An immunopotentiating polypeptide from houttuynia cordata thunb and its preparation method and application

Abstract

The present application relates to the technical field of peptide preparation, and specifically discloses a Houttuynia cordata-derived immune-enhancing polypeptide as well as a preparation method and application thereof.The amino acid sequence of the polypeptide is shown as SEQ ID NO.1.The polypeptide is obtained through enzymatic extraction, chromatographic separation and purification, activity tracking screening and mass spectrometry identification.Zebrafish experiments prove that the polypeptide can significantly improve alkaline phosphatase, acid phosphatase, superoxide dismutase activity and lysozyme content, and has excellent immune-enhancing activity.In a Pseudomonas plecoglossicida attack experiment, the survival rate of zebrafish in a 200 mg / kg dose group reaches 90.00%, which is much higher than 6.67% of the model control group.The Houttuynia cordata-derived immune-enhancing polypeptide has a clear structure and significant activity, and can be safely and efficiently used to enhance the immunity of aquatic animals and resist Pseudomonas plecoglossicida infection, without residue and drug resistance, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of peptide preparation technology, specifically to an immune-enhancing polypeptide derived from Houttuynia cordata, its preparation method, and its application. Background Technology

[0002] Houttuynia cordata (Houttuynia cordata) It is a traditional Chinese medicinal and edible plant with effects such as clearing heat and detoxifying, reducing swelling and draining pus. Modern research shows that houttuynia cordata contains rich active ingredients such as protein, polypeptides, and polysaccharides, and has potential applications in immune regulation, antioxidation, antibacterial and antiviral properties.

[0003] However, the development and utilization of peptides derived from Houttuynia cordata still faces the following technical shortcomings: 1. Traditional extraction methods are inefficient and prone to loss of activity. Conventional water extraction or organic solvent extraction methods are time-consuming and high-temperature, which easily damages the spatial structure of peptides, and the protein dissolution rate is less than 60%, leaving a large amount of active ingredients in the residue; 2. Insufficient structural identification and functional verification of single peptides. Existing studies mostly focus on crude extracts, and the analysis of amino acid sequences, structure-activity relationships, and mechanisms of action of single peptides is not systematic, leading to difficulties in controlling product quality; 3. There is an urgent need for disease control in aquaculture. Bacterial diseases caused by pathogens such as Shigella spp. are frequent, and antibiotic overuse has led to prominent problems of drug resistance and drug residues. The market urgently needs green and safe natural immune enhancers.

[0004] Therefore, developing Houttuynia cordata functional peptides with clear structures and well-defined activities is of great significance for overcoming existing technological bottlenecks, promoting the high-value utilization of Houttuynia cordata resources, and advancing green aquaculture. Summary of the Invention

[0005] This invention provides an immune-enhancing polypeptide derived from Houttuynia cordata with a defined amino acid sequence, its preparation method, and its application, in order to solve the technical problems of unclear structure and unsystematic functional verification of the active ingredients of Houttuynia cordata in the prior art.

[0006] The technical solution of the present invention is as follows: On one hand, this invention provides an immune-enhancing polypeptide derived from Houttuynia cordata, the amino acid sequence of which is shown in SEQ ID NO.1: Trp-Ile-Tyr-Asn-Asp-Gly-Glu-Asp-Arg-Leu-Thr-Ile-Val, consisting of 13 amino acids. This polypeptide is derived from the enzymatic hydrolysis product of Houttuynia cordata protein or obtained through solid-phase chemical synthesis.

[0007] On the other hand, the aforementioned houttuynia cordata-derived immune-enhancing peptides can be used to prepare immune-enhancing products for aquatic animals. These products are used to prevent or treat Shigella-like infections in aquatic animals. The immune-enhancing products can be used to improve the resistance of aquatic animals to Shigella-like infections. (Plesiomonas shigelloides) Infectivity.

[0008] On the other hand, the present invention provides an aquatic feed additive comprising the aforementioned houttuynia cordata-derived immune-enhancing polypeptide. The present invention provides an aquatic feed comprising the aforementioned houttuynia cordata-derived immune-enhancing polypeptide or comprising the aforementioned additive. The present invention also provides an aquatic animal immune-enhancing preparation comprising the aforementioned houttuynia cordata-derived immune-enhancing polypeptide.

[0009] On the other hand, the present invention provides a method for preparing the above-mentioned houttuynia cordata-derived immune-enhancing polypeptide, comprising the following steps: (1) After drying and crushing Houttuynia cordata, mix it with water for enzymatic hydrolysis, inactivate the enzyme, centrifuge and collect the supernatant to obtain crude polypeptide; (2) The crude polypeptide was separated and purified by chromatography, the immunologically active components were collected, and the Houttuynia cordata-derived immunomodulatory polypeptide was obtained by mass spectrometry.

[0010] Preferably, the enzymatic hydrolysis conditions are: a material-to-liquid ratio of 1:30 to 1:50, an enzymatic hydrolysis temperature of 45°C to 60°C, an enzymatic hydrolysis time of 2 to 5 hours, and the enzyme used is a compound flavor protease, with the enzyme addition amount being 1.5% to 3.5% of the mass of the dried Houttuynia cordata powder.

[0011] The preferred conditions are a material-to-liquid ratio of 1:40, 52°C, 3.5 h, and an enzyme addition of 2.5%.

[0012] Preferably, the chromatographic separation and purification uses a C18 preparative column for gradient elution, collecting 5%, 15%, 25%, 35%, 50%, and 80% methanol eluent fractions sequentially, wherein the 35% methanol eluent fraction (i.e., F4 fraction) is the target active fraction.

[0013] The beneficial effects achieved by this invention are as follows: 1. This invention obtains a single active polypeptide sequence derived from Houttuynia cordata: Trp-Ile-Tyr-Asn-Asp-Gly-Glu-Asp-Arg-Leu-Thr-Ile-Val (SEQ ID NO.1), which facilitates product quality control and standardized production; 2. Zebrafish experiments using the single active polypeptide sequence of this invention showed that the high-dose group (SEQ ID NO.1 polypeptide 200 mg / kg) significantly increased the activities of alkaline phosphatase (AKP), acid phosphatase (ACP), superoxide dismutase (SOD), and lysozyme content (P<0.01). Specifically, SOD activity reached 36.09 U / mg, and lysozyme content reached 22.57 μg / mL, representing increases of 188% and 166% respectively compared to the blank control group. In the challenge experiment with *Shigella spp.*, the survival rate of the high-dose group reached 90.00%, while the model control group only had a survival rate of 6.67%. 3. Molecular docking of this invention shows that the polypeptide has a strong binding affinity to key targets in the TLR4 / NF-κB immune regulatory pathway; 4. The polypeptides of this invention are derived from the medicinal and edible plant Houttuynia cordata, leaving no residue and being less likely to induce drug resistance, making them suitable for aquaculture. Attached Figure Description

[0014] Figure 1 HPLC separation chromatogram of crude Houttuynia cordata polypeptide (detection wavelength 220 nm) shows the peak positions of the six eluted components F1-F6.

[0015] Figure 2 Effects of different elution fractions (F1-F6) on the activities of AKP, ACP, SOD and lysozyme content in zebrafish.

[0016] Figure 3 : A molecular docking model diagram of the peptide SEQ ID NO.1 and the TLR4 protein.

[0017] Figure 4 Effects of different doses (50, 100, 200 mg / kg) of the SEQ ID NO.1 peptide on SOD activity and lysozyme content in zebrafish.

[0018] Figure 5 Survival curves of zebrafish infected with Shigella spp. in different treatment groups (x-axis: 1-blank control, 2-model control, 3-low dose, 4-medium dose, 5-high dose; y-axis: survival rate %). Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0021] Example 1: Preparation and Screening of Immune-Enhancing Peptides Derived from Houttuynia cordata 1.1 Enzymatic preparation of crude peptides from Houttuynia cordata Dried whole Houttuynia cordata was pulverized and passed through a 60-mesh sieve to obtain Houttuynia cordata powder. Distilled water was added at a material-to-liquid ratio of 1:40 (w / v), along with 2.5% (by weight of the Houttuynia cordata powder) of a compound flavor protease (Novozymes, enzyme activity 50,000 U / g). Enzymatic hydrolysis was performed at a constant temperature of 52℃ with shaking in a water bath for 3.5 hours. After hydrolysis, the hydrolysate was placed in a 95℃ water bath for 10 minutes to inactivate the enzyme, followed by centrifugation at 10,000 r / min for 20 minutes. The supernatant was collected and freeze-dried to obtain crude Houttuynia cordata polypeptide powder.

[0022] 1.2 High-performance liquid chromatography separation and purification Accurately weigh 10.00 g of crude Houttuynia cordata polypeptide, dissolve it in ultrapure water, and dilute to 100 mL to prepare a 100 mg / mL polypeptide stock solution. Separation and purification were performed using a high-performance liquid chromatography (HPLC) system equipped with a Dubhe C18 preparative column (250 × 20 mm, 10 μm), with a flow rate controlled at 8 mL / min, monitored in real-time at room temperature and a detection wavelength of 220 nm. Six fractions of different polarities were collected sequentially using a gradient elution program: 5% methanol elution fraction (F1), 15% methanol fraction (F2), 25% methanol fraction (F3), 35% methanol fraction (F4), 50% methanol fraction (F5), and 80% methanol fraction (F6) for later use. The HPLC chromatogram is shown below. Figure 1 As shown.

[0023] 1.3 Activity tracking screening Experimental animals: Healthy zebrafish (weight 0.3±0.05 g), purchased from an aquaculture farm, were domesticated in the laboratory for one week before use. Rearing conditions: water temperature 26±2℃, continuous aeration, 12 / 12 hour light / dark cycle, free access to feed.

[0024] Grouping and Administration: Zebrafish were randomly divided into 8 groups: blank control group, crude polypeptide mother liquor group, F1 group, F2 group, F3 group, F4 group, F5 group, and F6 group, with 30 fish in each group. Each component was dissolved in physiological saline and administered via gavage at a dose of 100 mg / kg for 7 consecutive days. The blank control group received an equal volume of physiological saline. Twenty-four hours after the last administration, zebrafish tissue samples were collected, and the activities of alkaline phosphatase (AKP), acid phosphatase (ACP), superoxide dismutase (SOD), and lysozyme (LZM) were measured according to the kit instructions.

[0025] Result: As Figure 2 As shown, compared with the blank control group, the F4 component significantly increased the activities of AKP, ACP, and SOD, as well as the lysozyme content in zebrafish (P<0.01), and its immune-enhancing effect was significantly better than that of other components and the crude polypeptide mother liquor group. This result indicates that the F4 component (35% methanol eluted component) is enriched with highly active immune-enhancing polypeptides, which is the target active component of this invention.

[0026] 1.4 Mass Spectrometry Identification and Sequence Analysis The F4 fraction was analyzed by primary and secondary mass spectrometry using an LCMS-9030 quadrupole-time-of-flight LC-MS / MS system. Chromatographic conditions: Inertsil HILIC C18 reversed-phase column (150 × 2.1 mm, 3 μm), column temperature 30℃, flow rate 0.3 mL / min; mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid. The gradient elution program was: 0–5 min 5% B, 5–40 min 5%–60% B, 40–45 min 60% B, 45–50 min 5% B. Mass spectrometry conditions: electrospray ionization (ESI), positive ion mode, scan range m / z 100–2000. Data were retrieved from the NCBI Viridiplantae database (January 2024 version) using ProteinPilot software. The precursor ion mass tolerance was ±10 ppm, the fragment ion mass tolerance was ±0.05 dB, and the confidence threshold was ≥95% (Unused Score ≥1.3). De novo sequencing analysis was then performed. A total of 28 high-confidence peptide sequences were identified, including a 13-amino acid peptide sequence named Trp-Ile-Tyr-Asn-Asp-Gly-Glu-Asp-Arg-Leu-Thr-Ile-Val, designated SEQ ID NO.1.

[0027] 1.5 Molecular docking analysis To verify the interaction mechanism between the active peptide and key targets of the TLR4 / NF-κB immune regulatory pathway, a molecular docking strategy combining AutoDock FR and Rosetta FlexPepDock was employed to comprehensively evaluate the binding mode and affinity between the peptide and the target. The results are shown in Table 1. Peptide SEQ ID NO.1 had the highest overall score and the most stable binding to the immune target, suggesting it possesses the optimal immunomodulatory activity. The molecular docking diagram of peptide SEQ ID NO.1 with TLR4 is shown below. Figure 3 As shown.

[0028] Table 1. Comprehensive Score Table for Peptide Molecular Docking from Houttuynia cordata

[0029] Example 2: Determination of the immunomodulatory activity of Houttuynia cordata-derived polypeptide (SEQ ID NO.1) (1) Synthesis of target polypeptide sequence The target peptide was synthesized using the Fmoc solid-phase synthesis method, with Rink Amide MBHA resin as a carrier. Amino acids were sequentially coupled from the C-terminus to the N-terminus according to the sequence SEQ ID NO.1. After synthesis, the peptide was cleaved, precipitated with diethyl ether, and purified by HPLC to obtain a purity ≥95%, which was confirmed by mass spectrometry.

[0030] (2) Laboratory animals and materials Experimental materials: physiological saline, electronic balance, aquaculture system, biochemical test kits, and gavage syringes.

[0031] Animals: Healthy zebrafish, similar weight, without injury or disease.

[0032] Rearing environment: Standard aquaculture conditions, water temperature 26±2℃, continuous aeration, 12 / 12 hour light / dark cycle, free feeding.

[0033] (3) Grouping and administration Healthy zebrafish were randomly divided into four groups: 1. a blank control group; 2. a low-dose group (50 mg / kg) of Houttuynia cordata-derived polypeptide; 3. a medium-dose group (100 mg / kg); and 4. a high-dose group (200 mg / kg). The blank control group was given an equal volume of physiological saline. All groups were treated for 7 consecutive days. After treatment, zebrafish tissue samples were collected, and the activities of AKP, ACP, SOD, and lysozyme were detected.

[0034] The results are as follows Figure 4As shown, compared with the blank control group, the activities of AKP, ACP, SOD, and lysozyme content in each dose group of Houttuynia cordata polypeptide were significantly increased (P<0.01), and the immune enhancement effect gradually increased with increasing dose, showing a clear dose-response relationship. The high-dose group (200 mg / kg) had an SOD activity of 36.09±2.65 U / mg and a lysozyme content of 22.57±1.85 μg / mL, which were 188% and 166% higher than the blank control group, respectively.

[0035] Example 3: Experiment on the resistance of Houttuynia cordata-derived peptides to Shigella simonii challenge. The zebrafish were grouped and administered the drug in the same manner as in Example 2.

[0036] Shigella-like enomonas (Plesiomonas shigelloides) The strain was isolated from diseased fish in our laboratory, purified and cultured, and then identified and preserved using 16S rDNA sequencing.

[0037] After 7 days of continuous administration, a concentration of 1×10⁻⁶ was used. 8 Intraperitoneal injection of CFU / mL Shigella spp. culture was used to challenge the virus. The patients were observed for 7 consecutive days after the challenge, and the mortality and survival rates of each group were recorded.

[0038] The results are as follows Figure 5 As shown, the mortality rate of zebrafish in the model control group was extremely high, with a survival rate of only 6.67%. All doses of Houttuynia cordata polypeptide significantly improved the survival rate of zebrafish, and the survival rate increased with increasing dosage, demonstrating a clear dose-response relationship. The high-dose group (200 mg / kg) achieved a survival rate of 90.00%, with a relative protection rate of 89.3%.

[0039] Example 4: Preparation of aquatic feed additives The polypeptide synthesized in Example 2 (SEQ ID NO. 1) was mixed with a feed-grade carrier (corn starch, dextrin, etc.) at a mass ratio of 1:9 and thoroughly ground to obtain an aquatic feed additive. The additive contains 10% polypeptide. When using, it can be added to the basal feed at a ratio of 0.01wt%-10wt% of the final feed polypeptide content.

[0040] Example 5: Preparation of aquatic feed containing peptides The basic feed ingredients (20% fishmeal, 25% soybean meal, 25% wheat flour, 15% wheat bran, 3% fish oil, 2% vitamin premix, 2% mineral premix, and the remainder being water) were added to the aquatic feed additive prepared in Example 4, so that the final concentration of the polypeptide of SEQ ID NO.1 in the feed was 0.1 wt%. Functional aquatic feed containing immune-enhancing polypeptides derived from Houttuynia cordata was obtained through conventional processes such as crushing, mixing, conditioning (85°C), granulation, drying, and cooling.

[0041] Example 6: Preparation of immune-enhancing agents for aquatic animals The polypeptide synthesized in Example 2 (SEQ ID NO. 1) was dissolved in sterile physiological saline to prepare a solution with a concentration of 10 mg / mL. The solution was then filtered through a 0.22 μm filter membrane for sterilization, dispensed into sterile injection vials, and freeze-dried to obtain the injectable immunostimulant. Before use, it was reconstituted with sterile water for injection and administered to aquatic animals.

[0042] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An immune-enhancing polypeptide derived from Houttuynia cordata, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. The application of the houttuynia cordata-derived immune-enhancing polypeptide of claim 1 in the preparation of an immune-enhancing product for aquatic animals; the immune-enhancing product for aquatic animals is used to improve the ability of aquatic animals to resist Shigella-like infection.

3. An aquatic feed additive, characterized in that: The aquatic feed additive contains the houttuynia cordata-derived immune-enhancing polypeptide as described in claim 1.

4. An aquatic feed, characterized in that: The aquatic feed contains the houttuynia cordata-derived immune-enhancing polypeptide of claim 1 or the aquatic feed additive of claim 3.

5. An immune-enhancing agent for aquatic animals, characterized in that: The formulation comprises the houttuynia cordata-derived immune-enhancing polypeptide of claim 1.

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