CN118562026B - Preparation method and application of golden squid ink peptidoglycan - Google Patents
Preparation method and application of golden squid ink peptidoglycan Download PDFInfo
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- CN118562026B CN118562026B CN202411060049.6A CN202411060049A CN118562026B CN 118562026 B CN118562026 B CN 118562026B CN 202411060049 A CN202411060049 A CN 202411060049A CN 118562026 B CN118562026 B CN 118562026B
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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Abstract
本发明提供了一种金乌贼墨肽聚糖的制备方法及用途，属于海洋生物医药领域。该技术方案取乌贼墨先后进行匀浆和脱脂，将脱脂乌贼墨超声处理，低温浸泡离心取上清，超滤膜超滤除杂质，无水乙醇沉淀，sevag法去除蛋白，冷冻干燥得到金乌贼墨多糖粗提物；采用DEAE‑Sepharose Fast Flow阴离子交换层析柱对粗提物进行分离纯化，收集蛋白‑糖峰值洗脱液，透析法脱盐浓缩，冷冻干燥，得到金乌贼墨肽聚糖。该制备方法不仅简便、高效，而且所得产物具有显著的卵巢修复作用。此外，试验发现本发明制备的金乌贼墨肽聚糖能改善肠道蠕动，修复肝脏氧化性损伤，还具有抑制胰腺癌增殖、迁移及侵袭的作用，基于以上有益的发现，进一步扩展了金乌贼墨肽聚糖的用途范围。
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
The invention belongs to the field of marine biological medicines, and particularly relates to a preparation method and application of sepia peptidoglycan.
Background
The sepia is a main cephalopod animal in the offshore of China, is mainly distributed in northern sea areas such as Shandong Japanese mountain head, qingdao and the like, is black viscous liquid synthesized by secretory glands in sepia capsules, has main chemical components of melanin and proteoglycan complex, and has higher nutrition and multiple medicinal functions. It has wide pharmacological actions of resisting oxidation, improving immunity, inhibiting cancer cell growth, etc., and thus becomes one of the current related research and development hot spots. On the other hand, when people eat and process cuttlefish, the black ink bag is generally abandoned, so that not only is the great waste of medical and edible resources caused, but also the environment pollution is caused, and therefore, the efficient extraction of the active ingredients in the cuttlefish ink has great significance for developing and utilizing the cuttlefish ink resources.
Peptidoglycan is a polysaccharide and amino acid polymer, which is an important constituent of the cell wall of prokaryotes. Studies have shown that peptidoglycans have biological activities such as antioxidant, antitumor, anticoagulant and antiviral. The sepia peptidoglycan is a proteoglycan bioactive substance separated and extracted from sepia, and the composition, activity and structure of the sepia peptidoglycan are slightly reported, but the research on the biological efficacy of the sepia peptidoglycan is still relatively limited.
Disclosure of Invention
The first technical problem to be solved by the invention is how to prepare sepia peptidoglycan more conveniently and efficiently.
The second technical problem to be solved by the invention is how to prepare sepia peptidoglycan with higher biological activity.
The third technical problem to be solved by the invention is how to expand the application range of sepia peptidoglycan.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
The preparation method of the sepia peptidoglycan comprises the following steps:
s1, cutting cuttlefish ink out of fresh cuttlefish ink bags, homogenizing by a homogenizer, degreasing, and filtering to obtain defatted cuttlefish ink;
S2, carrying out ultrasonic treatment on the defatted cuttlefish ink, leaching, centrifuging, and taking the supernatant to obtain a cuttlefish ink polysaccharide primary extract;
s3, filtering the initial extract of the cuttlefish ink polysaccharide by an ultrafiltration membrane to remove impurities, so as to obtain a cuttlefish Mo Duotang ultrafiltrate;
s4, adding absolute ethanol into the ultra-filtrate of the squid Mo Duotang for precipitation to obtain a squid ink polysaccharide precipitate;
S5, precipitating the sepia polysaccharide, adding a sevag reagent, removing free protein impurities, and freeze-drying to obtain the sepia polysaccharide;
S6, purifying the sepia polysaccharide by adopting a DEAE-Sepharose Fast Flow anion exchange chromatographic column, performing gradient elution by adopting a 0.1-0.5M NaCl solution, detecting a protein peak by using a nucleic acid protein detector, detecting a sugar peak by using a phenol sulfuric acid method, collecting a protein-sugar peak eluent, desalting and concentrating by using a dialysis method, and freeze-drying to obtain the sepia peptidoglycan.
Preferably, in step S1, degreasing conditions are 5% (m/v) SDS solution, mixing uniformly, water-bathing at 80 ℃ for 25min, centrifuging, re-suspending in 5% SDS solution, water-bathing at 80 ℃ for 15 min, and washing with ultrapure water until no foam is generated.
Preferably, in the step S2, the ultrasonic condition is that the ultrasonic power is 200w, the ultrasonic extraction time is 5h, the ratio of the extracted liquid to the extracted liquid is 1:20, the centrifugal condition is 10000 r/min, and the centrifugal time is 30min.
Preferably, in step S3, ultrafiltration conditions are 30 KD ultrafiltration membranes, a centrifugation temperature of 4 ℃, a centrifugation speed of 4000 r/min and a centrifugation time of 20 min.
Preferably, in step S4, ethanol is added at a concentration of 95% and a volume of 4 times that of the ultrafiltrate, the precipitation temperature is 4 ℃, and the precipitation time is 24 hours.
Preferably, in the step S5, the added sevag reagent is mixed with chloroform and n-butanol in a volume ratio of 3:1, and the shaking time is 30min.
Preferably, in step S5, the freeze-drying temperature is-40 ℃, the vacuum degree is 10pa, and the freeze-drying time is 12 hours.
Preferably, in step S6, the elution rate of the gradient elution is 1ml/min, 5ml per tube.
Preferably, in step S2, the temperature of the leaching is 4 ℃ and the duration of the leaching is 24 hours.
Preferably, the method further comprises the step S7 of detecting the purity of the sepia peptidoglycan by SDS-PAGE.
On the basis of the technical scheme, the invention further provides the application of the sepia peptidoglycan prepared by the preparation method in preparing digestion promoting medicines. Preferably, the dosage of the drug is 12mg/kg/d.
On the basis of the technical scheme, the invention further provides the application of the sepia peptidoglycan prepared by the preparation method in preparing liver oxidative damage repair drugs. Preferably, the dosage of the medicine is 10-50 mg/kg/d.
On the basis of the technical scheme, the invention further provides the application of the sepia peptidoglycan prepared by the preparation method in preparing pancreatic cancer therapeutic drugs.
In the present invention, VC refers to vitamin C and POF refers to premature ovarian failure model. This is described in detail.
The invention provides a preparation method and application of sepia peptidoglycan, belonging to the field of marine biological medicine. The technical scheme is that sepia is taken to carry out homogenate and degreasing successively, the defatted sepia is subjected to ultrasonic treatment, the supernatant is obtained by low-temperature soaking and centrifugation, the ultrafiltration membrane is used for ultra-filtering impurities, absolute ethyl alcohol precipitation is carried out, the sevag method is used for removing protein, the crude sepia polysaccharide extract is obtained by freeze drying, the DEAE-Sepharose Fast Flow anion exchange chromatographic column is used for separating and purifying the crude extract, the protein-sugar peak eluent is collected, the dialysis method is used for desalting and concentrating, and the freeze drying is carried out, so that sepia peptidoglycan is obtained. The preparation method is simple, convenient and efficient, and the obtained product has obvious ovarian repair effect. In addition, experiments show that the sepia peptidoglycan prepared by the invention can improve intestinal peristalsis, repair oxidative damage of liver, and inhibit proliferation, migration and invasion of pancreatic cancer, and based on the beneficial findings, the application range of the sepia peptidoglycan is further expanded.
Compared with the prior art, the invention has the following beneficial effects:
1. The invention selects the ink of the sepia as the raw material, extracts and prepares a new sepia polypeptide glycan with the molecular weight of about 71kD.
2. Compared with common cuttlefish ink peptidoglycan, the product of the invention has stronger antioxidant activity, can more effectively improve fertility such as estrus cycle, ovary index, ovulation quantity, fertilized oocyst embryo formation rate and the like, and has biological activity obviously higher than that of common cuttlefish ink peptidoglycan. Thanks to the technical advantages, the invention can be used for developing auxiliary medicines or health products for curing premature ovarian failure and fills the blank of the products in the current market.
3. The research of the invention shows that the prepared sepia peptidoglycan can improve intestinal peristalsis and promote digestion process, so that the sepia peptidoglycan can be used for developing digestion promoting medicines.
4. The research of the invention shows that the prepared cuttlefish Mo Tai glycan has definite repairing effect on liver tissues and cells of oxidative damage animals, so that the cuttlefish Mo Tai glycan can be used for developing liver oxidative damage repairing medicines.
5. In vitro experiments show that the prepared sepia peptidoglycan can inhibit proliferation, migration and invasion of pancreatic cancer cells, and is expected to be used for preparing pancreatic cancer therapeutic drugs.
6. The risk of side effects of the product of the invention is obviously lower than that of similar medicines after long-term use, and the product has higher safety.
Drawings
FIG. 1 is a physical diagram of each stage of the extraction process of sepia peptidoglycan, wherein the upper left diagram is a physical diagram of sepia, the upper right diagram is a physical diagram of sepia ink sac, the lower left diagram is a physical diagram of the black precipitate, and the lower right diagram is a physical diagram of crude sepia peptidoglycan extract.
FIG. 2 is a graph showing elution of sepia peptidoglycan.
FIG. 3 is a graph showing the peak elution of DEAE-Sepharose Fast Flow of sepia peptidoglycan.
FIG. 4 is a SDS-PAGE electrophoresis band of sepia peptidoglycan.
FIG. 5 is a graph showing changes in the ABTS radical clearance of the Sepiella maindroni Mo Tai glycan.
Fig. 6 is a graph showing the influence of the Sepia Mo Tai glycan on the estrus cycle under the cyclophosphamide injury state, wherein the left side is a microscopic view of the vaginal smear of the different estrus cycles of the mice, and the right side is a graph showing the estrus cycle change of each group.
FIG. 7 is a graph showing the effect of the Sepia esculenta Mo Tai glycan on the ovarian tissue in the cyclophosphamide damaged state, wherein A is the undamaged control ovarian tissue state, B is the cyclophosphamide damaged ovarian tissue state, and C is the gastric-lavage Sepia esculenta peptidoglycan ovarian tissue state.
FIG. 8 is a graph showing the effect of the Sepiella maindroni Mo Tai glycan on the ovary index under cyclophosphamide injury, wherein the left side is a physical graph of the ovary, and the right side is a statistical graph of the ovary index.
FIG. 9 is a graph showing the effect of a Sepiella maindroni Mo Tai glycan on fertility such as ovulation number and embryo formation rate of fertilized oocysts under cyclophosphamide injury, wherein the upper side is a microscopic image in which A, B shows the second pole discharge after fertilization, C, D shows the 2-cell stage, E, F shows the blastula stage, and the lower side is a statistical image.
FIG. 10 is a graph (x ̄.+ -.s) showing the effect of the Sepiella maindroni Mo Tai glycan on the liver weight, body weight and liver volume ratio of mice, wherein three groups of data are shown, and each group of data comprises a control group, a model group, a 10mg/kg peptidoglycan group, a 25mg/kg peptidoglycan group and a 50mg/kg peptidoglycan group in order from left to right.
FIG. 11 is a graph showing the effect of the Mo Tai glycans of Sepia esculenta on repairing oxidative damage of mouse liver tissue and cells (x ̄ + -s).
FIG. 12 is a graph showing the effect of CCK8 detection of squid Mo Tai glycan on pancreatic cancer PANC-1 cell activity.
FIG. 13 is a graph showing the effect of flow assay of Sepiella maindroni Mo Tai glycan on pancreatic cancer PANC-1 apoptosis.
FIG. 14 is a graph showing the effect of the Sepiella maindroni Mo Tai glycan on pancreatic cancer PANC-1 cell scratch healing and migration.
FIG. 15 is a graph showing the effect of the Sepiella maindroni Mo Tai glycan on pancreatic cancer PANC-1 cell invasion.
Detailed Description
Hereinafter, embodiments of the present invention will be described in detail. In order to avoid unnecessary detail, well-known structures or functions will not be described in detail in the following embodiments. Approximating language, as used in the following examples, may be applied to create a quantitative representation that could permissibly vary without resulting in a change in the basic function. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Cutting cuttlefish ink from fresh cuttlefish ink bag, placing in mortar, adding 2 times volume of phosphate buffer solution (PBS, 0.01mol/L, pH 7.4), repeatedly grinding and suspending, filtering with 8 layers of medical gauze, centrifuging filtrate at 4deg.C and 8000rpm for 30min, removing supernatant, and collecting black precipitate.
The black precipitate was dissolved by adding 20 volumes of degreasing solution (0.02M sodium acetate: chloroform: methanol ratio 4:5:10), stirred at room temperature for 24h, centrifuged at 8000rpm for 20min at 4℃and the precipitate was collected. And (3) centrifugally cleaning the sediment for 2 times by methanol, centrifugally cleaning the sediment for 3 times by deionized water, collecting the sediment, and freeze-drying to obtain the sepia fat-removed powder.
Adding 20 times volume of phosphate buffer solution (PBS, 0.01mol/L, pH 7.4) into the precipitate, performing ultrasonic treatment on defatted cuttlefish ink powder with 200w power, repeatedly performing ultrasonic treatment (15 s each time, 50 times of ultrasonic treatment), soaking at 4deg.C for 24h, centrifuging at 10000 rpm for 30min, collecting supernatant, and repeating for 2 times to obtain cuttlefish ink polysaccharide primary extract.
Centrifuging 20 min with 100 KD filter membrane at 4deg.C and 4000 rpm, removing impurities, collecting residual liquid on the filter membrane to obtain herba Sepiae Mo Duotang ultrafiltrate, adding 95% ethanol with volume of 4 times, precipitating at 4deg.C for 24 hr, discarding supernatant, and collecting precipitate. The precipitate was dissolved in PBS buffer, and 3 volumes of sevag reagent were added, extracted at 4℃for 24 hours, the supernatant was carefully collected, and freeze-dried to obtain sepia polysaccharide extract.
Purifying by adopting a DEAE-Sepharose Fast Flow anion exchange chromatography column, loading 2.5mg/ml of sepia peptidoglycan at a flow rate of 0.2ml/min, and performing gradient elution by adopting 0.1-0.6M NaCl solution to obtain 5 elution peaks. Detecting absorbance at 280nm by ultraviolet, monitoring in real time by a nucleic acid protein detector, and stopping eluting when no obvious protein peak exists in the eluent. And simultaneously detecting a sugar peak at 490nm by adopting a phenol sulfuric acid method, and collecting the optimal protein-sugar peak eluent. The solution collected from the corresponding peak was subjected to SDS-PAGE electrophoresis, respectively, and the result showed that the single protein band with a molecular weight of about 71KD was obtained from the elution peak of the 0.4M NaCl solution.
Collecting eluate at eluting peak of 0.4M NaCl solution, centrifuging at 4500rpm for 20min at 4deg.C with ultrafiltration tube with cutoff molecular weight of 30KD, desalting and concentrating, and freeze drying to obtain sepia peptidoglycan.
10Ml of ABTS solution (2 mg/ml) and 0.1ml of potassium persulfate solution (140 mmol/L) were mixed and allowed to stand overnight at room temperature in the dark to form an ABTS free radical stock working solution. The samples were diluted with 95% ethanol to absorbance 0.70 prior to the experiment. Adding 2ml of peptidoglycan solution with the mass concentration of 1-20 mg/ml and 5ml of ABTS solution into a reaction system, fully and uniformly mixing, placing in a dark place at room temperature, measuring absorbance at 734nm, and marking as a sample. The same treatment as above was performed with distilled water, and the absorbance label was measured as a control. The blank was zeroed with distilled water. Experiments were independently repeated three times to express antioxidant capacity in terms of clearance. The results show that the ability of the squid Mo Tai glycan to scavenge ABTS free radicals increases with mass concentration. The ABTS free radical scavenging capacity at the mass concentration of 2.5 mg/ml is equivalent to that of VC at the mass concentration of 0.5 mg/ml, which shows that the sepia polysaccharide obtained by the invention has a certain antioxidant capacity.
A chemotherapeutical premature ovarian failure animal model is prepared by single intraperitoneal injection of 75mg/kg cyclophosphamide, modeling effects are evaluated by indexes such as estrus cycle observation, hormone level detection, ovarian morphology structure observation, ovulation quantity, embryo development condition after fertilization and the like, and conditions for preparing the chemotherapeutical premature ovarian failure model of the rat by cyclophosphamide are optimized. On day 7 of cyclophosphamide injection, the repair group started to perfuse the stomach with a dose of 25mg/kg of sepia peptidoglycan, and the experiment was performed for 2 weeks. Through indexes such as estrus cycle observation, hormone level detection, ovary morphological structure observation, ovulation quantity, embryo development condition after fertilization and the like, the result shows that the squid Mo Tai glycan with the dosage of 25mg/kg has remarkable protection effect on the chemotherapeutical ovarian function injury.
The 40 mice were randomly divided into four groups, namely a normal group, a model control group, a positive drug, namely, a pricarboride succinate group and a sepia peptidoglycan group, and 10 mice each. The normal group was given 0.1mL of 0.9% physiological saline per lavage, and during the molding, the model control group, the sepia peptidoglycan group, and the positive drug, pramipexole succinate, were given 0.1mL of aqueous atropine sulfate solution by lavage twice a day for two consecutive days. On day 1 after molding, mice were given medication, normal and control groups were each time gastrically dosed with 0.1mL of 0.9% physiological saline, the sepia peptidoglycan group was gastrically dosed at a dose of 12mg/kg/d, and the positive drug, pramipexole succinate, was gastrically dosed at a dose of 0.03 mg/kg/d. The next day carbon powder is given, after 1.5 hours, the carbon powder propulsion distance in the intestines of the mice is observed and calculated, the length of the whole section of small intestine and the length of the small intestine propelled by the carbon powder are recorded respectively, and the carbon powder propulsion rate of the intestines is calculated, wherein the carbon powder propulsion rate= (the whole length of the small intestine-the length of the small intestine propelled by the carbon powder)/the whole length of the small intestine is multiplied by 100 percent.
The results of the mice intestinal carbon powder propulsion rate are shown in the following table 1, compared with the mice in the intestinal peristalsis debilitation model control group, the sepia peptidoglycan prepared by the invention can obviously improve the gastrointestinal peristalsis capability, and the effect is better than that of the prucalopride succinate.
Table 1 carbon powder Propulsion ratio of each experimental group
| Normal group | Model control group | Positive drug prucalopride succinate group | Sepia peptidoglycan set | |
| Carbon powder thrust rate | 93.72% | 76.62% | 85.27% | 91.48% |
Three test animals were selected, new Zealand white rabbits, box dogs, rhesus monkeys. After administration by gavage, the last feces before gavage and the first feces after gavage are collected, and the feces are weighed and counted.
By comparing the weight and the quantity of animal feces before and after the stomach irrigation, the weight (18.0%, 22.3.0% and 19.5% respectively) and the quantity of the feces of the experimental animal can be obviously increased after the administration of the sepia peptidoglycan, and meanwhile, the feces discharged by the administration group are softer and no water sample feces appears. The experiment shows that the sepia peptidoglycan can obviously enhance the intestinal vermicular ability of experimental animals and has the function of promoting digestion.
1. Test method
1.1 Animal experiments
40 Mice were randomly divided into 5 groups, namely a blank control group, a hydrogen peroxide injury group, a low-dose (10 mg/kg) of sepia peptidoglycan repair group, a medium-dose (25 mg/kg) repair group and a high-dose (50 mg/kg) repair group, 8 mice per group. After the components are kept in cages for one week to adapt to the environment, a 3% hydrogen peroxide solution is prepared, and the hydrogen peroxide solution is filled in the stomach from the first day of the experiment, and each mouse is filled in the stomach by 1 ml. In addition to the blank control group, the damaged group, the low, medium and high dose buffer groups are all filled with 3% H 2O2 solution, and the stomach is continuously filled for 10 days, so that drinking water is fed freely. After the liver injury model is established successfully, the blank group and the injury group are respectively filled with gastric distilled water, and the repair group is filled with gastric sepia peptidoglycan according to the low, medium and high doses, and the continuous period is 10 days, so that drinking water can be ingested freely. After each group of mice was weighed and recorded, the whole liver was rapidly isolated by cervical dislocation and fixed in 4% formaldehyde solution for 24: 24 h by cutting about 0.5cm3 of tissue after weighing and recording. Conventional paraffin embedding and 5 μm thick sections were made, HE stained and photographed by microscopic observation.
1.2 Cell experiments
After the isolated mouse liver cells are cultured for 24 hours, a complete culture medium is added into a control group, a complete culture medium containing 200 mu mol/L H O2 is added into a model group and an experimental group, and the culture is continued for 3 h, so that a mouse liver cell oxidative stress damage model is established. The control group and the model group are replaced by complete culture media, the repair group is respectively added with low concentration (100 ug/ml), medium concentration (250 ug/ml) and high concentration (500 ug/ml) of sepia peptidoglycan on the basis of the complete culture media for continuous culture of 48 h, the MTT method is used for detecting the cell proliferation activity, hochest is used for dyeing and observing the apoptosis condition, and the apoptosis rate is calculated.
2. Results
2.1 Effect of Sepia Mo Tai glycan on the growth State of Oxidation damaged mice
The mice in the control group have normal living state, good spirit and normal diet, the mice in the injured group have listlessness and poor appetite, and the mice in the low-medium-high dose repair group of the sepia peptidoglycan have more obvious state of spirit and diet along with the increase of the dose. The result shows that the liver injury caused by hydrogen peroxide can affect the normal life of mice, and the sepia peptidoglycan has a certain repairing effect on the injury caused by hydrogen peroxide.
2.2 Effect of Sepiella maindroni Mo Tai glycan on oxidative damage mice weight and liver ratio
Oxidative damage can lead to significant reductions in mice liver weight, body weight, and liver mass ratio. Compared with the model group, the squid ink peptidoglycan of 25mg/kg of stomach irrigation can significantly repair the liver weight and the liver-body ratio of mice, and has positive correlation with the dosage.
2.3 Repair action of Sepiella maindroni Mo Tai glycan on liver tissue and cells of mice with oxidative damage
The liver tissue of the control group has normal morphological structure, the liver cells are arranged in a compact and ordered way, a large number of cracks appear among the liver tissue cells of the H 2O2 oxidation injury group, the water is sampled by severe cavitation, and the sepia peptidoglycan can repair the pathological changes of the liver tissue in a dose-dependent way. Cell experiment results show that the cell oxidation injury model of the mouse liver cells can be established by culturing 3 h at 200 mu M/L H 2O2, and the oxidation injury of the liver cells can be effectively repaired by the cuttlefish ink peptidoglycan at 100-500 mu g/mL.
1. Test method
1.1 CCK8 assay of cellular Activity
Pancreatic cancer PANC-1 cells in logarithmic growth phase were inoculated at 3X 10 3 cells/well into 96-well plates and placed in an incubator overnight. The control group was replaced with serum-free medium, and the experimental group was further cultured with the addition of low concentration (100 ug/ml), medium concentration (250 ug/ml) and high concentration (500 ug/ml) of sepia peptidoglycan, and 4 replicate wells were set for each treatment. CCK-8 reagent was added at 24, 48 h, incubated at 37℃for 2h, absorbance at wavelength 450 nm was measured and relative cell viability was calculated.
1.2 Flow cytometry to detect apoptosis
Pancreatic cancer PANC-1 cells in logarithmic growth phase were inoculated in 6-well plates at 3×10 5 cells/well, and subjected to grouping treatment according to 1.1 method, and further cultured for 48 hours. Each group of cells was collected by pancreatin digestion without EDTA, centrifuged at 1000 Xg at 5 min, washed 3 times with pre-chilled PBS and resuspended in 400. Mu.L of Annexin V binding buffer. Annexin V-FITC and PI reagent are added into the mixture to be incubated at room temperature and in dark place for 15 min mu L respectively, and apoptosis rates of all groups are detected by a flow cytometer. The FlowJo flow cytometric analysis software obtains analysis data.
1.3 Cell scratch experiments
Ruler for circulating back of 6-hole plate straight lines are evenly drawn at intervals of 0.5-1 cm. Taking pancreatic cancer PANC-1 cells in logarithmic growth phase, inoculating 5×105 cells/well into a 6-well plate, culturing for 24 hours in a constant temperature incubator, vertically scratching along a straight line by using a 200ul gun head, washing for 2-3 times by using PBS, and carrying out grouping treatment according to a 1.1 method. Scratch width (L) was observed under a microscope, photographed, and measured using Image J software to calculate the scratch healing rate.
1.4 Cell migration and invasion experiments
The logarithmic growth phase pancreatic cancer PANC-1 cells were starved cultured for 24 h, resuspended in serum-free medium and adjusted to 1X 10 5/mL. In the cell invasion experiment, 60 mu L of matrigel diluted in a ratio of 1:10 is added into the upper chamber of a small chamber, a 37 ℃ incubator incubates 3 h, a serum-free culture medium is added, and the mixture is placed at 37 ℃ for 30 min for matrix membrane hydration. 200. Mu.L of the cell suspension was added to the Transwell upper chamber, 500. Mu.L of a medium containing 20% fetal bovine serum and exosomes of different concentrations was added to the lower chamber, and the mixture was subjected to a group treatment according to the method 1.1, and horizontally placed in a constant temperature incubator for culturing 48 h.4% paraformaldehyde is fixed for 15 min,0.05% crystal violet dye solution is used for dyeing for 5 min%, cells do not penetrate out of the cotton swab erasing chamber, 5 fields are randomly selected under a microscope to take pictures, and Image J software counts the number of invasion cells and migration cells in each field.
The foregoing describes the embodiments of the present invention in detail, but the description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the scope of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. The preparation method of the sepia peptidoglycan is characterized by comprising the following steps of:
cutting cuttlefish ink from fresh cuttlefish ink sac, placing in a mortar, adding 2 times volume of phosphate buffer solution, repeatedly grinding and suspending, filtering with medical gauze, centrifuging filtrate at 4deg.C and 8000rpm for 30min, pouring supernatant, and collecting black precipitate;
Adding 20 times of degreasing solution to dissolve black precipitate, stirring at room temperature for 24h, centrifuging at 8000rpm for 20min at 4 ℃, collecting precipitate, centrifuging with methanol, cleaning precipitate for 2 times, centrifuging with deionized water for 3 times, collecting precipitate, and lyophilizing to obtain sepia oil-removed powder;
Adding 20 times of phosphate buffer solution into the sepia fat-removed powder, wherein the concentration of the phosphate buffer solution is 0.01mol/L, the pH value is 7.4, performing ultrasonic treatment on the sepia fat-removed powder by 200w power for 15s each time, performing ultrasonic treatment for 50 times at intervals of 15s, soaking at 4 ℃ for 24h, centrifuging at 10000rpm for 30min, and collecting supernatant to obtain sepia peptidoglycan initial extract;
Centrifuging with 100KD filter membrane at 4deg.C and 4000rpm for 20min, removing impurities, collecting residual liquid on the filter membrane to obtain herba Equiseti hiemalis Mo Tai glycan ultrafiltrate, adding 4 times volume of 95% ethanol, precipitating at 4deg.C for 24 hr, discarding supernatant, collecting precipitate, dissolving the precipitate in PBS buffer, adding 3 times volume of sevag reagent, extracting at 4deg.C for 24 hr, carefully collecting supernatant, and lyophilizing to obtain herba Equiseti hiemalis peptidoglycan extract;
purifying by adopting a DEAE-Sepharose Fast Flow anion exchange chromatographic column, loading 2.5mg/ml of sepia peptidoglycan extract at a flow rate of 0.2ml/min, performing gradient elution by adopting a 0.1-0.6M NaCl solution to obtain 5 elution peaks, detecting absorbance at ultraviolet 280nm, monitoring in real time by a nucleic acid protein detector, stopping eluting when no obvious protein peak exists in the eluent, detecting a sugar peak at 490nm by adopting a phenol sulfuric acid method, collecting the optimal protein-sugar peak eluent, and performing SDS-PAGE electrophoresis detection on the solutions collected by the corresponding peaks respectively;
Collecting eluate at eluting peak of 0.4M NaCl solution, centrifuging at 4500rpm for 20min at 4deg.C using ultrafiltration tube with cutoff molecular weight of 30KD, desalting and concentrating, and freeze drying to obtain sepia peptidoglycan.
2. The use of sepia peptidoglycan prepared by the method of claim 1 for preparing digestion promoting medicine.
3. The use of sepia peptidoglycan prepared by the preparation method of claim 1 for preparing liver oxidative damage repair drugs.
4. The use of sepia peptidoglycan prepared by the method of claim 1 for preparing a medicament for inhibiting proliferation, migration and invasion of pancreatic cancer cells.
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