Neustatin cyclic peptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to a neurostatin cyclic peptide and a preparation method and application thereof. Comprising the following steps: preparing a peptide chain; washing the peptide chain, adding a mixed solution of trifluoroethanol and dichloromethane, oscillating, and filtering to obtain a linear peptide crude product; adding a dichloromethane solution containing hydroxybenzotriazole, benzotriazole-1-yl-oxytripyrrolidinyl phosphorus hexafluorophosphate and diisopropylethylamine into the linear peptide crude product, carrying out cyclization reaction, and carrying out vacuum concentration and purification to obtain a cyclized peptide crude product; and carrying out C-terminal amidation and side chain deprotection on the cyclized peptide crude product, and separating and purifying to obtain the neuroinhibitory cyclopeptide. By constructing the NST cyclopeptide which is more stable and higher in activity, the convenience of subsequent NST related researches is improved, and meanwhile, the NST cyclopeptide can be used as a research basis for developing novel potential medicines for treating obesity and eating disorder (such as violent eating).

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a neurostatin cyclic peptide and a preparation method and application thereof. Background Art

[0002] Neuronostatin (NST) is an endogenous neuropeptide with certain physiological functions that was finally identified by Samson et al. using a new bioinformatics method by searching for cleavage sites in the precursor of the known polypeptide Somatostatin (SST), screening for evolutionarily conserved sequences of more than 200 different potential peptides discovered, and then conducting experimental verification.

[0003] NST and SST have been shown to be co-expressed in many types of tissue cells, including hypothalamic neurons, pancreatic delta cells, and oxyntic mucosal parietal cells. However, although NST and SST are co-expressed in many types of tissue cells and are encoded by the same gene, their content in various tissues shows significant differences. NST has the highest content in the spleen, followed by the pancreas, brain, and hypothalamus, respectively, while SST is most abundant in the hypothalamus. Therefore, it can be speculated that they have different processing processes in different tissues. Given their co-expression in multiple different tissues and significant expression differences, it is suggested that NST may also play an important and different function from SST, just like SST.

[0004] However, NST does not interact with the five known SST receptors. Therefore, NST may play some other important functions in brain nerve function, which is different from SST. And preliminary studies have proved this: in cultured rat hypothalamic neurons, SST can reduce calcium influx, while NST can increase calcium concentration in neuronal cells. And NST induces the expression of early response genes c-Jun and c-Fos in many different tissues, and has been shown in vitro to participate in the discharge of hypothalamic neurons, regulate nerve migration, and has the ability to increase mean arterial pressure and inhibit light-induced rat eating and water intake after intraventricular administration. Eating and drinking are related to the expression of satiety molecules Nefatin-1 and NST.

[0005] The pancreas plays a very important role in the regulation of blood glucose. In the pancreas, NST can be produced in δ cells, thereby stimulating the production and release of glucagon, especially under low glucose conditions. In addition, intraperitoneal injection of NST in male mice can cause the accumulation of pancreatic c-Jun, which is consistent with the activation of α cells. In cultured pancreatic α cells, impaired expression of GPR107 caused NST to lose the ability to increase the level of proglucagon mRNA, and phosphorylation of PKA was also blocked. Therefore, in isolated rat islets, NST must promote the release of glucagon and the expression of glucagon mRNA through GPR107, and through endocrine interaction between pancreatic α cells and β cells to regulate glucose homeostasis. Consistent with this, in isolated rat pancreatic αTC1-9 cell line, high doses of neurostatin (4000nmol / kg) can promote low glucose-induced glucagon release and significantly increase the level of glucagon mRNA. Furthermore, NST was shown to significantly increase the level of proglucagon mRNA in cultured α cells and promote the release of glucagon in a concentration-dependent manner under low-glucose conditions. In addition, NST administration delayed the clearance of glucose in rat models and significantly inhibited the insulin response in vivo. Therefore, at least one role of NST in blood glucose regulation is to limit insulin secretion, promote glucagon secretion, and maintain blood glucose levels.

[0006] Commonly used anxiety assessment tests include the forced swim test and the elevated plus maze test. In the forced swim test in mice, intracerebroventricular injection of NST (4-40nmol / kg) leads to increased immobility time in mice and depressant-like effects on central melanocortin and γ-aminobutyric acid type A receptor (GABAA). In rats, intracerebroventricular injection of NST (24nmol / kg) produces an anxiolytic effect.

[0007] In addition to the physiological functions mentioned above, NST plays an important role in appetite, pain, neuroendocrine, etc. It is even a promising new therapeutic target for prostate cancer.

[0008] As a new type of drug carrier and therapeutic molecule with its unique advantages, peptides have been widely used in many fields such as tumor imaging, immunotherapy and cancer treatment in recent years. However, since proteases can easily recognize the amino and carboxyl groups at both ends of the peptide backbone and degrade the peptide, the peptide can easily lose its function. However, compared with easily degradable linear peptides, cyclic peptides have higher stability. Because the formation of cyclic peptides through disulfide bonds can eliminate the amino or carboxyl groups at both ends of the peptide, it can prevent it from being degraded, thereby extending the half-life of the peptide. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a neurostatin cyclic peptide and a preparation method and application thereof. This method not only improves the convenience of subsequent NST-related research by constructing a more stable and more active NST cyclic peptide, but also can serve as a research basis for developing new potential drugs for the treatment of obesity and eating disorders (such as bulimia).

[0010] To this end, the present invention provides the following technical solutions:

[0011] In a first aspect, the present invention provides a method for preparing a neurostatin cyclic peptide in an optional embodiment, comprising the following steps:

[0012] S1: adding a dichloromethane solution containing Fmoc-amino acid, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate and diisopropylethylamine to the resin for condensation reaction, and removing the Fmoc protecting group after the reaction to obtain a peptide chain;

[0013] S2: After washing the peptide chain, a mixed solution of trifluoroethanol and dichloromethane is added, shaken, and filtered to obtain a crude linear peptide;

[0014] S3: adding a dichloromethane solution containing hydroxybenzotriazole, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate and diisopropylethylamine to the crude linear peptide, performing a cyclization reaction, concentrating under reduced pressure and purifying to obtain a crude cyclized peptide;

[0015] S4: The crude cyclic peptide is subjected to C-terminal amidation and side chain deprotection, and then separated and purified to obtain the neuroinhibitory cyclic peptide.

[0016] Furthermore, in step S4, the method for C-terminal amidation is: adding N,N-dimethylformamide, hydroxybenzotriazole and diisopropylcarbodiimide to dissolve the crude cyclic peptide, incubating at room temperature for 30 minutes to activate the crude cyclic peptide, and then adding N,N-dimethylformamide solution containing aminobutyric acid and reacting for 4 hours.

[0017] Furthermore, in step S4, the side chain deprotection method is: adding the crude product after C-terminal amidation to a mild solution of trifluoroacetic acid and dichloromethane, and reacting for 4 hours.

[0018] Preferably, the molar ratio of trifluoroacetic acid to dichloromethane is 1:3.

[0019] Furthermore, in step S4, the separation and purification method is: dissolving the crude product after side chain deprotection in a 40% by mass acetonitrile aqueous solution, and then performing HPLC purification.

[0020] Preferably, the mobile phase A for HPLC purification is a trifluoroacetic acid aqueous solution with a mass fraction of 0.1%, and the mobile phase B is a trifluoroacetic acid acetonitrile solution with a mass fraction of 0.1%.

[0021] Preferably, in step S1, the Fmoc-amino acid includes one or more of Fmoc-S-trityl-L-cysteine, Fmoc-L-alanine, Fmoc-L-leucine, Fmoc-O-tert-butyl-L-serine, Fmoc-N-trityl-L-glutamine, Fmoc-L-phenylalanine, N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine and Fmoc-L-proline; and / or the molar ratio of the Fmoc-amino acid, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate and diisopropylethylamine is 1:1:2.

[0022] Preferably, in step S2, the molar ratio of trifluoroethanol to dichloromethane is 1:4; and / or, in step S3, the molar ratio of hydroxybenzotriazole, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate and diisopropylethylamine is 1:1:2.

[0023] In a second aspect, the present invention provides a neurostatin cyclic peptide in an optional embodiment, which is prepared by the above-mentioned preparation method.

[0024] In a third aspect, the present invention provides, in an optional embodiment, a neurostatin cyclic peptide prepared by the above preparation method or the use of the above neurostatin cyclic peptide in the preparation of drugs for treating obesity and eating disorders (such as bulimia).

[0025] Compared with the prior art, the present invention has one of the following beneficial effects:

[0026] This method not only improves the convenience of subsequent NST-related research by constructing a more stable and more active NST cyclic peptide, but also serves as a research basis for developing new potential drugs for the treatment of obesity and eating disorders (such as bulimia). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The synthetic route flow chart of Example 1 of the present invention is as follows;

[0028] Figure 2 It is the HPLC chromatogram in Example 1 of the present invention;

[0029] Figure 3is the structural formula of the neurostatin cyclic peptide in Example 1 of the present invention, wherein A indicates the sites for adding cysteine ​​at both ends of the linear NST. Since C-terminal amidation is very important for the function of the NST linear peptide, the constructed cyclic peptide still retains the C-terminal amidation, and the box indicated by B marks the C-terminal amidation that is still retained.

[0030] Figure 4 The HPLC chromatogram of the neurostatin cyclic peptide in Example 2 of the present invention;

[0031] Figure 5 The effects of neurostatin and neurostatin cyclic peptide on blood glucose in zebrafish in Example 2 of the present invention, where * represents P<0.05, ** represents P<0.01, and *** represents P<0.001;

[0032] Figure 6 The effects of neurostatin and neurostatin cyclic peptide on blood glucose in zebrafish in Example 2 of the present invention, where * represents P<0.05, ** represents P<0.01, and *** represents P<0.001;

[0033] Figure 7 The effects of neurostatin and neurostatin cyclic peptide on the open field behavior of zebrafish in Example 2 of the present invention, wherein * represents P<0.05, ** represents P<0.01, and *** represents P<0.001;

[0034] Figure 8 This is a thermal imaging image of the effects of neurostatin and neurostatin cyclic peptide on the open field behavior of zebrafish in Example 2 of the present invention;

[0035] Fig. 9 This is the effect of neurostatin and neurostatin cyclic peptide in Example 2 of the present invention on the behavior of zebrafish in the light and dark box, wherein * represents P<0.05, ** represents P<0.01, *** represents P<0.001, # represents P<0.05, ## represents P<0.01, and ### represents P<0.001. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] In the following examples, the Fmoc-amino acid is Fmoc-S-trityl-L-cysteine ​​(Fmoc-Cys(Trt)-OH), Fmoc-L-alanine (Fmoc-Ala-OH), Fmoc-L-leucine (Fmoc-Leu-OH), Fmoc-O-tert-butyl-L-serine (Fmoc-Ser(tBu)-OH), Fmoc-N-trityl-L-glutamine (Fmoc-Gln(Trt)-OH), Fmoc-L-phenylalanine (Fmoc-Phe-OH), N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-Arg(Pbf)-OH) and Fmoc-L-proline (Fmoc-Pro-OH).

[0040] Example 1

[0041] This example provides a method for preparing a neurostatin cyclic peptide. For the specific process, see Figure 1 , including the following steps:

[0042] S1: (1) Weigh a certain amount of resin, place it in a synthesis reaction tube, soak it with DCM to cover the resin, and stir it slowly for 30 minutes. Finally, drain it.

[0043] (2) Add appropriate amount of Fmoc-Cys(Trt)-OH and DIPEA in DCM solution and shake at room temperature overnight.

[0044] (3) Wash with DCM (10 ml) and DMF (10 ml) three times, each time for 3 minutes.

[0045] (4) Add appropriate amount of methanol and react for 20 min to seal the unreacted resin.

[0046] (5) Wash with DCM (10 ml) and DMF (10 ml) three times, each time for 3 minutes.

[0047] (6) Deprotection: Add 20% piperidine in DMF solution (10 ml), shake at room temperature for 10 minutes, repeat twice to remove the Fmoc protecting group on the amino acid amino group.

[0048] (7) Wash with DCM (10 ml) and DMF (10 ml) three times, each time for 3 minutes.

[0049] (8) Condensation: According to the polypeptide sequence, Fmoc-amino acid, HCTU and DIPEA were dissolved in an appropriate amount of NMP in a molar ratio of 1:1:2, added to a reaction tube, and reacted at 35°C with shaking for 1 h.

[0050] (9) Pick up a small amount of resin, add ninhydrin reagent and heat at 110°C for 5 minutes. If the resin is colorless, it means that the condensation reaction is complete. You can proceed to the next step, otherwise continue condensation. This step is to ensure the generation of the correct peptide chain.

[0051] (10) Repeat steps (6)-(9) until all amino acids are connected. Repeat step (6) to remove the Fmoc protecting group.

[0052] S2: (1) After the peptide chain is synthesized, it is thoroughly washed with anhydrous ether and dried, and an appropriate amount of TFE-DCM solution (molar ratio of 1:4) is added and shaken at room temperature overnight.

[0053] (2) Filter, collect the filtrate and concentrate under reduced pressure to obtain a crude linear peptide, and perform HPLC analysis.

[0054] S3: (1) HOBt (5-fold molar amount), PyBOP (5-fold molar amount) and DIPEA (10-fold molar amount) were dissolved in an appropriate amount of DCM and slowly added dropwise to the DCM solution of the linear peptide.

[0055] (2) After the dropwise addition was completed, the temperature was raised to room temperature under natural conditions and the reaction was slowly stirred for 1 h.

[0056] (3) The product was concentrated under reduced pressure, purified by gel column, and concentrated under reduced pressure.

[0057] S4: (1) After the last Cys is added and deprotected, perform the acylation reaction. First, add an appropriate amount of DMF, then add an appropriate amount of HOBt and DIC, stir and mix to completely dissolve. Incubate at room temperature for 30 minutes to fully activate the peptide.

[0058] (2) Add a DMF solution containing an appropriate amount of aminobutyric acid into the reaction tube and stir evenly. Stir and react at room temperature for 4 hours, or at 4°C overnight. Concentrate under reduced pressure.

[0059] (3) The crude cyclic peptide was added to a TFA-DCM (1:3) solution and reacted at room temperature with shaking for 4 h to remove the side chain protecting groups.

[0060] (4) The obtained crude C-terminal amidated cyclic peptide was dissolved in a 40% acetonitrile aqueous solution and purified by preparative RPHPLC. The analytical column type was SHIMADZUInertsilODS-SP (4.6*250mm*5um), the mobile phase A was 0.1% TFA aqueous solution, B was 0.1% TFA acetonitrile solution, the detection wavelength was 220nm, and the flow rate was 1ml / min. Elution mode: gradient elution, the initial concentration of phase B was 15%. The gradient elution program is shown in Table 1, and the HPLC chromatogram is shown in Table 1. Figure 2 The results of NST cyclic peptide separation and elution are as follows: Figure 2As shown in Table 2: the target peak of NST cyclic peptide appears at 11.632 min, and the peak area reaches 98.30%, while the non-NST cyclic peptide only accounts for 1.70%. Finally, freeze-drying to obtain a pure white powder, which is neurostatin cyclic peptide (NST cyclic peptide).

[0061] The structural formula of neurostatin cyclic peptide can be found in Figure 3 , A indicates the sites for adding cysteine ​​at both ends of the linear NST. Because C-terminal amidation is very important for the function of the NST linear peptide, the constructed cyclic peptide still retains the C-terminal amidation. The box indicated by B is marked with the C-terminal amidation that is still retained.

[0062] Table 1 Gradient elution program

[0063] Time Module Action Value 0 Pumps B.Conc 15 25 Pumps B.Conc 75 25.01 Pumps B.Conc 100 30 Pumps B.Conc 100 30.01 Pumps Stop

[0064] Table 2 Gradient elution results

[0065] RT Area %Area Height 1 11.5 7307 0.10 6378 2 11.632 7037157 98.30 819266 3 12.036 114725 1.6 11161

[0066] Example 2

[0067] Zebrafish intraperitoneal injection

[0068] Prepare anesthesia platform: Make a groove in the center of a soft sponge block with a length and width of about 5 cm. The size should be large enough to accommodate an adult zebrafish. Soak it in an anesthesia box for later use. Low temperature anesthesia: Put the zebrafish to be injected into an anesthesia box with a water temperature of 26-28℃, take out an adult zebrafish to be injected, and add crushed ice in small amounts and multiple times to gradually lower the water temperature. At this time, the zebrafish shows a slowdown in movement. As the temperature continues to drop, the zebrafish's gill cover breathing gradually weakens and slows down, and the swimming weakens. When the water temperature drops to about 12℃, the movement stops completely and the gill cover movement slows down until it stops. Finally, the zebrafish loses balance and floats to the surface.

[0069] Place the prepared groove sponge in the anesthesia box in advance. After the zebrafish floats to the surface and stops swimming, place the zebrafish with its back facing down in the sponge groove. Then quickly and steadily insert the microsyringe into the midline between the zebrafish's pelvic fins. The insertion depth should ensure that the peptide can be injected into the zebrafish's abdominal cavity. After the injection, quickly remove the fish from the sponge groove and put it back into independent warm water (26-28℃), and restore its movement ability within seconds. The same batch of fish must be injected quickly in a short time to prevent long-term low-temperature anesthesia from affecting the normal activity of the fish's body. When performing the second batch of intraperitoneal injections, replace the water in the anesthesia box with warm water and cool it down again.

[0070] Zebrafish blood sugar test

[0071] Zebrafish raised under the same conditions were weighed, and zebrafish with similar weights were selected and placed separately and fasted for 72 hours, so that the blood sugar of all zebrafish was reduced to the baseline level and their initial blood sugar was kept consistent as much as possible.

[0072] The experiment was divided into 3 groups, namely, the PBS-injected control group, the NST-injected group, and the NST-cyclic peptide-injected group, with 10 fish in each group.

[0073] During the experiment, after hypothermia anesthesia, PBS, NST (10mM, 4uL) or NST cyclic peptide (10mM, 4uL) were injected intraperitoneally, and the tail artery was cut 15min, 30min, 1h, 2h, 3h, and 4h after the injection to allow the blood to flow out, and then the blood sugar was measured using SANNUO blood glucose meter and Sinocare blood glucose test strips.

[0074] Results: Intraperitoneal injection of zebrafish with different concentrations of NST cyclic peptide (0.1mM, 1mM, 10mM, 100mM / 4μL) produced a dose-dependent blood glucose increase in the blood glucose measurement experiment, and there was a significant difference compared with the control group (PBS injection group). Intraperitoneal injection of 10mM, 4μL NST cyclic peptide showed the greatest blood glucose increase effect 15min after injection, while lower and higher doses did not show a better blood glucose increase effect (see Figure 4 ).

[0075] Compared with the group injected with NST, the intraperitoneal injection of 10mM, 4μL NST cyclic peptide had a longer duration of action, with significant differences at 2 and 3h after injection (see Figure 5 ).

[0076] Zebrafish feeding experiment

[0077] The experimental setup consisted of six circular fish tanks, each filled with 500 ml of distilled water to establish a uniform experimental baseline. Zebrafish were evenly divided into two main groups of 15: control, NST-injected, and NST-cyclotide-injected, with three replicates of 5 fish each. Equal amounts of freshly hatched brine shrimp were placed in each fish tank. The initial number of brine shrimp was determined as follows: First, the fish tank water was thoroughly stirred to evenly distribute the brine shrimp, and then 1 ml of water was drawn from five randomly selected locations using a pipette, for a total of 5 ml. The initial total number of brine shrimp in the entire fish tank was calculated by counting the number of brine shrimp in these 5 ml water samples, and this step was plotted as the starting total number graph. During the experiment, the above water sampling and counting process was repeated after 1 hour, 2 hours, 3 hours, 4 hours, and 5 hours to determine the number of remaining brine shrimp. By subtracting the remaining amount at each time point from the initial total amount, the actual food intake of zebrafish at each time interval can be accurately obtained.

[0078] Results: After zebrafish were intraperitoneally injected with 10mM, 4μL NST cyclic peptide, the experimental results showed that NST cyclic peptide had the same inhibitory effect on feeding as NST, and there was a significant difference between the control group and the NST injection group 1h and 2h after injection, while the NST cyclic peptide injection group was significantly different from the NST injection group 1h after injection (see the results). Figure 6 ).

[0079] Zebrafish behavioral assay to observe the effects of NST cyclotide on cognitive behavior

[0080] (1) Open field

[0081] S1: Grouping: The zebrafish were divided into groups, with 15 zebrafish in each group, and divided into experimental group 1 (NST injection treatment), experimental group 2 (NST cyclic peptide injection treatment) and control group (PBS injection treatment).

[0082] S2: Drug treatment: The zebrafish were adapted to standard conditions for a period of time to ensure that they were in a stable state. The zebrafish in experimental group 1 were intraperitoneally injected with 4 μL of NST solution, the zebrafish in experimental group 2 were intraperitoneally injected with 4 μL of NST cyclic peptide solution, and the control group was injected with an equal amount of PBS (phosphate buffer solution) as an experimental control.

[0083] S3: Open field experiment: Make sure that the open field experimental device is located in a quiet and evenly lit environment. After debugging the equipment, the treated zebrafish are placed one by one from the breeding container into the center of the open field experimental device. After a short adaptation period, the behavior within 10 minutes is recorded. During the experimental observation, the zebrafish treated with PBS in the control group are observed first. After the observation is completed, the zebrafish in the experimental group are observed. A high-definition camera is used to shoot the swimming trajectory of the zebrafish in the tank from the top, and the whole process is recorded to ensure that all behavioral details can be traced. After that, the behavior is analyzed through the dedicated video analysis software EthoVision.

[0084] Results: By studying the effects of Neuronostatin on zebrafish's autonomous behavior, exploratory behavior and tension in a novel environment, the results of the open field experiment showed that NST and NST cyclic peptide had an inhibitory effect on the behavioral activities of zebrafish. Figure 7 and Figure 8 The figure shows the relationship between the swimming speed and swimming distance of zebrafish after injection of NST and NST cyclic peptide.

[0085] (2) Light and dark box

[0086] S1: Grouping: The zebrafish were divided into groups, with 15 zebrafish in each group, and divided into experimental group 1 (NST injection treatment), experimental group 2 (NST cyclic peptide injection treatment) and control group (PBS injection treatment).

[0087] S2: Drug treatment: The zebrafish were adapted to standard conditions for a period of time to ensure that they were in a stable state. The zebrafish in experimental group 1 were intraperitoneally injected with 4 μL of NST solution, the zebrafish in experimental group 2 were intraperitoneally injected with 4 μL of NST cyclic peptide solution, and the control group was injected with an equal amount of PBS (phosphate buffer solution) as an experimental control.

[0088] S3: Light-dark box experiment: It was conducted in a quiet and well-light-controlled environment to ensure that the external light of the light-dark box was stable and would not cause additional stimulation to the zebrafish. The zebrafish was gently scooped out of the breeding container and gently placed in the dark box using a fishing net. The curtain of the experimental station was quickly closed to place the zebrafish in a quiet and independent environment to reduce external interference to the zebrafish. When placing the zebrafish in the light-dark box, the zebrafish were placed in the light-dark box device one by one. After a short adaptation period, the video recording device was started and the behavior within 10 minutes was recorded, including the time of entering the light and dark areas and the number of crossings. During the experimental observation, the zebrafish injected with PBS in the control group were observed first, and then the zebrafish in the experimental group were observed after the observation was completed. A high-definition camera was used to shoot the swimming trajectory of the zebrafish in the light-dark box from the top throughout the process, and the behavior of each zebrafish in the light-dark box for 10 minutes was recorded to ensure that all behavioral details were traceable, and then the behavior was analyzed by the dedicated video analysis software EthoVision.

[0089] Results: By studying the effects of Neuronostatin and Neuronostatin cyclic peptide on zebrafish in the light-dark box experiment, the anxiety level and light avoidance behavior of zebrafish can be analyzed. The results of the light-dark box experiment can be found in Fig. 9 , Fig. 9 It shows that there is no significant difference between cyclotide injection and PBS injection, and nst injection can cause anxiety.

[0090] Although the principle of the present invention has been described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely explanations of the exemplary implementations of the present invention, and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute limitations on the scope of the present invention, and any obvious changes such as equivalent transformations, simple replacements, etc. based on the technical solution of the present invention, without departing from the spirit and scope of the present invention, fall within the scope of protection of the present invention.

Claims

1. A method for preparing a neurostatin cyclic peptide, characterized in that: The following steps are involved: S1: adding a dichloromethane solution containing Fmoc-amino acid, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate and diisopropylethylamine to the resin for condensation reaction, and removing the Fmoc protecting group after the reaction to obtain a peptide chain; S2: After washing the peptide chain, a mixed solution of trifluoroethanol and dichloromethane is added, shaken, and filtered to obtain a crude linear peptide; S3: adding a dichloromethane solution containing hydroxybenzotriazole, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate and diisopropylethylamine to the crude linear peptide, performing a cyclization reaction, concentrating under reduced pressure and purifying to obtain a crude cyclized peptide; S4: The crude cyclic peptide is subjected to C-terminal amidation and side chain deprotection, and then separated and purified to obtain the neuroinhibitory cyclic peptide.

2. The method for preparing the neurostatin cyclic peptide according to claim 1, characterized in that: In step S4, the method for C-terminal amidation is: The crude cyclic peptide was dissolved in N,N-dimethylformamide, hydroxybenzotriazole and diisopropylcarbodiimide, incubated at room temperature for 30 minutes to activate the crude cyclic peptide, and then added with N,N-dimethylformamide solution containing aminobutyric acid and reacted for 4 hours.

3. The method for preparing the neurostatin cyclic peptide according to claim 1, characterized in that: In step S4, the side chain deprotection method is: The crude product after C-terminal amidation was added to a mild solution of trifluoroacetic acid and dichloromethane and reacted for 4 hours.

4. The method for preparing the neurostatin cyclic peptide according to claim 3, characterized in that: The molar ratio of trifluoroacetic acid to dichloromethane is 1:

3.

5. The method for preparing the neurostatin cyclic peptide according to claim 1, characterized in that: In step S4, the separation and purification method is: The crude product after side chain deprotection was dissolved in 40% by mass acetonitrile aqueous solution and then purified by HPLC.

6. The method for preparing the neurostatin cyclic peptide according to claim 5, characterized in that: The mobile phase A of the HPLC purification is a trifluoroacetic acid aqueous solution with a mass fraction of 0.1%, and the mobile phase B is a trifluoroacetic acid acetonitrile solution with a mass fraction of 0.1%.

7. The method for preparing the neurostatin cyclic peptide according to claim 1, characterized in that: In step S1, the Fmoc-amino acid includes one or more of Fmoc-S-trityl-L-cysteine, Fmoc-L-alanine, Fmoc-L-leucine, Fmoc-O-tert-butyl-L-serine, Fmoc-N-trityl-L-glutamine, Fmoc-L-phenylalanine, N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine and Fmoc-L-proline; and / or, The molar ratio of the Fmoc-amino acid, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate and diisopropylethylamine is 1:1:

2.

8. The method for preparing the neurostatin cyclic peptide according to claim 1, characterized in that: In step S2, the molar ratio of trifluoroethanol to dichloromethane is 1:4; and / or, In step S3, the molar ratio of hydroxybenzotriazole, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate and diisopropylethylamine is 1:1:

2.

9. A neurostatin cyclic peptide, characterized in that The preparation method is described in any one of claims 1 to 8.

10. Use of the neurostatin cyclic peptide prepared by the preparation method according to any one of claims 1 to 8 or the neurostatin cyclic peptide according to claim 9 in the preparation of drugs for treating obesity and eating disorders.

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