Linseed cyclic peptide and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to flaxseed cyclic peptide and application thereof. The invention provides a novel fluorescence labeling strategy of flaxseed cyclic peptide to solve the problems of low connection efficiency, poor stability and the like of a fluorescent probe and the cyclic peptide. A stable thiourea bond connection structure is formed by selecting flaxseed cyclic peptide of free amino on a lysine side chain and performing specific reaction on the free amino and an isothiocyanate group in an isothiocyanate fluorescent compound, so that effective marking of the fluorescent probe on the cyclic peptide is realized. Experimental results show that the connection mode has high reaction efficiency and chemical stability, the connection structure can be kept complete in the physiological pH and in the environment with multiple enzymes, and the operation stability of a labeled product in a complex biological system is remarkably improved.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a flaxseed cyclic peptide with fluorescent properties. Background Art

[0002] Cyclic peptides are cyclic compounds formed by amino acids linked by peptide bonds, with their head, tail or side chains closed by covalent bonds to form a cyclic configuration. Compared with linear peptides, cyclic peptide structures have significant advantages. The cyclic topology eliminates terminal charges, reduces molecular polarity, and rigid conformational constraints reduce structural freedom. At the same time, cyclization hides the enzyme cleavage sites on the peptide chain or increases steric hindrance, thereby giving cyclic peptides stronger chemical stability and resistance to enzymatic hydrolysis. In addition, the cyclic structure fixes the active conformation, allowing key functional groups to bind to targets more accurately and have higher bioavailability in the body. These characteristics give them great advantages in clinical therapeutic applications and have also attracted a lot of attention from scholars at home and abroad. Studies have shown that cyclic peptides have multiple biological activities such as anti-hypertension, anti-inflammatory, anti-cancer, and immunomodulatory.

[0003] As a type of cyclic peptide, flaxseed cyclic peptide not only possesses the aforementioned advantages of cyclic peptides but is also classified into multiple types based on their amino acid composition and sequence. These different types of flaxseed cyclic peptides demonstrate unique potential and efficacy in anti-inflammatory and anti-cancer applications. It not only reduces the transmembrane resistance of inflammatory cells but also inhibits the increase in ROS levels in RAW264.7 cells through the TLR4 and NF-κB signaling pathways, reducing the production of malondialdehyde (MDA) by lipopolysaccharide (LPS)-induced oxidative stress. Flaxseed cyclic peptide also exhibits anti-cancer effects, specifically by decreasing gastric cancer cell survival and increasing apoptosis. Flaxseed cyclic peptides inhibit the spread of gastric cancer, breast cancer, and malignant gliomas by activating the expression of genes and proteins in the pro-apoptotic Caspase family and suppressing the activation of the signal transduction factor Stat3.

[0004] However, the in vivo absorption mechanism of flaxseed cyclic peptides remains unclear, hindering their development and utilization. The complex internal environment of the body, with its various small peptide fragments, interferes with the absorption and detection of cyclic peptides. Furthermore, the absorption of cyclic peptides in the body is minimal, making it difficult to detect with existing chromatography and biological assays. Furthermore, it is impossible to distinguish whether the substance is absorbed unchanged or as a metabolite, nor is it possible to track the dynamic absorption process of cyclic peptides in the body. Currently, researchers are actively exploring the use of fluorescent probes to label cyclic peptides, hoping to visualize and dynamically monitor their absorption process at the cellular and even whole-body levels, thereby revealing their absorption pathways. However, this technology still faces numerous challenges: the current linking efficiency between fluorescent probes and cyclic peptides is low, and the linked structure is prone to breakage in the complex environment of the body, particularly in areas prone to hydrolysis or enzymatic degradation, such as the digestive tract, where stability is particularly prominent. Furthermore, the specific spatial structure of cyclic peptides makes finding suitable labeling sites without affecting their original biological activity a major challenge. Therefore, how to achieve efficient and stable connection between cyclic peptides and fluorescent probes without affecting the properties of the cyclic peptides themselves and the fluorescent properties of the fluorescent probes remains a key issue that needs to be broken through. Summary of the Invention

[0005] To solve the above technical problems, the present application provides a flaxseed cyclic peptide with high fluorescent group connection stability and good tracing effect, which is achieved specifically through the following technical solutions: A flaxseed cyclic peptide is obtained by reacting a flaxseed cyclic peptide with an isothiocyanate fluorescent compound, and the amino group on the lysine side chain of the flaxseed cyclic peptide reacts with the isothiocyanate group in the isothiocyanate fluorescent compound to form a thiourea bond.

[0006] Preferably, the structural formula of the flaxseed cyclic peptide is: .

[0007] Preferably, the thiourea bond is obtained under catalytic conditions of a base catalyst.

[0008] Preferably, the base catalyst comprises diethylenetriamine.

[0009] Preferably, the thiourea bond is obtained by reaction in a light-proof environment.

[0010] Preferably, the reaction conditions are: temperature 20-30° C., magnetic stirring 5-7 h.

[0011] Preferably, the molar ratio of the flaxseed cyclic peptide to the isothiocyanate fluorescent compound is 1:1.2.

[0012] Preferably, the purity of the flaxseed cyclic peptide is 95-99%.

[0013] Use of any of the above-mentioned flaxseed cyclic peptides in the preparation of anti-inflammatory or anti-cancer drugs.

[0014] Use of any of the above-mentioned flaxseed cyclic peptides in the study of intestinal absorption of flaxseed cyclic peptides.

[0015] Compared with the prior art, this application has the following beneficial effects: In response to the problems of low efficiency and poor stability in the connection between fluorescent probes and cyclic peptides, this application proposes a new fluorescent labeling strategy for flaxseed cyclic peptides. By selecting flaxseed cyclic peptides with free amino groups on the side chain of lysine, and using the free amino groups to react specifically with the isothiocyanate groups in isothiocyanate fluorescent compounds, a stable thiourea bond connection structure is formed, thereby achieving effective labeling of the cyclic peptide by the fluorescent probe. The experimental results show that this connection method has high reaction efficiency and chemical stability, and can maintain the integrity of the connection structure under physiological pH and in the presence of multiple enzymes, significantly improving the operational stability of the labeled product in complex biological systems. At the same time, since most lysine residues are located in non-critical functional regions of cyclic peptides, their modification has little effect on the original spatial conformation and biological activity, so that the labeled cyclic peptide can maintain its own biological function while having good fluorescence tracking ability. This method provides a stable and reliable technical means for the dynamic monitoring of cyclic peptides in vivo and the study of their absorption mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] To clearly introduce the embodiments, the following briefly introduces the drawings: Figure 1 This is the mass spectrum of the flaxseed cyclic peptide in Example 1; Figure 2 is the reaction formula of the flaxseed cyclic peptide in Example 1; Figure 3 This is a graph of cell survival rates in Example 2, with the DMSO group on the left and the flaxseed cyclic peptide group on the right; Figure 4 This is a diagram of the fluorescence distribution in Caco-2 cells taken using a fluorescence microscope in Example 3, wherein: Blue is the cell nucleus, green is flaxseed cyclic peptide; Figure 5 This is a graph showing the fluorescence intensity of cells in each group detected by flow cytometry in Example 4; Figure 6 This is a graph showing the fluorescence intensity of cells in each group detected by flow cytometry in Example 5; Figure 7 This is a graph showing the fluorescence intensity of cells in each group detected by flow cytometry in Example 6; Figure 8 This is the real-time optical imaging fluorescence distribution in Example 7. DETAILED DESCRIPTION

[0017] The present application will be further described below in the form of specific embodiments. Those skilled in the art will be able to implement the present application based on these descriptions. In addition, the embodiments of the present application involved in the following description are generally only embodiments of a portion of the present application, rather than all embodiments. Therefore, based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making any creative efforts should fall within the scope of protection of the present application.

[0018] Example 1 This embodiment discloses a method for preparing a flaxseed cyclic peptide. The reaction structure of the flaxseed cyclic peptide is shown in the attached Figure 2 . Specifically, the following steps are included: dissolving 121.1 mg of flaxseed cyclopeptide (cyclo-Met-Leu-Lys-Pro-Phe-Phe-Phe-Trp-Ile) raw material in 7 mL of dimethylformamide solution to obtain flaxseed cyclopeptide original solution; dissolving 46.7 mg of isothiocyanate fluorescent compound in 3 mL of dimethylformamide solution to obtain isothiocyanate fluorescent compound solution; under light-proof conditions, adding the isothiocyanate fluorescent compound solution dropwise to the flaxseed cyclopeptide original solution, and adding 52.3 μL of diethylenetriamine DIEA as a base catalyst, and magnetically stirring at room temperature for 6 h to promote the reaction of the amino group on the lysine side chain of the flaxseed cyclopeptide with the isothiocyanate group in the isothiocyanate fluorescent compound to form a thiourea bond, thereby obtaining a flaxseed cyclopeptide mixture; purifying the reaction mixture and collecting the main peak fraction, and freeze-drying to obtain the flaxseed cyclopeptide (purity > 95%). For mass spectrometry identification of flaxseed cyclopeptide, please refer to the attached. Figure 1 , whose molecular formula is C 87 H 98 N 12 O 14 S2, molecular weight is 1599.93.

[0019] Example 2 In this example, the CCK-8 method was used to detect the effects of solvent DMSO and CLQ-FITC on Caco-2 cell viability. The specific steps were as follows: Caco-2 cell suspension was prepared at 5×10 4The cells were inoculated at a density of cells / mL in a 96-well plate. They were cultured in a 37°C incubator for 24 hours and the drugs were administered after they attached to the wall. Caco-2 cells with good growth were randomly divided into a blank group (containing culture medium, CCK-8 solution, but not cells or drugs), a control group (containing cells, culture medium, CCK-8 solution, but not drugs), a DMSO solvent group (containing cells, culture medium, CCK-8 solution and DMSO solution) and a flaxseed cyclic peptide group (containing cells, culture medium, CCK-8 solution and flaxseed cyclic peptide solution). The DMSO concentration was set to 0.1%, 0.3%, 0.5%, 1%, 2% and 3%, and the flaxseed cyclic peptide solution concentration was 30, 60, 90, 120, 150 and 180 μM. After culturing for 24 hours, 10 μL of the solution was added to the 96-well plate and incubated in a 37°C incubator for 1.5 hours. The OD value was detected at 450 nm using a multifunctional microplate reader. The survival rate was calculated according to the following formula and summarized. Figure 3 .

[0020] Calculate the cell survival rate = (OD average of each experimental group - OD average of the blank group) / (OD average of the control group - OD average of the blank group) * 100%.

[0021] according to Figure 3 The results show that at lower concentrations (0.1%, 0.3%, and 0.5%), DMSO has almost no effect on cell viability, and the survival rate is close to that of the control group (about 98%-100%). However, when the DMSO concentration increased to 1% and above, the cell survival rate decreased significantly and continued to decrease with increasing concentration (down to about 80% at a concentration of 3%). For the flaxseed cyclic peptide group, the experimental results showed that compared with the DMSO group, the flaxseed cyclic peptide group had no significant negative impact on the survival rate of Caco-2 cells. This shows that under the conditions of this experiment, flaxseed cyclic peptide has basically no cytotoxic effect and can provide nutrition for cell reproduction.

[0022] Example 3 This example tests the fluorescence of flaxseed cyclic peptide in Caco-2 cells, specifically including the following steps: Caco-2 cells in good growth state are seeded in a 12-well plate, and after the cells adhere, a 90 μM flaxseed cyclic peptide solution is added to the plate and cultured for 1, 4, and 6 hours, and then washed twice with 0.5 mL PBS. After fixing with 4% paraformaldehyde for 15 minutes, the plate is washed twice with 0.5 mL PBS, and DAPI staining solution is added to each well for staining at room temperature for 10 minutes. After that, the plate is washed twice with PBS and photographed under a fluorescence microscope to obtain the attached fluorescence. Figure 4Fluorescence microscopy results show the absorption of flaxseed cyclic peptide in Caco-2 cells. The blue color represents the cell nucleus stained with DAPI, and the green color represents the flaxseed cyclic peptide absorbed into the cells. As the incubation time increases, the distribution of green fluorescence in the cells increases significantly, indicating that the absorption of flaxseed cyclic peptide by Caco-2 cells increases with time.

[0023] Example 4 This example compares the fluorescence intensity of flaxseed cyclic peptide in Caco-2 cells after incubation for 6 h, specifically including the following steps: Caco-2 cell suspension was incubated at 1×10 5 The cells were seeded at a density of 1000 / well in a 6-well plate. The cells were cultured in an incubator at 37°C for 24 hours. When the density grew to more than 80%, the drug was administered, and a blank control group and a flaxseed cyclic peptide group were set up. The flaxseed cyclic peptide groups were added with 2 mL of 30, 90, and 150 μM flaxseed cyclic peptide solutions, respectively, and cultured for 6 hours. After the culture was completed, the old culture medium was discarded, and the cells were washed three times with PBS. 0.5 mL of trypsin was added to each well and digested in an incubator for 5 minutes. After the digestion was completed, 1 mL of fresh culture medium was added to each well to terminate the digestion. The suspension was collected in a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The old culture medium was discarded, and the precipitate was washed three times with PBS. Finally, 500 μL of PBS was added to blow the precipitate evenly, and the fluorescence intensity of each group of cells was detected by flow cytometry. Figure 5 .observe Figure 5 It can be found that as the amount of flaxseed cyclic peptide added continues to increase, the fluorescence intensity gradually increases.

[0024] Example 5 This example compares the fluorescence intensity of flaxseed cyclic peptide CLQ combined with different fluorescent groups in Caco-2 cells after incubation for 6 h, specifically including the following steps: Caco-2 cell suspension was incubated at 1×10 5 The cells were seeded at a density of 1000 / well in a 6-well plate. They were cultured in an incubator at 37°C for 24 h. When the density grew to more than 80%, the drug was administered, and a blank control group, a CLQ-Cy5 group, and a CLQ-FITC group were set up. 2 mL of 90 μM CLQ-Cy5 solution and CLQ-FITC solution were added, respectively, and cultured for 6 h. After the culture was completed, the old culture medium was aspirated and discarded, and the cells were washed 3 times with PBS. 0.5 mL of trypsin was added to each well and digested in the incubator for 5 minutes. After the digestion was completed, 1 mL of fresh culture medium was added to each well to terminate the digestion, and the suspension was collected into a centrifuge tube and centrifuged at 1000 rpm for 5 min. The old culture medium was aspirated and discarded, and the precipitate was washed 3 times with PBS. Finally, 500 μL of PBS was added to blow the precipitate evenly, and the fluorescence intensity of each group of cells was detected by flow cytometry. Figure 6 .observe Figure 6It can be found that the absorption of flaxseed cyclic peptide fluorescently labeled with Cy5 in cells is significantly lower than that of the FITC-labeled group.

[0025] Example 6 This example studies the effect of chlorpromazine treatment on the fluorescence intensity of flaxseed cyclic peptide in Caco-2 cells, which specifically includes the following steps: a certain concentration of Caco-2 cell suspension is added at 1×10 5 The cells were seeded at a density of 100 cells / well in a 6-well plate. They were cultured in a 37°C incubator for 24 hours and then given drugs when the density grew to more than 80%. A blank control group (Black), a chlorpromazine-treated group (CLQ-FITC+CPZ) and a flaxseed cyclic peptide group (CLQ-FITC) were set up. The flaxseed cyclic peptide group was added with 2 mL of 90 μM flaxseed cyclic peptide solution and cultured for 6 hours. 20 μM chlorpromazine and flaxseed cyclic peptide were added to the chlorpromazine group and cultured for 6 hours. After the end, the old culture medium was discarded and the cells were washed 3 times with PBS. 0.5 mL of trypsin was added to each well and digested in the incubator for 5 minutes. After the digestion was completed, 1 mL of fresh culture medium was added to each well to terminate the digestion, the suspension was collected in a centrifuge tube, and centrifuged at 1000 rpm for 5 minutes. The old culture medium was discarded, the precipitate was washed 3 times with PBS, and finally 500 μL of PBS was added to blow the precipitate evenly, and the fluorescence intensity of the cells in each group was detected by flow cytometry. Figure 7 .observe Figure 7 It can be found that in the chlorpromazine-treated group, since chlorpromazine CPZ is a clathrin inhibitor, when it is added, the clathrin-dependent endocytosis pathway will be completely or partially blocked, causing the absorption rate of large molecules such as cyclic peptides to decrease, and thus the intracellular fluorescence intensity will decrease.

[0026] Example 7 This example studies the effect of flaxseed cyclic peptide on the fluorescence distribution of various intestinal segments of rats, and specifically includes the following steps: 18 SD rats were divided into three groups, namely, flaxseed cyclic peptide group (CLQ-FITC), chlorpromazine treatment group (CLQ-FITC+CPZ), and distilled water group. SD rats that had been fasted for 12 hours (with free access to water) were taken. According to the above grouping, each group of rats was gavaged with flaxseed cyclic peptide (10 mg / kg), chlorpromazine (10 mg / kg), flaxseed cyclic peptide (10 mg / kg), and distilled water. 4 and 6 hours after gavage, the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate solution (4 mL / kg). Real-time optical imaging of small animals was used to observe the fluorescence distribution of flaxseed cyclic peptide in various intestinal segments of rats, and the fluorescence of the flaxseed cyclic peptide in various intestinal segments of rats was obtained. Figure 8 .from Figure 8It can be seen that 4 hours after rats were gavaged with flaxseed cyclic peptide, the fluorescence was mainly in the front half of the rat's jejunum, and the fluorescence of the rats in the group that were gavaged with chlorpromazine and flaxseed cyclic peptide at the same time was significantly weaker than that of the rats in the group that were gavaged with flaxseed cyclic peptide only. 6 hours after gavage, the fluorescence intensity in the rat intestine shifted to the back half of the jejunum and ileum, and the fluorescence intensity was significantly weaker than that after gavage for 4 hours, indicating that the flaxseed cyclic peptide may be absorbed into the blood in the intestine. It can be seen that the flaxseed cyclic peptide with fluorescent properties prepared in this application still shows good stability and continuous fluorescence response ability in a complex intestinal environment, and can truly and dynamically reflect its absorption process in intestinal cells, providing a reliable technical means and experimental basis for studying the transport pathway and absorption mechanism of active peptides in the body.

Claims

1. A flaxseed cyclic peptide, characterized in that The flaxseed cyclic peptide is obtained by reacting the flaxseed cyclic peptide with an isothiocyanate fluorescent compound, and the amino group on the lysine side chain of the flaxseed cyclic peptide reacts with the isothiocyanate group in the isothiocyanate fluorescent compound to form a thiourea bond.

2. The flaxseed cyclic peptide according to claim 1, characterized in that The structural formula of the flaxseed cyclic peptide is: 。 3. The flaxseed cyclic peptide according to claim 1, characterized in that The thiourea bond is obtained under base catalyst conditions.

4. The flaxseed cyclic peptide according to claim 3, characterized in that The base catalyst includes diethylenetriamine.

5. The flaxseed cyclic peptide according to claim 1, characterized in that The thiourea bond is obtained by reaction under a light-proof environment.

6. The flaxseed cyclic peptide according to claim 1, characterized in that The reaction conditions are: temperature 20-30° C., magnetic stirring 5-7 h.

7. The flaxseed cyclic peptide according to claim 1, characterized in that The molar ratio of the flaxseed cyclic peptide to the isothiocyanate fluorescent compound is 1:1.

2.

8. The flaxseed cyclic peptide according to claim 7, characterized in that The purity of the flaxseed cyclic peptide is 95-99%.

9. Use of the flaxseed cyclic peptide according to any one of claims 1 to 8 in the preparation of anti-inflammatory or anti-cancer drugs.

10. Use of the flaxseed cyclic peptide according to any one of claims 1 to 8 in a study on intestinal absorption of the flaxseed cyclic peptide.

Images