore tlje antioxidant
potential of the KKI4 peptide, derived from LEAP 28 of Cyprinus carpio, and its applications in
antioxidant therapy and the development of novel pep tides for biomedical use.
SUMMARY OF THE INVENTION
The invention focuses on investigating the antioxidant properties of the KK14 peptide derived from
LEAP 28 using a zebrafish larvae model exposed to oxidative stress. Bioinformatics tools like
HeliQuest were utilized to retrieve the KK 14 sequence and generate peptides with alpha-helical
structures, while PeptideRanker was used to screen and select KK 14 based on its predicted
bioactivity. The effects of the KK14 peptide on intracellular ROS levels were assessed through
DCFDA and acridine orange staining. Zebrafish larvae, after being exposed to H20 2 to induce
oxidative stress, were treated with the KK 14 peptide. The effectiveness of the peptide in enhancing
antioxidant activity is assessed by measuring changes in key antioxidant enzyme activities, such as
superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GPx), and
glutathione S-transferase (GST), which are essential components of the antioxidant defense system .
The results showed a notable reduction in intracellular ROS and apoptosis levels, indicating a strong
antioxidant effect of the KKI4 peptide. To further understand the response at the transcriptional level,
the expression of antioxidant genes such as SOD, CAT, GPx, and glutathione reductase (GSR) is
examined in zebrafish larvae using the RT-PCR technique. This study highlights the potential of the
KK 14 peptide from Cyprinus carpio as a candidate for· antioxidant therapy, with promising
applications in biomedical research and drug development.
COMPLETE SPECIFICATION
Specifications
• The sequence ofKK14 peptide was obtained from the eDNA sequence of LEAP 2B peptide,
and predictive tools like HeliQuest were utilized to generate peptides with alpha-helical
structures.
• Subsequently, PeptideRanker was employed to assess and rank these sequences based on their
predicted bioactivity. Among the sequences generated, KK14 (KlRMSPLWRIMGFK) was
selected as it exhibited promising bioactive potential. Utilizing Chimera, the 3D structure of
the KK14 peptide was constructed to provide insights into its molecular architecture.
• The KK14 peptide was produced in GenScript, USA, and its purity was verified to be >98%
pure. The peptide was dissolved in HPLC water and a stock concentration of 1 mM was
prepared; it was then preserved at -20°C for future use in research.
• Synthesis of the KK14 peptide ensued, followed by its evaluation for antioxidant potential in
an in vivo zebrafish embryo and larvae model. Oxidative stress induction was achieved using
hydrogen peroxide (H202), simulating physiological conditions relevant to oxidative stressrelated
ailments.·
• Assessment of antioxidant enzyme levels like superoxide dismutase (SOD) and catalase
(CAT) glutathione S-transferase (GST), glutathione (GSH), and glutathione peroxidase (GPx)
enzymes in zebrafish larvae following treafment with KC 14 peptide was carried out.
• Zebrafish larvae subjected to stress were treated with KK14 peptide in a semi-static exposure
manner for up to 96 hours post fertilization (hpf). Assessment of intracellular reactive oxygen
species (ROS) levels and stress-induced apoptosis was conducted using DC FDA and acridine
orange (AO) staining, respectively.
• In-silica molecular docking analysis supported the in-vitro cell free and in-vivo zebrafish
enzyme estimation results. KC 14 peptide's molecular docking results revealed strong binding
energies and amino acid interactions with antioxidant proteins.
• This research signifies a significant step forward in antioxidant therapy, demonstrating the
potential of Cyprinus carpio-derived KK14 peptide to mitigate oxidative stress-related
ailments .
DESCRIPTION
The study focuses on evaluating the impact of KK 14 peptide derived from the LEAP 28 on
intracellular reactive oxygen species (ROS) levels within an oxidative stress-induced zebra fish larvae
model, aiming to elucidate its antioxidant properties. Leveraging computational tools such as
HeliQuest and PeptideRanker, the sequence of LEAP 28 was analysed and optimized, leading to the
selection of KKI4 based on its predicted bioactivity. The 30 structure of the KK14 peptide was
subsequently generated using computational modelling techniques. Following the synthesis of the
KKI4 peptide, an in vivo zebrafish embryo and larvae model was employed to assess its antioxidant
potential under conditions of oxidative stress induced by hydrogen peroxide (H202) exposure.
Zebrafish larvae were treated with KKI4 peptide in a semi-static exposure manner for a duration of
up to 96 hours post fertilization (hpf). Evaluation of intracellular ROS levels was conducted utilizing
DCFDA staining, providing insights into the ability of KKI4 peptide to mitigate oxidative stressinduced
ROS production. Additionally, stress-induced apoptosis levels were assessed using acridine
orange (AO) staining, offering further understanding of KK 14 peptide's impact on cellular responses
to oxidative stress. This study represents a significant advancement in antioxidant research,
highlighting the potential of Cyprinus carpio-derived KK 14 peptide as a promising candidate for
antioxidant therapy. Its efficacy in modulating intracellular ROS levels and mitigating oxidative
stress-induced cellular damage underscores its potential applications in-biomedical research and drug development for oxidative stress-related disorders.
CLAIM
We Claim
l. Claim: A process for synthesizing KKI4 peptide derived from LEAP 2B of Cyprinus carpio,
optimized for antioxidant therapy through computational modeling techniques.
2. Claim: A composition containing the KKI4 peptide designed for antioxidant therapy, aimed at
treating oxidative stress-induced disorders in zebra fish larvae, with potential applications for other
orgamsms.
3. Claim: A formulation containing the KK14 peptide for use in biomedical research, showcasing its
potential to reduce oxidative stress-induced cellular damage.
4. Claim: A pharmaceutical composition containing the KKI4 peptide for therapeutic applications
aimed at treating oxidative stress-related disorders, with promising implications for drug
development.
5. Claim: The KKI4 peptide, a sustainable and eco-friendly antioxidant agent, provides a
biodegradable solution for managing oxidative stress while supporting environmental conservation
efforts.

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