A gold thread cliff honey polypeptide with anti-enteritis activity

Abstract

The present application provides a kind of polypeptide with anti-inflammatory and intestinal barrier protection activity, and the amino acid sequence of the polypeptide is SEQ ID NO:1.The present application realizes the fine application of active components from natural product crude extract to single functional component by determining the biological activity of specific honey polypeptide in anti-inflammatory and intestinal barrier protection.The polypeptide provided by the present application has a clear chemical sequence and mechanism of action, which is beneficial to subsequent qualitative, quantitative characterization and quality control of related products, thereby providing a core material basis for realizing the standardized production and clinical conversion of functional products based on the polypeptide.

Description

Technical Field

[0001] This invention belongs to the field of functional peptide screening applications. Specifically, it relates to a peptide derived from *Gynostemma pentaphyllum* honey that exhibits anti-enteritis activity. Background Technology

[0002] Gut health is a crucial foundation for overall human health, and its core lies in maintaining the dynamic balance of the gut microenvironment, or "gut homeostasis." This homeostasis depends on the synergistic effects of intestinal barrier function, immune regulation, and the micro-ecosystem. Disruption of gut homeostasis often triggers inflammatory responses, impaired barrier function, and various metabolic disorders, leading to a range of gut-related diseases. Therefore, effectively maintaining or restoring gut homeostasis has become a significant research focus in the fields of life sciences and functional foods.

[0003] Existing methods for regulating gut health mainly include chemical drugs, probiotics, and antioxidants. However, these methods generally have certain limitations. For example, chemical drugs may cause toxic side effects and have low safety with long-term use; probiotics have limited colonization capacity in the body, and their effects are easily affected by individual differences and environmental factors, resulting in unstable performance; while traditional antioxidants mostly have non-specific effects and lack the ability to precisely regulate the function of the intestinal epithelial cell barrier. Therefore, developing a novel active substance that is safe in origin, has a clear function, and can regulate intestinal homeostasis in multiple dimensions is of great significance.

[0004] In recent years, small molecule bioactive peptides have gradually become ideal candidates for regulating intestinal function due to their advantages such as low molecular weight, easy absorption, high bioavailability, and ability to specifically act on target cells. However, it should be noted that screening functional peptides with clear biological activity that can directly act on intestinal epithelial cells from natural sources is technically challenging.

[0005] On the one hand, natural systems (such as honey) are extremely complex in composition, with peptides typically having low content and diverse structures. Target active peptides are often masked by large amounts of sugars and other components, making isolation and purification difficult. On the other hand, peptides exhibit highly specific biological activity; peptides with different sequences show significant functional differences, requiring systematic screening, identification, and functional verification to determine their actual effects. Furthermore, the stability, targeting ability, and direct regulatory capacity of peptides in the in vivo environment also need to be confirmed through rigorous experiments. Therefore, discovering and identifying active peptides with clear intestinal protective functions from natural foods is not only of significant application value but also a challenging research topic.

[0006] Honey, as a traditional natural food, has long been considered to have certain health benefits. However, existing research mainly focuses on its sugar composition and phenolic antioxidants. There is little research on specific polypeptide components with clear biological functions, especially on the mechanisms of intestinal epithelial cell protection and intestinal homeostasis regulation. Summary of the Invention

[0007] The purpose of this invention is to provide a polypeptide with anti-inflammatory and intestinal barrier protective activities, which has the effect of relieving intestinal inflammation and repairing the intestinal barrier.

[0008] This invention provides a polypeptide isolated from honey from Jin Si Cliff, the amino acid sequence of which is SEQ ID NO: 1.

[0009] The present invention also provides one use of the said polypeptide in the preparation of intestinal repair products.

[0010] In another aspect, the present invention provides an article for intestinal repair comprising a pharmacologically effective concentration of the aforementioned polypeptide.

[0011] The present invention also provides another use of the said polypeptide in the preparation of articles for inhibiting oxidative stress.

[0012] The present invention also provides an article for inhibiting oxidative stress, comprising the above-mentioned polypeptide.

[0013] This invention clarifies the bioactivity of specific honey peptides in anti-inflammatory and intestinal barrier protection, enabling the refined application of active components from crude natural product extracts to single functional components. The peptides provided by this invention possess well-defined chemical sequences and mechanisms of action, facilitating subsequent qualitative and quantitative characterization and quality control of related products. This provides a core material basis for the standardized production and clinical translation of functional products based on these peptides. Attached Figure Description

[0014] Figure 1 The diagram shows how the peptides from *Gynostemma pentaphyllum* protect Caco2 cells from H2O2-induced damage and apoptosis. In this diagram, A represents cell viability detection; B represents oxidative damage protection; C represents cell survival rate statistics; and D represents cell apoptosis rate statistics.

[0015] Figure 2 The diagram shows how the peptides from *Gynostemma pentaphyllum* protect Caco2 cells from H2O2-induced damage and apoptosis. In this diagram, A represents cell viability detection; B represents oxidative damage protection; C represents cell survival rate statistics; and D represents cell apoptosis rate statistics. Detailed Implementation

[0016] This invention addresses the challenges of screening functional peptides in natural systems and the lack of clarity regarding their mechanisms of action. Through systematic isolation and screening of honey, particularly Jin Si Ya honey, a peptide with definite biological activity was identified for the first time. Studies show that this peptide can significantly reduce oxidative stress damage to intestinal epithelial cells and enhance cell barrier function, thus playing a crucial role in maintaining intestinal homeostasis. This discovery not only fills a gap in research on functional peptides in honey but also provides a new theoretical basis and material source for developing novel intestinal health regulating products. The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0017] Example 1: Preparation of Polypeptides from Golden Silk Cliff Honey Spray-dried *Gynostemma pentaphyllum* honey powder was added to PBS (0.01 M, pH 7.4) at a material-to-liquid ratio of 1:30 and extracted at 4°C with stirring for 2 h. The extract was centrifuged at 10,000 r / min for 20 min at 4°C, and the supernatant was collected. Anhydrous ethanol was added to a final concentration of 80%, and the mixture was allowed to stand at 4°C for alcohol precipitation overnight. The precipitate was collected by centrifugation again, washed with anhydrous ethanol, and then freeze-dried to obtain crude *Gynostemma pentaphyllum* honey peptide powder.

[0018] 10.00 g of crude peptide powder was dissolved in 20 mM Tris-HCl buffer (pH 8.0) and brought to a final volume of 100 mL (100 mg / mL). After filtering through a 0.22 μm filter membrane, the sample was separated using a Zorbax SB-C18 preparative column (250 × 20 mm, 5 μm). The flow rate was 5 mL / min, the detection wavelength was 214 nm, and the mobile phase A was 0.1% trifluoroacetic acid aqueous solution, while phase B was 0.1% trifluoroacetic acid acetonitrile solution. Gradient elution was performed as follows: 0–10 min, 5% B; 10–50 min, 5%→50% B; 50–55 min, 50%→95% B (hold for 5 min); 60–65 min, 95%→5% B. Fractions with retention times of 8–12 min (HP-F1), 22–28 min (HP-F2), 35–40 min (HP-F3), and 48–52 min (HP-F4) were collected. Each fraction was removed by rotary evaporation to remove acetonitrile and then freeze-dried to obtain four different polarity Peptide fractions from *Hypericum esculentum* honey.

[0019] Example 2: Preparation of Golden Silk Cliff Honey Polypeptide with Anti-Enteritis Activity This study evaluated the protective effects of different components of the *Holothuria spp.* honey peptide (HP-F1 to HP-F4) against oxidative stress-induced damage in Caco-2 cells. Human colorectal adenocarcinoma cells (Caco-2) were used for activity screening. Caco-2 cells were routinely cultured in DMEM complete medium containing 10% fetal bovine serum. Logarithmic growth phase cells were harvested and cultured at a concentration of 1 × 10⁻⁶ cells / cell. 4Cells were seeded at a density of cells / well in 96-well plates and cultured for 24 hours until adherence. Then, the cells were divided into three groups: a normal control group, an H2O2 model group (treated with 100 μM H2O2 for 1 hour), and four peptide pretreatment groups (pretreated with 500 μg / mL HP-F1, HP-F2, HP-F3, and HP-F4 components for 24 hours, then replaced with medium containing 100 μM H2O2 for 1 hour). Cell viability was assessed using the CCK-8 assay after treatment. Results showed that at a concentration of 500 μg / mL, HP-F1, HP-F2, HP-F3, and HP-F4 were not cytotoxic, and cell viability was above 95%. Compared to the H2O2 model group (cell viability approximately 28.5%), only the HP-F2 pretreatment group showed a significantly increased cell viability of 53.9% (P<0.01), while the other components showed no significant difference from the model group. Further apoptosis verification experiments were conducted on the HP-F2 component: Caco-2 cells were seeded in 6-well plates (5 × 10⁻⁶ cells / well). 5 Cells were pretreated with 500 μg / mL H2O2 for 24 hours followed by 100 μM H2O2 for 1 hour. Apoptosis rates were detected by flow cytometry using Annexin V-FITC / PI double staining. Results showed that the apoptosis rate in the H2O2 model group was 27.0%, while the apoptosis rate in the HP-F2 pretreatment group significantly decreased to 10.3% (P<0.001). This study demonstrates that at a concentration of 500 μg / mL, HP-F2 is the only *Hymenochloa crus-galli* polypeptide component with significant anti-H2O2 oxidative stress damage and inhibitory activity against apoptosis.

[0020] The structures of HP-F2 fractions were identified using an LCMS-9030 Q-TOF liquid chromatography-mass spectrometry system. Chromatographic separation was performed using a Zorbax Eclipse Plus C18 reversed-phase column (35°C) with gradient elution at a flow rate of 0.3 mL / min (mobile phase A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile). Mass spectrometry was performed using an electrospray ionization source in positive ion mode, with data-dependent acquisition and a scan range of m / z 100–2000. The acquired data were imported into PEAKS Studio software, and De Novo sequencing combined with a search of bee protein databases ultimately identified 15 peptides with well-defined sequences from HP-F2.

[0021] Nrf2 is a core antioxidant regulatory target in the body's response to oxidative stress. In the inflammatory microenvironment of the gut, excessive ROS accumulation can disrupt the conformational stability of Keap1, leading to the dissociation of Nrf2 from Keap1 and its translocation to the cell nucleus. Once in the nucleus, Nrf2 binds to the ARE sequence, initiating the transcriptional expression of downstream antioxidant enzyme genes such as HO-1 and NQO1. This helps intestinal epithelial cells resist oxidative damage and maintain the integrity of the barrier function, ultimately enhancing the anti-inflammatory and repair capabilities of intestinal tissue.

[0022] To verify the interaction mechanism between honey peptides from *Gynostemma pentaphyllum* and the Nrf2 / Keap1 pathway, this invention employs a molecular docking strategy combining AutoDockFR and Rosetta FlexPepDock. This method first assesses the flexible conformation of the Keap1 protein's Kelch domain using AutoDockFR, then optimizes the peptide's conformation within the Keap1 binding pocket and calculates its binding free energy using Rosetta FlexPepDock. Finally, based on the binding energy and interaction mode, the competitive binding potential of the honey peptides to Keap1 is predicted, providing a molecular-level mechanistic basis for their antioxidant and anti-enteritis effects. The docking prediction results show that three peptides have high binding affinity to Keap1, with the peptide shown in SEQ ID NO.1 having the highest overall score, suggesting that it may exert optimal anti-enteritis activity by promoting Nrf2 nuclear translocation through highly competitive binding to Keap1.

[0023] Table 1: Comprehensive Score Table for Polypeptide Molecular Docking of Golden Silk Cliff Honey SEQ ID NO polypeptide sequence AutoDock Vina (kcal / mol) Rosetta I_score 1 PFVNWG -9.1 -13.1 2 GRYPSF -8.3 -9.5 3 AKFP DV -5.2 -5.7 Example 3: Determination of the anti-enteritis activity of peptides from Cliff Honey (SEQ ID NO: 1) 1) Synthesis of the target polypeptide sequence The target golden thread honey polypeptide sequence (SEQ ID NO.1) was successfully prepared using solid-phase synthesis technology.

[0024] 2) Evaluation of anti-enteritis activity Based on the above screening methods, the anti-enteritis activity of the target *Hylocereus undatus* honey peptide (SEQ ID NO.1) was evaluated. Cell viability was detected by the CCK-8 assay. The results showed that pretreatment with SEQ ID NO.1 peptide significantly alleviated the H2O2-induced decrease in cell viability, increasing cell viability from approximately 30.5% in the model group to 51.9% (P<0.01), indicating its significant cytoprotective effect. Further analysis using Annexin V-FITC / PI double staining combined with flow cytometry to detect cell apoptosis showed that SEQ ID NO.1 pretreatment significantly reduced the apoptosis rate from 30.0% in the model group to 11.3% (P<0.001), confirming its effective inhibition of oxidative stress-induced apoptosis. In conclusion, SEQ ID NO.1 *Hylocereus undatus* honey peptide exhibits significant anti-H2O2 oxidative damage and anti-apoptotic activity, providing cellular-level experimental evidence for its anti-enteritis efficacy.

[0025] In summary, this invention has prepared a *Hylocereus undatus* (golden thread honey) polypeptide with the function of maintaining the intestinal barrier. Regarding the alleviation of oxidative damage to intestinal epithelial cells, this polypeptide can effectively enhance the resistance of Caco-2 cells to H2O2-induced oxidative stress and significantly improve the survival rate of damaged cells. Simultaneously, this *Hylocereus undatus* polypeptide can significantly inhibit H2O2-induced apoptosis in Caco-2 cells, reducing the apoptosis rate, thereby protecting the integrity of intestinal epithelial cells and providing cellular-level experimental evidence for its anti-enteritis efficacy.

Claims

1. A polypeptide isolated from honey from Jin Si Cliff, characterized in that, The amino acid sequence of the polypeptide is SEQ ID NO:

1.

2. The use of the polypeptide according to claim 1 in the preparation of intestinal repair products.

3. A product for intestinal repair, characterized in that, The product contains a pharmacologically effective concentration of the polypeptide described in claim 1.

4. The use of the polypeptide of claim 1 in the preparation of articles that inhibit oxidative stress.

5. A product for inhibiting oxidative stress, characterized in that, The product contains the polypeptide of claim 1.

Images