Preparation method and application of golden squid ink peptidoglycan

Abstract

The present invention provides a preparation method and use of golden squid ink peptidoglycan, which belongs to the field of marine biomedicine. The technical solution takes squid ink and successively homogenizes and defats it, ultrasonically treats the defatted squid ink, soaks and centrifuges it at low temperature to take the supernatant, ultrafilters it to remove impurities with an ultrafiltration membrane, precipitates it with anhydrous ethanol, removes protein with the sevag method, and freezes and dries it to obtain a crude extract of golden squid ink polysaccharide; the crude extract is separated and purified by a DEAE-Sepharose Fast Flow anion exchange chromatography column, the protein-sugar peak eluate is collected, the dialysis method is used for desalting and concentrating, and freeze-drying is performed to obtain golden squid ink peptidoglycan. The preparation method is not only simple and efficient, but also the obtained product has a significant ovarian repair effect. In addition, the experiment found that the golden squid ink peptidoglycan prepared by the present invention can improve intestinal peristalsis, repair liver oxidative damage, and also has the effect of inhibiting pancreatic cancer proliferation, migration and invasion. Based on the above beneficial findings, the scope of use of golden squid ink peptidoglycan is further expanded.

Description

Technical Field

[0001] The invention belongs to the field of marine biomedicine, and specifically relates to a preparation method and application of golden cuttlefish ink peptidoglycan. Background Art

[0002] Golden cuttlefish is the main cephalopod in my country's coastal waters, mainly distributed in the northern waters of Rizhao, Shandong, Lanshantou, Qingdao, etc. Squid ink is a black viscous liquid synthesized by the secretory glands in the squid ink sac. Its main chemical components are melanin and proteoglycan complexes, which have high nutrition and multiple medicinal functions. It has become one of the current research and development hotspots because of its wide range of pharmacological effects such as anti-oxidation, improving immunity, and inhibiting the growth of cancer cells. On the other hand, people usually discard the black ink sac when eating and processing squid, which not only causes a great waste of medicinal and edible resources, but also causes environmental pollution. Therefore, efficient extraction of active ingredients in squid ink is of great significance to the development and utilization of squid ink resources.

[0003] Peptidoglycan is a polysaccharide and amino acid polymer, and is an important component of the cell wall of prokaryotes. Studies have shown that peptidoglycan has biological activities such as antioxidant, anti-tumor, anti-coagulation and antiviral. Cuttlefish ink peptidoglycan is a proteoglycan bioactive substance isolated and extracted from cuttlefish ink. There have been a few reports on the composition, activity and structure of cuttlefish ink peptidoglycan, but research on its biological efficacy is still relatively limited. Summary of the invention

[0004] The first technical problem to be solved by the present invention is: how to prepare golden cuttlefish ink peptidoglycan more conveniently and efficiently.

[0005] The second technical problem to be solved by the present invention is: how to prepare golden squid ink peptidoglycan with higher biological activity.

[0006] The third technical problem to be solved by the present invention is: how to expand the application scope of golden cuttlefish ink peptidoglycan.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The preparation method of golden cuttlefish ink peptidoglycan comprises the following steps:

[0009] S1. Cutting out the cuttlefish ink from the ink sac of fresh golden cuttlefish, homogenizing with a homogenizer, defatting, and filtering to obtain defatted cuttlefish ink;

[0010] S2, ultrasonically treating the defatted cuttlefish ink, extracting, centrifuging and collecting the supernatant to obtain a cuttlefish ink polysaccharide primary extract;

[0011] S3, filtering the squid ink polysaccharide primary extract through an ultrafiltration membrane to remove impurities, thereby obtaining a golden squid ink polysaccharide ultrafiltrate;

[0012] S4, adding anhydrous ethanol to the ultrafiltrate of golden cuttlefish ink polysaccharide for precipitation to obtain cuttlefish ink polysaccharide precipitate;

[0013] S5, precipitating the squid ink polysaccharide, adding sevag reagent to remove free protein impurities, and freeze-drying to obtain golden squid ink polysaccharide;

[0014] S6. Purify the cuttlefish ink polysaccharide by using a DEAE-Sepharose Fast Flow anion exchange chromatography column, using a 0.1-0.5 M Nacl solution for gradient elution, detecting the protein peak by a nucleic acid protein detector, and detecting the sugar peak by a phenol-sulfuric acid method, collecting the protein-sugar peak eluate, desalting and concentrating it by dialysis, and freeze-drying it to obtain golden cuttlefish ink peptidoglycan.

[0015] Preferably, in step S1, the degreasing conditions are: 5% (m / v) SDS solution, mixed and incubated in a water bath at 80°C for 25 min, centrifuged and resuspended in a 5% SDS solution, in a water bath at 80°C for 15 min, and washed with ultrapure water until there is no foam.

[0016] Preferably, in step S2, the ultrasonic conditions are: ultrasonic power 200w, ultrasonic extraction time 5h, extraction material-liquid ratio 1:20, centrifugal conditions 10000 r / min, and centrifugal time 30min.

[0017] Preferably, in step S3, the ultrafiltration conditions are: using a 30 KD ultrafiltration membrane, a centrifugal temperature of 4°C, a centrifugal speed of 4000 r / min, and a centrifugal time of 20 min.

[0018] Preferably, in step S4, the concentration of ethanol added is 95%, the volume added is 4 times that of the ultrafiltrate, the precipitation temperature is 4° C., and the precipitation time is 24 h.

[0019] Preferably, in step S5, the ratio of the added sevag reagent is 3:1 by volume of chloroform and n-butanol, and the shaking time is 30 min.

[0020] Preferably, in step S5, the freeze-drying temperature is -40°C, the vacuum degree is 10 Pa, and the freeze-drying time is 12 h.

[0021] Preferably, in step S6, the elution rate of gradient elution is 1 ml / min, and each tube has 5 ml.

[0022] Preferably, in step S2, the leaching temperature is 4°C and the leaching time is 24 hours.

[0023] Preferably, the method further comprises the following step S7: using SDS-PAGE to detect the purity of the golden cuttlefish ink peptidoglycan.

[0024] On the basis of the above technical solution, the present invention further provides the use of the golden cuttlefish ink peptidoglycan prepared by the above preparation method for preparing a digestive stimulating drug. Preferably, the dosage of the drug is 12 mg / kg / d.

[0025] On the basis of the above technical solution, the present invention further provides the use of the golden cuttlefish ink peptidoglycan prepared by the above preparation method for preparing a liver oxidative damage repair drug. Preferably, the dosage of the drug is 10-50 mg / kg / d.

[0026] On the basis of the above technical scheme, the present invention further provides the use of the golden cuttlefish ink peptidoglycan prepared by the above preparation method for preparing a drug for treating pancreatic cancer.

[0027] In the present invention, VC refers to vitamin C, and POF refers to premature ovarian failure model.

[0028] The present invention provides a preparation method and use of golden squid ink peptidoglycan, which belongs to the field of marine biomedicine. The technical scheme takes squid ink and successively homogenizes and defats it, ultrasonically treats the defatted squid ink, soaks and centrifuges it at low temperature to obtain the supernatant, ultrafilters it through an ultrafiltration membrane to remove impurities, precipitates it with anhydrous ethanol, removes protein through the sevag method, and freezes and dries it to obtain a crude extract of golden squid ink polysaccharide; a DEAE-Sepharose Fast Flow anion exchange chromatography column is used to separate and purify the crude extract, the protein-sugar peak eluate is collected, the dialysis method is used for desalting and concentration, and freeze-drying is used to obtain golden squid ink peptidoglycan. The preparation method is not only simple and efficient, but also the obtained product has a significant ovarian repair effect. In addition, the experiment found that the golden squid ink peptidoglycan prepared by the present invention can improve intestinal peristalsis, repair liver oxidative damage, and also has the effect of inhibiting the proliferation, migration and invasion of pancreatic cancer. Based on the above beneficial findings, the scope of use of golden squid ink peptidoglycan is further expanded.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The present invention uses the ink of golden cuttlefish as raw material to extract and prepare a new golden cuttlefish ink polypeptide polysaccharide with a molecular weight of about 71kD.

[0031] 2. Compared with common squid ink peptidoglycan, the product of the present invention has stronger antioxidant activity, can more effectively improve fertility such as estrous cycle, ovarian index, ovulation number and fertilized egg blastocyst formation rate, and its biological activity is significantly higher than that of common squid ink peptidoglycan. Thanks to this technical advantage, the present invention can be used to develop auxiliary treatment drugs or health products for premature ovarian failure, filling the gap of such products on the current market.

[0032] 3. The present invention has found that the prepared golden squid ink peptidoglycan can improve intestinal motility and promote the digestion process, and can therefore be used to develop digestion-promoting drugs.

[0033] 4. The present invention has found that the prepared golden squid ink peptidoglycan has a definite repairing effect on liver tissues and cells of animals with oxidative damage, and can therefore be used to develop drugs for repairing liver oxidative damage.

[0034] 5. In vitro experiments showed that the prepared golden squid ink peptidoglycan can inhibit the proliferation, migration and invasion of pancreatic cancer cells, and is expected to be used in the preparation of drugs for the treatment of pancreatic cancer.

[0035] 6. The risk of side effects from long-term use of the product of the present invention is significantly lower than that of similar drugs, and is safer. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The figures are physical pictures of each stage of the extraction process of golden cuttlefish peptidoglycan; among them, the upper left picture is a physical picture of golden cuttlefish; the upper right picture is a physical picture of the ink sac of golden cuttlefish; the lower left picture is a physical picture of the black precipitate; and the lower right picture is a physical picture of the crude extract of golden cuttlefish peptidoglycan.

[0037] Figure 2 This is the elution curve of golden squid ink peptidoglycan.

[0038] Figure 3 This is the DEAE-Sepharose Fast Flow elution peak diagram of golden squid ink peptidoglycan.

[0039] Figure 4 This is the SDS-PAGE electrophoresis band diagram of golden squid ink peptidoglycan.

[0040] Figure 5 This is a graph showing the changes in the scavenging rate of ABTS free radicals by golden squid ink peptidoglycan.

[0041] Figure 6 This is a diagram showing the effect of golden cuttlefish ink peptidoglycan on the estrous cycle under cyclophosphamide injury; the left side shows the microscopic examination of vaginal smears of mice in different estrous cycles; the right side shows the estrous cycle change curves of each group.

[0042] Figure 7 This is a diagram showing the effect of golden squid ink peptidoglycan on ovarian tissue in a state of cyclophosphamide damage; A is the state of undamaged control ovarian tissue, B is the state of cyclophosphamide damaged ovarian tissue, and C is the state of ovarian tissue after intravenous administration of golden squid ink peptidoglycan.

[0043] Figure 8 This is a diagram showing the effect of golden cuttlefish ink peptidoglycan on the ovarian index under cyclophosphamide damage; the left side is a physical picture of the ovary, and the right side is a statistical chart of the ovarian index.

[0044] Fig. 9 The figure is the effect of golden cuttlefish ink peptidoglycan on fertility such as the number of ovulations and the blastocyst formation rate of fertilized eggs under cyclophosphamide damage; the upper side is a microscopic examination picture, in which A and B show the discharge of the second polar body after fertilization, C and D show the 2-cell stage, and E and F show the blastocyst stage; the lower side is a statistical chart.

[0045] Fig.10 This is a graph showing the effect of golden squid ink peptidoglycan on liver weight, body weight and liver-to-body ratio of mice (x̄±s); the figure shows three groups of data on liver weight, body weight and liver-to-body ratio, and in each group of data, from left to right are control group, model group, 10 mg / kg peptidoglycan group, 25 mg / kg peptidoglycan group and 50 mg / kg peptidoglycan group.

[0046] Fig.11 This is the repair effect of golden squid ink peptidoglycan on oxidative damage of mouse liver tissue and cells (x̄±s).

[0047] Fig.12 This is a graph showing the effect of CCK8 on the activity of pancreatic cancer PANC-1 cells by detecting golden squid ink peptidoglycan.

[0048] Fig.13 This is a flow cytometry analysis of the effect of golden squid ink peptidoglycan on apoptosis of pancreatic cancer PANC-1 cells.

[0049] Fig.14 This is a diagram showing the effect of golden squid ink peptidoglycan on scratch healing and migration of pancreatic cancer PANC-1 cells.

[0050] Fig.15 This is a diagram showing the effect of golden squid ink peptidoglycan on the invasion of pancreatic cancer PANC-1 cells. DETAILED DESCRIPTION

[0051] The specific embodiments of the present invention will be described in detail below. In order to avoid too much unnecessary details, the well-known structures or functions will not be described in detail in the following examples. The approximate language used in the following examples can be used for quantitative expression, indicating that the quantity can be allowed to have a certain change without changing the basic function. Unless otherwise defined, the technical and scientific terms used in the following examples have the same meaning as those generally understood by those skilled in the art to which the present invention belongs.

[0052] The cuttlefish ink was removed from the ink sac of fresh golden cuttlefish, placed in a mortar, and 2 volumes of phosphate buffer solution (PBS, 0.01 mol / L, pH 7.4) were added to grind repeatedly to suspend evenly, filtered through 8 layers of medical gauze, and the filtrate was centrifuged at 4°C and 8000 rpm for 30 min. The supernatant was discarded and the black precipitate was collected.

[0053] Add 20 times the volume of defatting solution (0.02M sodium acetate: chloroform: methanol ratio 4:5:10) to dissolve the black precipitate, stir at room temperature for 24 hours, centrifuge at 8000rpm for 20 minutes at 4℃, and collect the precipitate. Wash the precipitate twice by centrifugation with methanol, and then wash the precipitate three times by centrifugation with deionized water, collect the precipitate, and freeze-dry to obtain golden squid ink defatted powder.

[0054] Add 20 times the volume of phosphate buffer solution (PBS, 0.01 mol / L, pH 7.4) to the precipitate, use 200 w power ultrasonic treatment to defatted squid ink powder, repeatedly ultrasonic treatment (each ultrasonic treatment for 15 s, interval of 15 s, ultrasonic treatment 50 times), soak at 4 ℃ for 24 h, centrifuge at 10000 rpm for 30 min, collect the supernatant, repeat twice to obtain the primary extract of squid ink polysaccharide.

[0055] Use 100 KD filter membrane, 4℃, 4000 rpm centrifugation for 20 min to remove impurities, collect the residual liquid on the filter membrane to obtain the golden squid ink polysaccharide ultrafiltrate, add 4 times the volume of 95% ethanol, precipitate at 4℃ for 24h, discard the supernatant, and collect the precipitate. Dissolve the precipitate in PBS buffer, add 3 times the volume of sevag reagent, extract at 4℃ for 24h, carefully collect the supernatant, and freeze-dry to obtain the golden squid ink polysaccharide extract.

[0056] DEAE-Sepharose Fast Flow anion exchange chromatography column was used for purification. 2.5 mg / ml of golden squid ink peptidoglycan was loaded at a flow rate of 0.2 ml / min, and 0.1-0.6 M NaCl solution was used for gradient elution, and 5 elution peaks were obtained. The absorbance was detected by ultraviolet 280 nm, and the nucleic acid protein detector was used for real-time monitoring. When there was no obvious protein peak in the eluate, the elution was stopped. At the same time, the sugar peak was detected at 490 nm by the phenol-sulfuric acid method, and the best protein-sugar peak eluate was collected. The solutions collected from the corresponding peaks were subjected to SDS-PAGE electrophoresis detection, and the results showed that a single protein band with a molecular weight of about 71 KD could be obtained from the 0.4 M NaCl solution elution peak.

[0057] The eluate at the elution peak of 0.4 M NaCl solution was collected, and centrifuged at 4°C, 4500 rpm for 20 min using an ultrafiltration tube with a molecular weight cutoff of 30 KD. This was repeated three times for desalting and concentration, and freeze-drying to obtain golden squid ink peptidoglycan.

[0058] 10 ml of 2 mg / ml ABTS solution and 0.1 ml of 140 mmol / L potassium persulfate solution were mixed and allowed to stand overnight at room temperature and in the dark to form an ABTS free radical reserve working solution. Dilute with 95% ethanol to an absorbance of 0.70 before the experiment. Add 2 ml of peptidoglycan solution with a mass concentration of 1 to 20 mg / ml and 5 ml of ABTS solution to the reaction system, mix thoroughly, keep away from light and at room temperature, measure the absorbance at 734 nm, and mark it as a sample. Use distilled water to do the same treatment as above, measure its absorbance and mark it as a control. The blank group was adjusted to zero with distilled water. The experiment was repeated three times independently, and the scavenging rate was used to represent the antioxidant capacity. The results showed that the scavenging ability of golden squid ink peptidoglycan for ABTS free radicals increased with the increase of mass concentration. The scavenging ability of ABTS free radicals at a mass concentration of 2.5 mg / ml was equivalent to that of VC at 0.5 mg / ml, indicating that the golden squid ink polysaccharide obtained by the present invention has a certain antioxidant capacity.

[0059] The animal model of chemotherapy-induced premature ovarian failure was prepared by a single intraperitoneal injection of 75 mg / kg cyclophosphamide. The modeling effect was evaluated by observing the estrous cycle, detecting hormone levels, observing the morphological structure of the ovaries, the number of ovulations, and the development of the embryos after fertilization, and the conditions for preparing the rat model of chemotherapy-induced premature ovarian failure by cyclophosphamide were optimized. On the 7th day after the injection of cyclophosphamide, the repair group began to be gavaged with 25 mg / kg of golden cuttlefish ink peptidoglycan for 2 weeks. The results showed that the 25 mg / kg dose of golden cuttlefish ink peptidoglycan had a significant protective effect on chemotherapy-induced ovarian function damage through observing the estrous cycle, detecting hormone levels, observing the morphological structure of the ovaries, the number of ovulations, and the development of the embryos after fertilization.

[0060] 40 mice were randomly divided into four groups: normal group, model control group, positive drug prucalopride succinate group, and golden cuttlefish ink peptidoglycan group, with 10 mice in each group. Each mouse in the normal group was given 0.1 mL of 0.9% saline by gavage; during the modeling period, the model control group, golden cuttlefish ink peptidoglycan group, and positive drug prucalopride succinate group were given 0.1 mL of atropine sulfate aqueous solution by gavage twice a day for two consecutive days. On the first day after modeling, mice were given drug treatment, and the normal group and the control group were gavaged with 0.1 mL of 0.9% saline each time, the cuttlefish ink peptidoglycan group was gavaged at a dose of 12 mg / kg / d, and the positive drug prucalopride succinate group was gavaged at a dose of 0.03 mg / kg / d. The mice were given carbon powder the next day and were killed 1.5 hours later. The distance of carbon powder advancement in the intestines of the mice was observed and calculated. The length of the entire small intestine and the length of the small intestine advanced by carbon powder were recorded respectively. The intestinal carbon powder advancement rate was calculated, carbon powder advancement rate = (total length of small intestine - length of small intestine advanced by carbon powder) / total length of small intestine × 100%.

[0061] The results of the intestinal carbon propulsion rate of mice are shown in the following Table 1: Compared with the mice in the intestinal motility fatigue control group, the golden cuttlefish ink peptidoglycan prepared by the present invention can significantly improve the gastrointestinal motility, and the effect is better than that of prucalopride succinate.

[0062] Normal group Model control group Positive drug prucalopride succinate group Golden squid ink peptidoglycan group Carbon powder advancement rate 93.72% 76.62% 85.27% 91.48%

[0063] Three test animals were selected: New Zealand white rabbits, Boxer dogs, and rhesus monkeys. After intragastric administration, the last feces before intragastric administration and the first feces after intragastric administration were collected, and the feces were weighed and counted.

[0064] By comparing the weight and quantity of animal feces before and after gavage, it was found that the administration of golden cuttlefish ink peptidoglycan significantly increased the weight (by 18.0%, 22.3.0%, and 19.5%, respectively) and quantity of feces of experimental animals; at the same time, the feces of the drug-treated group were softer and no watery stools appeared. The above experiments show that golden cuttlefish ink peptidoglycan can significantly enhance the intestinal motility of experimental animals and has the effect of promoting digestion.

[0065] 1. Test methods

[0066] 1.1 Animal experiments

[0067] Forty mice weighing 30±5g were randomly divided into five groups: blank control group, hydrogen peroxide injury group, golden squid ink peptidoglycan low-dose (10mg / kg) repair group, medium-dose (25mg / kg) repair group and high-dose (50mg / kg) repair group, with 8 mice in each group. After each group was caged for one week to adapt to the environment, a 3% hydrogen peroxide solution was prepared and gavaged with hydrogen peroxide solution from the first day of the experiment, with each mouse gavaged with 1 ml. Except for the blank control group, which was gavaged with distilled water, the injury group, low-, medium- and high-dose relief groups were gavaged with 3% H2O2 solution for 10 consecutive days, with free access to food and water. After the liver injury model was successfully established, the blank group and the injury group were gavaged with distilled water respectively, and the repair group was gavaged with golden squid ink peptidoglycan at low, medium and high doses as mentioned above for 10 consecutive days, with free access to food and water. After weighing and recording, mice in each group were killed by cervical dislocation, and the whole liver was quickly separated. After weighing and recording, about 0.5 cm3 of tissue was cut and fixed in 4% formaldehyde solution for 24 h. Conventional paraffin embedding and 5 μm thick sections were made, HE staining, and microscopic observation and photography were performed.

[0068] 1.2 Cell experiments

[0069] After culturing the isolated mouse hepatocytes for 24 hours, the control group was added with complete medium, and the model group and the experimental group were added with complete medium containing 200 μmol / L H2O2, and the culture was continued for 3 hours to establish the mouse hepatocyte oxidative stress injury model. The control group and the model group were replaced with complete medium, and the repair group was added with low concentration (100ug / ml), medium concentration (250ug / ml) and high concentration (500ug / ml) of golden cuttlefish ink peptidoglycan on the basis of complete medium and continued to culture for 48 hours. The cell proliferation activity was detected by MTT method, and the cell apoptosis was observed by Hochest staining and the cell apoptosis rate was calculated.

[0070] 2. Results

[0071] 2.1 Effects of golden squid ink peptidoglycan on the growth status of mice with oxidative damage

[0072] The mice in the control group had a normal life, good spirits, and a normal diet; the mice in the injury group were listless and had poor appetite; the mental state and diet of the mice in the low, medium, and high dose repair groups of golden cuttlefish ink peptidoglycan improved more significantly with the increase in doses. This shows that liver damage caused by hydrogen peroxide can affect the normal life of mice, and golden cuttlefish ink peptidoglycan has a certain repair effect on damage caused by hydrogen peroxide.

[0073] 2.2 Effects of golden squid ink peptidoglycan on body weight and liver-to-body ratio in mice with oxidative damage

[0074] Oxidative damage can lead to a significant decrease in liver weight, body weight and liver-to-body ratio in mice. Compared with the model group, oral administration of 25 mg / kg squid ink peptidoglycan can significantly restore the liver weight and liver-to-body ratio of mice, and is positively correlated with the dose.

[0075] 2.3 Repair effect of golden squid ink peptidoglycan on oxidatively damaged liver tissue and cells in mice

[0076] The morphology and structure of liver tissue in the control group were normal, and the arrangement of liver cells was tight and orderly; a large number of cracks appeared between liver tissue cells in the H2O2 oxidative damage group, and cavitation and wateriness were serious; golden cuttlefish ink peptidoglycan could repair liver tissue lesions in a dose-dependent manner. The results of cell experiments showed that 200μM / L H2O2 culture for 3 h could establish a mouse liver cell oxidative damage model, and 100-500μg / mL cuttlefish ink peptidoglycan could effectively repair liver cell oxidative damage.

[0077] 1. Test methods

[0078] 1.1 CCK8 assay for cell viability

[0079] Pancreatic cancer PANC-1 cells in logarithmic growth phase were cultured at 3×10 3The cells were inoculated into 96-well plates and incubated overnight in an incubator. The control group was replaced with serum-free medium, and the experimental group was added with low (100ug / ml), medium (250ug / ml) and high (500ug / ml) concentrations of golden squid ink peptidoglycan for continued culture. Four replicate wells were set for each treatment. CCK-8 reagent was added at 24 and 48 h, incubated at 37°C for 2 h, and the absorbance at 450 nm was measured to calculate the relative cell viability.

[0080] 1.2 Flow cytometry detection of cell apoptosis

[0081] Pancreatic cancer PANC-1 cells in logarithmic growth phase were cultured at 3×10 5 Cells were inoculated into 6-well plates, grouped according to method 1.1, and cultured for 48 hours. Cells in each group were collected by trypsin digestion without EDTA, centrifuged at 1000×g for 5 min at room temperature, washed 3 times with pre-cooled PBS, and resuspended in 400μL of Annexin V binding buffer. 5μL of Annexin V-FITC and PI reagents were added, incubated at room temperature in the dark for 15 min, and the apoptosis rate of cells in each group was detected by flow cytometry. FlowJo flow cytometry analysis software was used to obtain analysis data.

[0082] 1.3 Cell scratch assay

[0083] Draw straight lines evenly on the back of the 6-well plate with a ruler at intervals of 0.5 to 1 cm. Take pancreatic cancer PANC-1 cells in the logarithmic growth phase and inoculate them in a 6-well plate at 5x105 cells / well. Incubate in a constant temperature incubator for 24 hours, use a 200ul pipette tip to vertically scratch along a straight line, wash with PBS 2-3 times, and group and process according to the method in 1.1. Observe and take pictures under a microscope, and use Image J software to measure the scratch width (L) and calculate the scratch healing rate.

[0084] 1.4 Cell migration and invasion assay

[0085] Pancreatic cancer PANC-1 cells in logarithmic growth phase were starved for 24 h and resuspended in serum-free medium and adjusted to 1×10 5 / mL. In the cell invasion experiment, 60µL of matrix gel diluted at a ratio of 1:10 was added to the upper chamber of the chamber, incubated in a 37°C incubator for 3 h, and serum-free medium was added and placed at 37°C for 30 min to hydrate the matrix membrane. 200μL of cell suspension was added to the upper chamber of the Transwell, and 500μL of culture medium containing 20% ​​fetal bovine serum and different concentrations of exosomes was added to the lower chamber. The cells were grouped according to the method in 1.1 and placed horizontally in a constant temperature incubator for 48 h. 4% paraformaldehyde was used for fixation for 15 min, 0.05% crystal violet stain was used for staining for 5 min, and the cells that did not penetrate the chamber were wiped with a cotton swab. Five fields of view were randomly selected under a microscope for photography, and the number of invasive and migrating cells in each field of view was counted by Image J software.

[0086] The embodiments of the present invention are described in detail above, but the contents are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the scope of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing golden squid ink peptidoglycan, characterized in that: The following steps are involved: Cut out the squid ink from the fresh golden squid ink sac, put it in a mortar, add 2 times the volume of phosphate buffer solution, grind it repeatedly to suspend it evenly, filter it with medical gauze, centrifuge the filtrate at 4°C and 8000rpm for 30min, pour off the supernatant, and collect the black precipitate; Add 20 times the volume of defatting liquid to dissolve the black precipitate, stir at room temperature for 24 hours, centrifuge at 8000 rpm for 20 minutes at 4°C, collect the precipitate, wash the precipitate twice by methanol centrifugation, and then wash the precipitate three times by deionized water centrifugation, collect the precipitate, and freeze-dry to obtain golden squid ink defatted powder; Add 20 times volume of phosphate buffer solution to the golden cuttlefish ink defatted powder, the concentration of the phosphate buffer solution is 0.01 mol / L, and the pH is 7.4, and treat the golden cuttlefish ink defatted powder with ultrasound at a power of 200 W, each ultrasound for 15 seconds, an interval of 15 seconds, ultrasound for 50 times, soak at 4° C. for 24 hours, centrifuge at 10000 rpm for 30 minutes, collect the supernatant, and obtain the primary extract of cuttlefish ink peptidoglycan; Use 100KD filter membrane, 4℃, 4000rpm centrifugation for 20min to remove impurities, collect the residual liquid on the filter membrane to obtain the golden squid ink peptidoglycan ultrafiltrate, add 4 times the volume of 95% ethanol, precipitate at 4℃ for 24h, discard the supernatant, collect the precipitate, dissolve the precipitate in PBS buffer, add 3 times the volume of sevag reagent, extract at 4℃ for 24h, carefully collect the supernatant, and freeze-dry to obtain the golden squid ink peptidoglycan extract; DEAE-Sepharose Fast Flow anion exchange chromatography column was used for purification. 2.5 mg / ml golden squid ink peptidoglycan extract was loaded at a flow rate of 0.2 ml / min. 0.1-0.6 M NaCl solution was used for gradient elution. Five elution peaks were obtained. The absorbance was detected at 280 nm by ultraviolet. The nucleic acid protein detector was used for real-time monitoring. When there was no obvious protein peak in the eluate, the elution was stopped. At the same time, the sugar peak was detected at 490 nm by the phenol-sulfuric acid method. The best protein-sugar peak eluate was collected. The solutions collected from the corresponding peaks were respectively subjected to SDS-PAGE electrophoresis detection. The eluate at the elution peak of 0.4M NaCl solution was collected, and centrifuged at 4°C, 4500rpm for 20min using an ultrafiltration tube with a molecular weight cutoff of 30KD. This was repeated three times for desalting and concentration, and freeze-drying to obtain golden squid ink peptidoglycan.

2. Use of the golden cuttlefish ink peptidoglycan prepared by the preparation method of claim 1 for preparing digestive promoting drugs.

3. Use of the golden cuttlefish ink peptidoglycan prepared by the preparation method of claim 1 for preparing drugs for repairing liver oxidative damage.

4. Use of the golden cuttlefish ink peptidoglycan prepared by the preparation method of claim 1 in preparing a drug for inhibiting the proliferation, migration and invasion of pancreatic cancer cells.

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