Marine mangrove forest extracted active peptide and application thereof in tumor treatment

Abstract

The invention relates to active peptide extracted from marine mangrove forest and application of the active peptide in tumor treatment, and belongs to the technical field of biology. The active peptide FIYAGNNKLA (SEQ ID NO: 1) is extracted from kandelia candel in a mangrove forest natural reserve in Zhanjiang River of South China Sea, and the active peptide is free of cytotoxicity and good in safety. Experimental results show that the active peptide has an inhibition effect on the proliferation ability of breast cancer cells (MCF-7 cells, MDA-MB-231 cells and SK-BR-3 cells) under different concentrations, and scratch experiments can also significantly inhibit the migration ability of the breast cancer cells, so that a foundation is laid for preparation of drugs for treating breast cancer, and the active peptide has broad application prospects. A certain theoretical basis is also provided for the safety and effectiveness of the mangrove forest-sourced active peptide clinically used for breast cancer patients, and the mangrove forest-sourced active peptide can be widely used for developing related health-care foods or medicines for treating breast cancer, and has a wide market prospect in the fields of health-care foods, medicines and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the extraction of bioactive peptides from marine mangroves and their use in the treatment of tumors. Background Technology

[0002] Cancer, as a major disease posing a serious threat to human life and health worldwide, still faces numerous challenges in its clinical treatment. Currently, commonly used cancer treatments mainly include chemotherapy drugs, targeted therapies, and immunotherapies. Although these have achieved certain efficacy in cancer treatment, they generally suffer from many drawbacks: chemotherapy drugs lack targeting specificity, easily damaging normal tissue cells while killing tumor cells, leading to serious adverse reactions such as anemia, nausea, and vomiting; targeted therapies and immunotherapies face problems such as large individual differences in efficacy due to tumor heterogeneity and the easy development of drug resistance, and their high research and development and treatment costs significantly limit their clinical accessibility. Therefore, discovering novel anti-tumor active substances from natural products with advantages such as high efficiency, low toxicity, and strong targeting has become an important direction in cancer drug development.

[0003] The ocean covers 70% of the Earth's surface and fosters rich biodiversity. Marine organisms have evolved unique metabolic pathways to adapt to their challenging environments, such as high salinity, high pressure, and hypoxia, producing novel and bioactive natural products. Among these, marine-derived bioactive peptides, with their small molecular weight, easy absorption, significant bioactivity, and low toxicity, show great potential for development in the field of anti-tumor therapy. In recent years, researchers have isolated bioactive peptides with anti-tumor activity from various marine organisms, including invertebrates, algae, and microorganisms. Some of these peptides have already entered clinical trials, highlighting the promising application prospects of marine bioactive peptides as novel anti-tumor drugs.

[0004] Mangroves, as a key component of unique marine ecosystems growing in the intertidal zones of tropical and subtropical regions, are an important treasure trove of natural bioactive substances in the ocean. Their special growing environment endows mangrove plants with unique chemical defense mechanisms, enabling them to synthesize various chemical components with antibacterial, anti-inflammatory, antioxidant, and antitumor activities, such as flavonoids, terpenes, tannins, and alkaloids, making them a hot resource for marine drug research and development. Kandelia obovata, a typical mangrove plant belonging to the genus Kandelia in the family Rhizophoraceae, is widely distributed in mangrove reserves along the southeastern coast of my country and has a long history of medicinal use. Extracts from its roots, bark, leaves, and fruits have all been shown to possess various bioactivities. Previous studies have shown that the ethanol extract of Kandelia obovata roots has certain antitumor activity, but research on bioactive peptides in Kandelia obovata fruits is currently lacking. There are no reports of extracting and isolating small molecule peptides with antitumor activity from Kandelia obovata fruits growing in the Zhanjiang Mangrove Nature Reserve in the South China Sea and applying them to cancer treatment.

[0005] Based on the above situation, developing a technology to extract active peptides from the fruit of the Kandelia candel in the Zhanjiang Mangrove Nature Reserve in the South China Sea and clarifying its application in tumor treatment will not only fully tap the potential of my country's rich mangrove biological resources, but also provide new, safe and effective candidate drugs for clinical tumor treatment, which has important scientific significance and clinical application value. Summary of the Invention

[0006] To address the aforementioned technical issues, this invention extracts active peptides from Kandelia candel in the Zhanjiang Mangrove Nature Reserve in the South China Sea. These active peptides are non-cytotoxic, have good safety profiles, and can inhibit the proliferation and migration of breast cancer cells. They can be widely used to treat breast cancer and have a good therapeutic effect.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0008] The present invention provides an active peptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0009] Furthermore, the amino acid sequence of the active peptide is Phe-Ile-Tyr-Ala-Gly-Asn-Asn-Lys-Leu-Ala (FIYAGNNKLA).

[0010] Furthermore, the molecular weight of the active peptide is 1.11 kDa (1110.28 Da), and the theoretical isoelectric point PI is 9.30.

[0011] Furthermore, the active peptide is derived from Kandelion (Kandelia obovata) or is artificially synthesized.

[0012] Furthermore, the Kandelion (Kandelia obovata) mentioned originates from the Zhanjiang Mangrove Nature Reserve in the South China Sea; specifically, it is located in the Gaoqiao Mangrove Reserve Area, with coordinates: 109°45′E, 21°30′N.

[0013] The present invention also provides a pharmaceutical composition containing the aforementioned active peptide.

[0014] Furthermore, the pharmaceutical composition also contains pharmaceutically acceptable excipients.

[0015] Furthermore, the pharmaceutical composition can be formulated into any therapeutically acceptable dosage form.

[0016] Furthermore, the dosage form of the pharmaceutical composition includes an oral formulation.

[0017] Furthermore, the oral preparation is selected from any one of pills, powders, granules, oral liquids, decoctions, and tablets.

[0018] The present invention also provides the use of the said active peptide or the said pharmaceutical composition in the preparation of a drug for treating tumors.

[0019] Furthermore, the tumor is breast cancer.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This invention extracts the bioactive peptide FIYAGNNKLA (SEQ ID NO:1) from Kandelia candel in the Zhanjiang Mangrove Nature Reserve in the South China Sea. The bioactive peptide exhibits no cytotoxicity and good safety. Experimental results show that the bioactive peptide inhibits the proliferation of breast cancer cells (MCF-7, MDA-MB-231, and SK-BR-3 cells) at different concentrations, and also significantly inhibits the migration ability of breast cancer cells in scratch assays. This lays the foundation for drug preparation for breast cancer treatment and provides a theoretical basis for the safety and efficacy of mangrove-derived bioactive peptides in the clinical use of breast cancer patients. It can be widely used in the development of related health foods or drugs for breast cancer treatment, and has broad market prospects in many fields such as health foods and pharmaceuticals. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 These are the predicted three-dimensional structures (A and B) of the active peptides of this invention.

[0024] Figure 2 The results of the cytotoxicity test of the active peptide of this invention;

[0025] Figure 3 This is a statistical result showing the effect of different concentrations of the active peptides of this invention on the proliferation activity of different breast cancer cells;

[0026] Figure 4 This is a statistical result showing the effect of the active peptide of the present invention on the migration activity of different breast cancer cells. Detailed Implementation

[0027] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are all available from conventional commercial sources or can be obtained by existing known methods.

[0028] The terms “comprising,” “including,” “containing,” “having,” “comprising,” or other variations thereof are intended to cover non-closed inclusion, and no distinction is made between these terms. The term “comprising” means that other steps and components may be added without affecting the final result. The term “comprising” also includes the terms “consisting of” and “substantially consisting of”. The compositions and methods / processes of the present invention may comprise, consist of, and substantially consist of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0029] Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments without a specified manufacturer are commercially available, conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. Those skilled in the art should understand that the invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well-known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0030] Example 1: Preparation of bioactive peptides

[0031] The preparation of bioactive peptides includes the following steps:

[0032] (1) Raw material pretreatment

[0033] Mature, mold-free, and pest-free Kandelia candel fruits (collected from the Gaoqiao Mangrove Conservation Area in Zhanjiang, Guangdong, coordinates: 109°45′E, 21°30′N) were selected. The fruit stems and peel impurities were removed, and the fruits were quickly rinsed three times with deionized water. After draining the surface water, the fruits were crushed into pulp with a particle size of 1-3 mm in a high-speed crusher. Then, the fruits were placed in a freeze dryer and freeze-dried at -50℃ and vacuum degree ≤10Pa until the moisture content was ≤8% to obtain Kandelia candel fruit powder.

[0034] Add petroleum ether to the dried Kandelia candel fruit powder at a material-to-liquid ratio of 1:15 (g:mL), place it in a low-temperature shaker at 4℃, shake at 120r / min for 2 hours to defatting, and repeat the defatting process twice; filter to remove petroleum ether, and place the filter residue in a sterile fume hood to evaporate the residual solvent to obtain sterile defatted Kandelia candel fruit powder.

[0035] (2) Enzymatic hydrolysis

[0036] Add pH 6.5 phosphate buffer to sterile defatted eggplant fruit powder at a material-to-liquid ratio of 1:20 (g:mL), and homogenize in a sterile homogenizer at 2500 rpm for 10 min to form a uniform suspension. Add a compound enzyme (cellulase:neutral protease:flavor protease = 3:1:2, with a total enzyme amount of 1.5% of the raw material mass) to the suspension, stir evenly, and then transfer to a sterile enzymatic hydrolysis reactor. Hydrolyze at 40℃ and 100 rpm for 4 h, maintaining a stable pH of 6.5. After hydrolysis, raise the temperature to 95℃ and hold for 15 min.

[0037] (3) Purification

[0038] After cooling the enzymatic hydrolysate to room temperature, filter it through gauze to remove coarse residue. The filtrate is then filtered through a sterile plate and frame filter with a pore size of 0.45 μm. Collect the clear filtrate to obtain the crude enzymatic hydrolysate of Kandelia candel fruit. Inject the crude extract into an ultrafiltration apparatus and perform ultrafiltration using a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 2000 Da. Collect the permeate, discarding the large molecular weight proteins and impurities to obtain the ultrafiltration permeate. Load the permeate onto a Sephacryl S-200 HR gel filtration chromatography column, using deionized water as the eluent at a flow rate of 0.5 mL / min, and collect the eluted fraction.

[0039] (4) Purification by reverse high performance liquid chromatography

[0040] The eluted fraction was first filtered through a 0.22 μm filter membrane and then finely purified using preparative C18 reversed-phase high-performance liquid chromatography (HPLC). An Agilent Shim-pack Arata C18 column was used; the injection volume was 0.6 mL, the flow rate was 1 mL / min, the detection wavelength was 220 nm, and the column temperature was 30 °C. Gradient elution was performed using acetonitrile as mobile phase A and deionized water (containing 0.1% trifluoroacetic acid) as mobile phase B. The gradient elution program was: 0–8 min, 10–30% B; 8–12 min, 30–50% B; 12–20 min, 50–60% B; 20–30 min, 60–90% B. The eluted peaks were collected and their structures identified.

[0041] (5) Structural identification

[0042] Electrospray ionization (ESI+) scanning mode was used; the mass scan range was 100–2000 m / z; the drying gas flow rate (N2) was 9 L / min; the dryer temperature was 300 °C; the nebulization voltage was 35 psig; the capillary voltage was 3.5 kV; the fragmentation voltage was 175 V; the cone voltage was 65 V; the octetrel RF voltage was 250 V; and the collision voltage for secondary mass spectrometry was 12–15 eV. Mass spectrometry output data were resolved using Peaks 7.5 software (Bioinformatics Solutions Inc.). The results showed that the molecular weight of the active peptide was 1.11 kDa (1110.28 Da), the amino acid sequence was FIYAGNNKLA (SEQ ID NO:1), and the theoretical isoelectric point (PI) was 9.30.

[0043] (6) Structural prediction

[0044] The tertiary structure prediction tool PEP-FOLD (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD3 / ) was used to predict the bioactive peptide of SEQ ID NO:1. The prediction results are as follows: Figure 1 As shown.

[0045] The active peptide shown in SEQ ID NO: 1 of this invention was synthesized by Shanghai Jier Biochemical Co., Ltd. for later use. The polypeptide compound is a white powder with a purity of ≥98% and is stored at low temperature for later use.

[0046] Example 2 Safety evaluation of bioactive peptides

[0047] Fully adherent HeLa cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number: CL-0101) were digested with 0.25% trypsin and cultured in high-glucose DMEM containing 5% FBS to achieve a cell density of 1×10⁻⁶ cells / cells. 5Cell suspension at 100 μL / mL was seeded into 96-well plates and cultured at 37°C with 5% CO2 saturated humidity. After 24 h of culture, the complete culture medium was aspirated. The experimental groups were added with high-glucose DMEM medium diluted to concentrations of 0.5 mg / ml, 2 mg / ml, and 10 mg / ml, respectively, to the solutions of SEQ ID NO:1 peptide prepared in Example 1. The control group consisted of cells cultured in DMEM medium, and the blank group consisted of cell-free DMEM medium. The cells were cultured for another 24 h at 37°C with 5% CO2 saturated humidity. 10 μL of CCK-8 reagent (GLPbio) was added to each group, and the cells were incubated for 1–4 h. The absorbance (OD value) of each well was measured using an ELISA reader at 450 nm. Cell viability was calculated based on the mean absorbance of each group using the following formula:

[0048]

[0049] Experimental results are as follows Figure 2 As shown, the cell survival rates of the active peptides at different concentrations and the control group of the present invention are all above 100%, and the activity of the active peptide group is significantly improved compared with the blank group. It has no cytotoxicity and good safety.

[0050] Example 3: Effects of bioactive peptides on breast cancer cell proliferation

[0051] Breast cancer cell sources: MCF-7 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number: CL-0149), MDA-MB-231 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number: CL-0150), and SK-BR-3 cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number: CL-0211).

[0052] Experimental groups: Experimental group: breast cancer cells + 10% FBS medium (Sigma) + active peptide of the present invention; Control group: breast cancer cells + 10% FBS medium, no active peptide; Blank group: no cells, no active peptide, 10% FBS medium.

[0053] Experimental Methods: Three types of breast cancer cell lines (MCF-7, MDA-MB-231, and SK-BR-3) were seeded at appropriate densities in 96-well plates at a volume of 100 μL / well. After cell attachment, bioactive peptides were added to final concentrations of 0.1 mg / mL, 0.5 mg / mL, and 2 mg / mL, respectively. The control group received no bioactive peptide (0 mg / mL). After co-culturing the cells and bioactive peptides for 36 h, the 96-well plates were transferred to a clean bench in a cell culture room. The culture medium was discarded, and residual liquid was aspirated. Under light-protected conditions, 100 μL of serum-free culture medium and 10 μL of CCK-8 were added to each well. PBS was added around the perimeter of the 96-well plate to prevent reagent evaporation. The 96-well plates were incubated at 37°C in the dark for 1 hour. The absorbance (OD450 nm) was measured using a microplate reader, and the average value was taken after three repeated measurements. The absorbance was calculated according to the formula in the CCK-8 kit instructions.

[0054] .

[0055] Experimental results are as follows Figure 3 As shown, the bioactive peptides of this invention can inhibit the proliferative activity of breast cancer cells. After group culture, compared with the control group (0 mg / mL), the proliferative activity of the three types of breast cancer cells treated with bioactive peptides was significantly reduced, and the higher the concentration of bioactive peptides, the lower the cell survival rate, indicating that the bioactive peptides have an inhibitory effect on the proliferative capacity of breast cancer cells.

[0056] Example 4: Effects of bioactive peptides on the migration ability of breast cancer cells

[0057] The source of breast cancer cells and experimental grouping were the same as in Example 3.

[0058] Experimental Methods: Breast cancer cells were seeded into six-well plates at a volume of 2 mL / well. After cell adhesion, the experimental group (marked in the figure) was co-cultured with active peptides prepared in 10% FBS medium, with a final collagen concentration of 2 mg / mL. The control group (marked in the figure) was cultured in 10% FBS medium. When each well was 95% confluent and formed a monolayer, a 200 μL pipette tip was used to make a "I" shaped incision on the monolayer. The cells were washed three times with PBS. The experimental group was then cultured with active peptides prepared in serum-free medium, with a final active peptide concentration of 1 mg / mL. The control group was cultured in serum-free medium. After incubation for 24 h, the culture medium was aspirated, and the cells were washed three times with PBS. The cells were observed and photographed under an inverted fluorescence microscope, and the relative cell migration rate was recorded.

[0059] Relative migration rate (%) = (initial scratch width - 24h scratch width) / initial scratch width × 100%.

[0060] Experimental results are as follows Figure 4As shown, the active peptide of this invention can inhibit the migration ability of breast cancer cells. After group culture, compared with the control group, the migration rate of the three types of breast cancer cells treated with the active peptide was significantly reduced (P<0.01), indicating that the active peptide has an inhibitory effect on the migration ability of breast cancer cells.

[0061] The basic principles, main features, and advantages of this invention have been described above. Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection claimed by this invention is defined by the appended claims and their equivalents.

Claims

1. An active peptide, characterized in that, The amino acid sequence of the active peptide is shown in SEQ ID NO:

1.

2. The active peptide according to claim 1, characterized in that, The active peptides are derived from eggplant or are artificially synthesized.

3. The active peptide according to claim 2, characterized in that, The eggplant mentioned comes from the Zhanjiang Mangrove Nature Reserve in the South China Sea.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the active peptide as described in claim 1.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition also contains pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition can be formulated into any pharmacologically acceptable dosage form.

7. The pharmaceutical composition according to any one of claims 4-6, characterized in that, The dosage form of the pharmaceutical composition includes oral formulations.

8. The pharmaceutical composition according to claim 7, characterized in that, The oral preparation is selected from any one of pills, powders, granules, oral liquids, decoctions, and tablets.

9. Use of the active peptide according to any one of claims 1-3 or the pharmaceutical composition according to any one of claims 4-8 in the preparation of a medicament for treating tumors.

10. The use according to claim 9, characterized in that, The tumor is breast cancer.

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