Preparation method and application of cyclina sinensis oligosaccharide
The preparation of blue clam oligosaccharides by purification and acid hydrolysis of anion exchange column and gel permeation chromatography column was solved, and the problem of insufficient research on the biological activity of blue clam oligosaccharides was achieved, high antioxidant activity and anti-tumor effects were achieved, and human lung cancer cells were used to treat high antioxidant activity and anti-tumor effects. A549 has a significant inhibitory effect.
Patent Information
- Application Number
- CN202411230981.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-04
- Publication Date
- 2024-12-27
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
In the prior art, there are few studies on the preparation and biological activities of blue clam oligosaccharides, especially their antioxidant and anti-tumor activities have not been reported.
The anion exchange column and gel permeation chromatography column were used to purify the cyanobacter polysaccharides at the same time, and the cyanobacter oligosaccharides with high antioxidant activity were prepared by acid hydrolysis, and the enzymatic lysis of papain and multiple steps were used to remove the mixed proteins and different polar sugars. , protects the hydroxyl and amino groups in polysaccharides and ensures their biological activity.
The prepared blue clam oligosaccharide has significant antioxidant activity, and the scavenging rates of DPPH, hydroxyl radicals and superoxide anions reach 89.93%, 70.55% and 83.66%, respectively, and has a significant inhibitory effect on human lung cancer cell A549, and IC50 value. 1.13 mg/mL.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of deep processing of aquatic products, and specifically relates to a preparation method of Cynaroides oligosaccharide and application thereof. Background Art
[0002] Green clam Cyclina sinensis ) belongs to the class Lamellibranchia, the subclass Heterodonta, the order Venerida, the family Veneridae, the subfamily Cyclina, and the genus Cyclina. Cyclina ), commonly known as black clams, iron clams, round clams and ox-eye clams. my country has rich tidal flat resources, with an area of 3.07×10 5 The total production of green clams in Jiangsu Province in 2020 was close to 100,000 tons. It is one of the top ten leading seafood products in Jiangsu Province, and there is still a lot of room for expansion in its production and farming scale.
[0003] The nutritional value of green clams is high, and they are rich in protein, fat, carbohydrates, sugar, inorganic elements, etc. The clam-related medicines recorded in the Compendium of Materia Medica include sea clams, Manila clams, and clams. Among them, sea clams refer to all clams, which are mainly used to treat cough, shortness of breath, chest pain, cold and heat, bladder and large intestine, urination, and phlegm-resolving drinks. In the past decade, with the gradual maturity of artificial seedling technology, green clams have begun to occupy a relatively important economic position in the aquaculture of tidal flat shellfish in Jiangsu Province, and the production of green clams has also increased year by year. Therefore, it is imperative to study the subsequent processing characteristics and biological activity of large-scale aquaculture green clams. At present, the research on green clams mainly focuses on reproduction and aquaculture biology, population genetics and germplasm resource evaluation and immunity, while there are few reports on the biological activity and processing characteristics of green clams. Shellfish are rich in many kinds of bioactive polysaccharides. Many researchers have studied the structural analysis, biological activity and marine drugs of polysaccharides extracted from shellfish. For example, Patent Publication No. CN 110128560 A discloses a polysaccharide with enhanced immune activity and its preparation method and application. The composition of the polysaccharide obtained in the patent document is relatively complex, and the method of removing impurities and sugars of different polarity is not discussed in the method of the patent document. At the same time, the inventor searched related technologies and found that there are few technical studies on the preparation and biological activity of shellfish oligosaccharides, especially the preparation and activity of oligosaccharides from green clam. Based on this, the present invention optimizes and supplements the crude extraction and purification methods of green clam polysaccharides, adopts the method of simultaneous purification by anion exchange column and gel permeation chromatography column, and obtains a pure product of green clam polysaccharides with relatively uniform molecular weight; and uses acid hydrolysis to prepare green clam oligosaccharides, and evaluates the biological activities of green clam oligosaccharides, including antioxidant and anti-tumor activities, especially green clam oligosaccharides. Summary of the invention
[0004] In view of the technical problems involved in the background technology, the present invention provides a method for preparing Cynarostegium wilfordii oligosaccharides. The prepared Cynarostegium wilfordii oligosaccharides have good antioxidant activity and good inhibitory effect on human lung cancer cells A549.
[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for preparing oligosaccharides of Cynaroides cynaroides, characterized in that the method is as follows: S1: Preparation of crude polysaccharide from Cynarocephala sinensis: The fresh green clam meat was cleaned and minced, soaked in 75% ethanol, crushed and freeze-dried; acetone solution was added at a material ratio of 1:4 (w / v) to defat, and papain was used for enzymatic hydrolysis after ultrasonic treatment to obtain an enzymatic hydrolyzate, anhydrous ethanol was added to the enzymatic hydrolyzate, and the enzymatic hydrolyzate was allowed to stand in a chromatography cabinet, repeatedly precipitated with alcohol, and freeze-dried to obtain a crude green clam polysaccharide; S2: removing protein from the crude polysaccharide of Cyclamen sinensis: precipitate the above-mentioned lyophilized crude polysaccharide of Cyclamen sinensis, dissolve it with an appropriate amount of water to form a polysaccharide solution of Cyclamen sinensis, add calcium hydroxide to the polysaccharide solution to adjust the pH to 11, place it in a water bath, centrifuge, take the supernatant, add phosphoric acid to adjust the pH to 7, place it in a water bath, centrifuge again; take the supernatant, add ethanol to mix, precipitate the polysaccharide of Cyclamen sinensis overnight, discard the supernatant after centrifugation, and lyophilize to obtain the crude polysaccharide of Cyclamen sinensis with impurities removed; S3: Preliminary separation and purification of the crude polysaccharide of Cynarostegium wilfordii in S2 by anion exchange column method: S4: using gel permeation chromatography to further purify the peak components collected by anion exchange column method. Gel permeation chromatography is a method for separation and purification based on the molecular weight of polysaccharides, so as to obtain a pure product of Cynarocephala polysaccharide with relatively uniform molecular weight; S5: Preparation of Green Clam Oligosaccharide: Take 10 g of pure Green Clam polysaccharide, add 20.0 mL 2 M trifluoroacetic acid to dissolve the sample, seal and hydrolyze for 2 h; evaporate the hydrolyzed sample under low temperature and reduced pressure, add 10 mL methanol and evaporate it to remove excess trifluoroacetic acid; hydrolyze the sample with 1.5 mL double distilled water and transfer it to a 2.5 mL EP tube, centrifuge at 12000×g for 10 min, and take the supernatant to a new 2.5 mL EP tube, which is Green Clam Oligosaccharide.
[0006] Further, in the S1, fresh green clam meat is taken, washed 3 times and minced, soaked in 75% ethanol overnight to remove low-polymerization sugars and impurities, crushed with a tissue homogenizer, and dried by freeze-drying for later use; acetone solution is added according to a material ratio of 1:4 (w / v), degreased with a magnetic stirrer for 2 h, and removed from the oil and acetone after standing at 4°C overnight, and then the defatted green clam meat is freeze-dried for later use; the hydrolysis bottle mouth is sealed with plastic wrap, and papain is used for hydrolysis after ultrasonic treatment for 30 min, and enzymatic hydrolysis is performed for 8 h at a material-liquid ratio of 1:5 (w / v) and an enzyme amount of 3%, and then centrifuged to obtain an enzymatic hydrolyzate; 4 times the volume of anhydrous ethanol is added, and the mixture is allowed to stand in a chromatography cabinet at 4°C for 24 h to precipitate polysaccharides at the bottom; after centrifugation, deionized water is added to dissolve the precipitate, and the alcohol precipitation is repeated 2-3 times, and the crude green clam polysaccharide is obtained by alcohol evaporation and freeze-drying.
[0007] Further, in S2, the polysaccharide solution of the green clam was adjusted to pH 11 with calcium hydroxide, incubated in a water bath at 60°C for 20 min, centrifuged at 12,000×g for 20 min, and the supernatant was adjusted to pH 7 with phosphoric acid, incubated in a water bath at 80°C for 20 min, and centrifuged at 12,000×g for 20 min. The supernatant was taken, 4 times the volume of ethanol was added and mixed, and the green clam polysaccharide was precipitated at 4°C overnight, and the supernatant was discarded after centrifugation at 12,000×g for 5 min, and freeze-dried.
[0008] Further, in S3, 300 mg of the crude polysaccharide of Scutellaria quinquefolia was weighed, dissolved with appropriate amount of water, centrifuged at 12000×g for 10 min, and the supernatant was taken for sample loading; when loading, pay attention to controlling the flow rate so that the polysaccharide of Scutellaria quinquefolia is adsorbed on the column, and gradient elution was performed with water and different concentrations of NaCl solution, respectively. The required concentration gradient can be set after preliminary condition exploration; collect each component, collect 2 column volumes for each component, elute with 1 mol / L NaCl solution of twice the column volume after collection, remove the residual sample impurities in the filler, and finally elute with 2 column volumes of water before loading again; collect the obtained Scutellaria quinquefolia polysaccharide solution, detect the distribution of Scutellaria quinquefolia polysaccharide by absorbance at a wavelength of 625 nm by the sulfuric acid-anthrone method, and plot the absorbance against the tube number; collect and combine the same sugar-containing components, dialyze the collected components with double distilled water, change the water every 4 hours, dialyze for 3 days, and then dialyze with triple distilled water for 1 day, and the dialyzed polysaccharide sample is concentrated under reduced pressure and freeze-dried.
[0009] Furthermore, in S4, 60 mg of freeze-dried polysaccharide sample of Cynaroides sinensis was weighed, 1-2 mL of 0.2 M NH4HCO3 solution was added to dissolve, and the supernatant was sampled at 12000×g for 5 min, and the mobile phase was 0.2 M NH4HCO3 solution; after being collected by an automatic fraction collector, the distribution of Cynaroides sinensis polysaccharide was detected by absorbance at 490 nm using the phenol-sulfuric acid method, and the OD was calculated by the tube number.490nm The values were plotted; the collected solutions corresponding to the main sugar peak of the eluted peaks were combined, concentrated and evaporated to dryness under reduced pressure at 50 °C to decompose NH4HCO3; the pure polysaccharide of Cynaroides sinensis was obtained after dissolving in double distilled water and freeze-drying.
[0010] A preparation method is provided to obtain Cynarostegium wilfordii oligosaccharides with high antioxidant activity.
[0011] The invention discloses an application of Cynarostegium wilfordii oligosaccharide in the preparation of antioxidant and anti-tumor products, and has a good inhibitory effect on human lung cancer cells A549.
[0012] The following beneficial effects can be obtained through the above technical solution: Compared with the prior art, the green clam oligosaccharide obtained by the invention is free of impurities and sugars of different polarities, and trifluoroacetic acid can protect the hydroxyl groups or amino groups in the polysaccharide to ensure that the green clam oligosaccharide with good biological activity is obtained.
[0013] The green clam oligosaccharide prepared by the present invention has good antioxidant activity. When the concentration is 5.0 mg / mL, the scavenging rates of DPPH, hydroxyl radicals and superoxide anions are 89.93%, 70.55% and 83.66% respectively, and the total antioxidant capacity is 23.15 U / mg mass. It has significant cytotoxic activity against human lung cancer cells A549, and its IC 50 The value is 1.13 mg / mL. The invention provides new ideas and references for the deep processing and high value of green clam. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 A is the process flow chart of the extraction of oligosaccharides from Cynaroides; B is the extraction diagram of crude polysaccharides from Cynaroides.
[0015] Figure 2 A is a DEAE-Sepharose Fast Flow anion exchange column separation diagram of the crude polysaccharide of Scutellaria quinquefolia; B is a gel permeation chromatography column elution result diagram of the crude polysaccharide of Scutellaria quinquefolia; C is a freeze-dried sample diagram of Scutellaria quinquefolia oligosaccharide.
[0016] Figure 3 The figure shows the total antioxidant capacity of oligosaccharides from the green clam (A) and the scavenging rate of DPPH, hydroxyl radicals and superoxide anions (B).
[0017] Figure 4 The inhibition rate and IC 50 Value graph.
[0018] Figure 5This is a diagram showing the effect of Scutellaria oligosaccharide on the morphology of human lung cancer A549 cells in vitro, where A: negative control, culture medium treatment group; B: Scutellaria oligosaccharide 1.28 mg / mL treatment group; C: Scutellaria oligosaccharide 10.00 mg / mL treatment group. DETAILED DESCRIPTION
[0019] The following is combined with Figure 1-5 The present invention is further described with examples: A method for preparing oligosaccharides of Cynanchum cylindrica, wherein the crude polysaccharides of Cynanchum cylindrica are extracted from the meat of Cynanchum cylindrica by ultrasound-assisted combined protease, the crude polysaccharides of Cynanchum cylindrica are purified by anion exchange column and gel permeation chromatography column, and then the oligosaccharides of Cynanchum cylindrica are prepared by acid hydrolysis, and the application of the oligosaccharides of Cynanchum cylindrica in antioxidant activity and anti-tumor is disclosed. The specific method is as follows: the meat of the new Cynanchum cylindrica is washed 3 times and minced, soaked in 75% ethanol, crushed and freeze-dried by a tissue homogenizer, defatted for 2 h by adding acetone solution according to a material ratio of 1:4 (w / v), ultrasonically treated for 30 min, and then enzymatically hydrolyzed by papain for 8 h, 4 times the volume of anhydrous ethanol is added, and the mixture is allowed to stand in a chromatography cabinet at 4°C for 24 h, repeatedly precipitated with alcohol for 2-3 times, and freeze-dried by evaporating alcohol to obtain the crude polysaccharides of Cynanchum cylindrica. The crude polysaccharides of Cynanchum cylindrica are purified by anion exchange column and gel permeation chromatography column, and then the oligosaccharides of Cynanchum cylindrica are prepared by acid hydrolysis. The prepared Cynarostegium wilfordii oligosaccharide has good antioxidant activity and has a good inhibitory effect on human lung cancer cells A549. Example
[0020] This example provides a method for preparing oligosaccharides of Cynarocephala, and the specific method is as follows: Step 1: Pretreatment of Clam Meat and Preparation of Polysaccharides: Preparation of crude green clam polysaccharide: fresh green clam (purchased from Lianyungang Zhongchuang Aquaculture Co., Ltd.) meat was washed 3 times and minced, soaked in 75% ethanol overnight to remove oligosaccharides and impurities, crushed with a tissue homogenizer, and freeze-dried to dry the green clam meat homogenate for later use; acetone solution was added according to a material ratio of 1:4 (w / v), and degreased with a magnetic stirrer for 2 h, and the oil and acetone were removed after standing at 4 °C overnight, and then the defatted green clam meat was freeze-dried for later use; the hydrolysis bottle mouth was sealed with plastic wrap, and papain was used for hydrolysis after ultrasonic treatment for 30 min, and the material-liquid ratio was 1:5 (w / v) and the enzyme amount was 3% for 8 h. After the end, the enzymatic hydrolyzate was obtained by centrifugation; 4 times the volume of anhydrous ethanol was added, and the mixture was allowed to stand in a chromatography cabinet at 4 °C for 24 h to allow the polysaccharide to precipitate at the bottom; after centrifugation, deionized water was added to dissolve the precipitate, and the alcohol precipitation was repeated 2-3 times, and the alcohol was evaporated and freeze-dried to obtain the crude green clam polysaccharide.
[0021] Step 2: Polysaccharide protein removal process: The freeze-dried precipitate is dissolved in an appropriate amount of water to form a polysaccharide solution of Cyclamen sinensis. The Cyclamen sinensis polysaccharide solution is adjusted to pH 11 by adding calcium hydroxide, and then placed in a water bath at 60°C for 20 min, centrifuged at 12,000×g for 20 min, and the supernatant is added with phosphoric acid to adjust to pH 7, and then placed in a water bath at 80°C for 20 min, and centrifuged at 12,000×g for 20 min. The supernatant is taken, 4 times the volume of ethanol is added and mixed, and Cyclamen sinensis polysaccharide is precipitated at 4°C overnight. After centrifugation at 12,000×g for 5 min, the supernatant is discarded, and the solution is freeze-dried to obtain a crude Cyclamen sinensis polysaccharide with impurities removed. In this step, the impurities in the crude Cyclamen sinensis polysaccharide can be effectively removed, thereby avoiding the influence on the subsequent purification of the crude Cyclamen sinensis polysaccharide, the preparation of oligosaccharides, and the activity evaluation of oligosaccharides. The Cyclamen sinensis polysaccharide also has certain biological activity.
[0022] Step 3: Anion exchange column purification of green clam polysaccharide: The present invention uses DEAE-Sepharose FastFlow anion exchange column to perform preliminary separation and purification of green clam polysaccharide crude product. Weigh 300 mg of green clam polysaccharide crude product, add appropriate amount of water to dissolve, centrifuge at 12000×g for 10 min, and take the supernatant for sample loading. When loading, pay attention to control the flow rate so that the green clam polysaccharide is adsorbed on the column, and gradient elution is performed with water and different concentrations of NaCl solution, respectively. The required concentration gradient can be set after preliminary condition exploration (i.e., elution is performed with double distilled water, 0.1 M NaCl, 0.2 M NaCl, 0.3 M NaCl, 0.4 M NaCl, and 0.5 M NaCl solution, respectively). Collect each component, collect 2 column volumes for each component, and after collection, elute with twice the column volume of 1 mol / L NaCl solution to remove residual sample impurities in the filler, and finally elute with 2 times the column volume of water before loading again. The obtained polysaccharide solution of the green clam was collected, and the distribution of the green clam polysaccharide was detected by absorbance at a wavelength of 625nm using the sulfuric acid-anthrone method, and the absorbance was plotted against the tube number. The same sugar-containing components were collected and combined, and the collected components were dialyzed with double distilled water, and the water was changed every 4 hours for 3 days, and then dialyzed with triple distilled water for 1 day. The dialyzed polysaccharide sample was concentrated under reduced pressure and freeze-dried. The composition of green clam polysaccharide is relatively complex, similar to a mixture, so this step can separate green clam polysaccharide and other sugar substances, and can separate polysaccharides or proteins of different polarities. At the same time, it can separate and purify biological macromolecules without causing changes in molecular structure, and does not affect the activity of green clam polysaccharide. That is, this step can remove polysaccharides and non-sugar substances with different polarities from green clam polysaccharide, and does not affect the structure and biological activity of green clam polysaccharide.
[0023] Step 4: Gel permeation chromatography column purification of green clam polysaccharide: The present invention uses Sephacryl S-100 gel permeation chromatography column to further purify the peak components collected by DEAE-Sepharose Fast Flow anion exchange column method. Gel permeation chromatography is a method for separation and purification based on the molecular weight of polysaccharides, and can obtain pure polysaccharides with relatively uniform molecular weight. Weigh 60 mg of freeze-dried green clam oligosaccharide sample, add 1-2 mL of 0.2 M NH4HCO3 solution to dissolve, centrifuge at 12000×g for 5 min, take the supernatant and load the sample, and the mobile phase is 0.2 M NH4HCO3 solution. After collection by an automatic fraction collector, the distribution of green clam polysaccharides is detected by absorbance at 490 nm using the phenol-sulfuric acid method, and the OD is calculated according to the tube number. 490nm The values were plotted. The collected solutions corresponding to the main sugar peak of the elution peaks were combined and concentrated at 50 °C under reduced pressure to dryness to decompose NH4HCO3. After dissolving in double distilled water, the pure green clam polysaccharide was freeze-dried to obtain a relatively uniform molecular weight of the obtained green clam polysaccharide. Gel permeation chromatography is a method for separation and purification based on the molecular weight of polysaccharides, and a relatively uniform molecular weight of green clam polysaccharide can be obtained.
[0024] Step 5: Preparation of Green Clam Oligosaccharide: Take 10 g of purified Green Clam polysaccharide, place it in a 50 mL conical flask, add 20.0 mL 2 M trifluoroacetic acid to dissolve the sample, seal and hydrolyze for 2 h. The hydrolyzed sample is evaporated to dryness under low temperature and reduced pressure, and 10 mL of methanol is evaporated to dryness to remove excess trifluoroacetic acid. Use 1.5 mL of double distilled water to hydrolyze the sample and transfer it to a 2.5 mL EP tube, centrifuge at 12000×g for 10 min, and take the supernatant to a new 2.5 mL EP tube, which is Green Clam Oligosaccharide. In this step, trifluoroacetic acid can protect the hydroxyl or amino groups in the polysaccharide to ensure that Green Clam Oligosaccharide with good biological activity is obtained, and trifluoroacetic acid is relatively easy to remove. The above functions and effects are not recorded or disclosed in the existing conventional technology.
[0025] Experimental analysis was performed on the oligosaccharides of Cynarostegium wilfordii obtained in the above examples: Total antioxidant capacity determination: The total antioxidant capacity (T-AOC) test kit (Cat. No. BC1315) purchased from Beijing Solebow Technology Co., Ltd. was used for the determination. The principle is as follows: There are many antioxidant substances in the test object, which can make Fe 3+ Reduction to Fe 2+, which can form a stable blue complex with tripyridine triazine TPTZ, and its antioxidant capacity can be measured by colorimetry. The green clam oligosaccharide solution was dissolved and diluted by 1×PBS, and the green clam oligosaccharide was divided into 5 groups according to different concentrations (concentrations were 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 3.0 mg / mL and 5.0 mg / mL), and a blank control group (1×PBS) was set up at the same time. The micro-method was used for determination. The specific operation refers to the instructions of the kit.
[0026] Hydroxyl radical (·OH) scavenging ability determination: The hydroxyl radical scavenging ability is one of the important indicators of the antioxidant capacity of the sample, and the hydroxyl radical scavenging ability detection kit (Cat. No.: BC1325) purchased from Beijing Solebow Technology Co., Ltd. was used for determination. The principle is as follows: H2O2 / Fe 2+ The Fenton reaction generates hydroxyl radicals and converts o-phenanthroline-Fe 2+ Fe in aqueous solution 2+ Oxidized to Fe 3+ , resulting in a decrease in the absorbance at 536 nm. The degree of inhibition of the rate of decrease in the absorbance at 536 nm of the sample reflects the ability of the sample to scavenge hydroxyl free radicals. The solution of green clam oligosaccharide was dissolved and diluted by 1×PBS. Green clam oligosaccharide was divided into 5 groups according to different concentrations (concentrations were 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 3.0 mg / mL and 5.0 mg / mL), and a blank control group (1×PBS) was set up. The micro-method was used for determination. For specific operations, refer to the instructions of the kit.
[0027] Superoxide anion (O2 - ) Determination of scavenging capacity The superoxide anion content detection kit (Cat. No.: BC1295) purchased from Beijing Solebow Technology Co., Ltd. was used for determination. The principle is as follows: superoxide anion reacts with hydroxylamine hydrochloride to generate NO 2- , NO 2- Under the action of p-aminobenzenesulfonamide and naphthylethylenediamine hydrochloride, a purple-red azo compound is generated with a characteristic absorption peak at 530 nm. 530 The value can be used to calculate the O2 － The green clam oligosaccharide solution was dissolved and diluted by 1×PBS, and the green clam oligosaccharide was divided into 5 groups according to different concentrations (the concentrations were 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 3.0 mg / mL and 5.0 mg / mL), and a blank control group (1×PBS) was set up at the same time. The micro-method was used for determination. For specific operations, please refer to the instructions of the kit.
[0028] DPPH free radical scavenging ability assay DPPH free radical is a very stable nitrogen-centered free radical, which is one of the important indicators of the antioxidant capacity of the sample. It was measured by the DPPH free radical scavenging ability test kit (Cat. No.: BC4755) purchased from Beijing Solebow Technology Co., Ltd. The principle is as follows: DPPH free radical has a single electron, its alcohol solution is purple, and it has strong absorption at 515 nm. When an antioxidant is present, the DPPH free radical is scavenged, the color of its solution becomes lighter, and the absorbance at 515 nm decreases. Within a certain range, the change in its absorbance is proportional to the degree of free radical scavenging. In this kit, the degree of decrease in absorbance reflects the ability of the sample to scavenge DPPH free radicals. The green clam oligosaccharide solution was dissolved and diluted by 1×PBS, and the green clam oligosaccharide was divided into 5 groups according to different concentrations (concentrations were 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 3.0 mg / mL and 5.0 mg / mL, respectively). At the same time, a blank control group (1×PBS) was set up, and the micro-method was used for determination. For specific operations, refer to the instructions of the kit.
[0029] CCK-8 staining to detect cell viability: The cells with a live cell ratio of more than 90% were selected for the experiment. The cell proliferation inhibition test was performed using the EnoGeneCell™ Counting Kit-8 (CCK-8) cell viability detection kit. The cells were digested, counted, and prepared into a concentration of 1×10 5 Add 100 μL of cell suspension (1×10 cells / mL) to each well of a 96-well plate. 4 cells); the 96-well plate was placed in a 37°C, 5% CO2 incubator for 24 hours; 100 μL of culture medium with different concentrations of Cynaropicrin oligosaccharides (0-10 mg / mL) was added to each well, and a negative control group and a positive control group (paclitaxel injection, Taiji Group Sichuan Taiji Pharmaceutical Co., Ltd., 5mL:30mg, batch number: 14121201) were set up at the same time, with 5 replicate wells in each group; the 96-well plate was placed in a 37°C, 5% CO2 incubator for 48 hours; 10 μL of CCK-8 solution was added to each well, the culture plate was incubated in the incubator for 4 hours, and the OD value at 450 nm was measured with an enzyme marker to calculate the inhibition rate of human lung cancer cell A549 and IC 50 The morphological changes of cells were recorded using an inverted microscope.
[0030] The experimental results are as follows: Preparation of Clam Oligosaccharide: This embodiment provides a method for preparing oligosaccharides of Cynarodisiac based on ultrasound-assisted combined protease and acid hydrolysis. Figure 1 A. The extraction process of polysaccharide from Cynarostegium wilfordii is as follows Figure 1As shown in B, the fresh green clam meat was washed three times and minced, soaked in 75% ethanol, crushed and freeze-dried using a tissue homogenizer, defatted for 2 h with an acetone solution at a material ratio of 1:4 (w / v), ultrasonically treated for 30 min, and then enzymatically hydrolyzed with papain for 8 h, and 4 times the volume of anhydrous ethanol was added, and the mixture was allowed to stand in a 4 ℃ chromatography cabinet for 24 h, and repeatedly precipitated with alcohol for 2-3 times, and the lyophilized product was the crude green clam polysaccharide. After defatting, deproteinizing and repeatedly precipitating with alcohol, the green clam polysaccharide was in a light yellow translucent colloidal form.
[0031] The obtained polysaccharide from the green clam was separated by a DEAE-Sepharose Fast Flow anion exchange column, eluted with distilled water, 0.1 M NaCl solution and 0.3 M NaCl solution, respectively, and the distribution of the polysaccharide from the green clam was detected by the sulfuric acid-anthrone method at a wavelength of 625 nm. The results are as follows Figure 2 As shown in A. The crude polysaccharide was subjected to gradient elution with a DEAE-Sepharose Fast Flow anion exchange column to obtain a component, which was then dialyzed through a dialysis bag and concentrated and freeze-dried to obtain a preliminarily purified polysaccharide sample.
[0032] Weigh 1g of the purified polysaccharide from the anion exchange column, dissolve it in distilled water, and run it at 12000 r / min for 15 min. Take the supernatant and pass it through a Shodex Ohpak SB 803HQ, 8.0*300mm gel column, and elute it with ultrapure water. After being collected by an automatic fraction collector, the sugar distribution of each tube fraction was detected by the phenol-sulfuric acid method, and the OD was calculated based on the tube number. 490 The nm values are plotted. The results are shown in Figure 2 B. The gel permeation chromatography column elution results showed that there was only a single elution peak, indicating that it contained only one polysaccharide with uniform molecular weight. After vacuum concentration and drying, the pure product of the green clam polysaccharide was obtained. Figure 2 As shown in C, it is an off-white powder.
[0033] Antioxidant activity of oligosaccharides from Cynarocephala sinensis: In order to better apply the oligosaccharide of Cynaroides sinensis, the present invention measured the total antioxidant capacity index of the oligosaccharide of Cynaroides sinensis and the scavenging capacity of DPPH, hydroxyl radicals and superoxide anions. Figure 3 As shown, with the increase of the concentration of the green clam oligosaccharide, its total antioxidant capacity and the scavenging liquid of DPPH, hydroxyl free radicals and superoxide anions continue to increase. When the concentration of the green clam oligosaccharide is 5.0 mg / mL, the scavenging rates of DPPH, hydroxyl free radicals and superoxide anions are 89.93%, 70.55% and 83.66% respectively, and the total antioxidant capacity is 23.15 U / mg mass. It shows that the green clam oligosaccharide prepared by the present invention has good biological activity.
[0034] The activity of oligosaccharides from Cynarostegium wilfordii in inhibiting the proliferation of human lung cancer cells A549: The inhibitory effect of oligosaccharides from Cynarostegma cylindrica on the proliferation of human lung cancer A549 cells was studied by MTT method. 50 The results are as follows. Figure 4 As shown in the figure, with the increase of the concentration of C. cylindrica oligosaccharide, the inhibitory effect on the proliferation activity of human lung cancer cell A549 became more and more significant. When the concentration of C. cylindrica oligosaccharide was 10.00 mg / mL, the inhibition rate of human lung cancer cell A549 was as high as 91.33%. It had obvious cytotoxic activity against human liver cancer cell HepG2, and its IC 50 The value is 1.13 mg / mL.
[0035] Under a 100x fluorescence inverted microscope, the effects of C. cylindrica oligosaccharides on the morphology of human lung cancer A549 cells in vitro were recorded. Figure 5 As shown in the figure, the morphology of human lung cancer cell A549 in the negative control group was vigorous growth with adherent growth. In the medium-dose 1.28 mg / mL treatment group, the number of surviving human lung cancer cell A549 was significantly reduced, there were more floating cells, the cell volume shrank, and the cell morphology was wrinkled and deformed. In the high-dose 10.00 mg / mL treatment group, most of the cell growth was inhibited, the cells died, and cell fragments were observed. This shows that the 1.28 mg / mL treatment group has significant cytotoxic activity on human lung cancer cell A549, which not only inhibits growth but also changes the morphology of tumor cells.
[0036] The above are all preferred embodiments of the present invention. For ordinary technicians in this technical field, without departing from the principle of the present invention, various equivalent modifications to the present invention belong to the protection scope of the claims attached to this application.
Claims
1. A method for preparing oligosaccharides of Cynaropoda cylindrica, characterized in that: The method is as follows: S1: Preparation of crude polysaccharide from Cynarocephala sinensis: The fresh green clam meat was cleaned and minced, soaked in 75% ethanol, crushed and freeze-dried; acetone solution was added at a material ratio of 1:4 (w / v) to defat, and papain was used for enzymatic hydrolysis after ultrasonic treatment to obtain an enzymatic hydrolyzate, anhydrous ethanol was added to the enzymatic hydrolyzate, and the enzymatic hydrolyzate was allowed to stand in a chromatography cabinet, repeatedly precipitated with alcohol, and freeze-dried to obtain a crude green clam polysaccharide; S2: removing protein from the crude green clam polysaccharide: precipitate the above-mentioned freeze-dried crude green clam polysaccharide, dissolve it with an appropriate amount of water to form a green clam polysaccharide solution, add calcium hydroxide to the green clam polysaccharide solution to adjust the pH to 11, water bath, centrifuge, take the supernatant and add phosphoric acid to adjust the pH to 7, water bath, centrifuge again; take the supernatant, add ethanol and mix well, precipitate the green clam polysaccharide overnight, discard the supernatant after centrifugation, and freeze-dry to obtain the green clam polysaccharide crude product after protein removal; S3: Preliminary separation and purification of the crude polysaccharide of Cynarostegium wilfordii in S2 by anion exchange column method: S4: using gel permeation chromatography to further purify the peak components collected by anion exchange column method. Gel permeation chromatography is a method for separation and purification based on the molecular weight of polysaccharides, so as to obtain a pure product of Cynarocephala polysaccharide with relatively uniform molecular weight; S5: Preparation of Green Clam Oligosaccharide: Take 10 g of the pure Green Clam polysaccharide purified in S4, add 20.0 mL 2 M trifluoroacetic acid to dissolve the sample, seal and hydrolyze for 2 h; evaporate the hydrolyzed sample under low temperature and reduced pressure, add 10 mL methanol and evaporate it to remove excess trifluoroacetic acid; hydrolyze the sample with 1.5 mL double distilled water and transfer it to a 2.5 mL EP tube, centrifuge at 12000×g for 10 min, and take the supernatant to a new 2.5 mL EP tube, which is Green Clam Oligosaccharide.
2. The method for preparing oligosaccharides of Cilantro according to claim 1, characterized in that: In the S1, fresh green clam meat is taken, washed 3 times and minced, soaked in 75% ethanol overnight to remove oligosaccharides and impurities, crushed with a tissue homogenizer, and dried by freeze-drying for standby use; acetone solution is added according to a material ratio of 1:4 (w / v), degreased for 2 h using a magnetic stirrer, and removed grease and acetone after standing at 4°C overnight, and then the defatted green clam meat is freeze-dried for standby use; the hydrolysis bottle mouth is sealed with plastic wrap, and papain is used for hydrolysis after ultrasonic treatment for 30 min, and enzymatic hydrolysis is performed for 8 h at a material-liquid ratio of 1:5 (w / v) and an enzyme amount of 3%, and then centrifuged to obtain an enzymatic hydrolyzate; 4 times the volume of anhydrous ethanol is added, and the mixture is stood in a chromatography cabinet at 4°C for 24 h to precipitate polysaccharides at the bottom; after centrifugation, deionized water is added to dissolve the precipitate, and the alcohol precipitation is repeated 2-3 times, and the alcohol is evaporated and freeze-dried to obtain a crude green clam polysaccharide.
3. The method for preparing oligosaccharides of Cilantro according to claim 1, characterized in that: In S2, the polysaccharide solution of Cyclamen sinensis was adjusted to pH 11 by adding calcium hydroxide, incubated in a water bath at 60°C for 20 min, centrifuged at 12,000×g for 20 min, and the supernatant was adjusted to pH 7 by adding phosphoric acid, incubated in a water bath at 80°C for 20 min, and centrifuged at 12,000×g for 20 min. The supernatant was taken, 4 times the volume of ethanol was added and mixed, and Cyclamen sinensis polysaccharide was precipitated at 4°C overnight. The supernatant was discarded after centrifugation at 12,000×g for 5 min, and the crude Cyclamen sinensis polysaccharide after protein removal was obtained by freeze drying.
4. The method for preparing oligosaccharides of Cilantro according to claim 1, characterized in that: In S3, 300 mg of crude polysaccharide from the Clam Clam after protein removal was weighed, dissolved in appropriate amount of water, centrifuged at 12000×g for 10 min, and the supernatant was taken for sample loading; When loading the sample, pay attention to controlling the flow rate so that the polysaccharide of the green clam is adsorbed onto the column, and gradient elution is performed with water and different concentrations of NaCl solution. The required concentration gradient can be set after preliminary condition exploration; collect each component, collect 2 column volumes for each component, and after collection, elute with 1 mol / L NaCl solution of twice the column volume to remove the residual sample impurities in the filler, and finally elute with 2 column volumes of water before loading the sample again; The obtained Cynaroides polysaccharide solution was collected, and the distribution of Cynaroides polysaccharide was detected by absorbance at a wavelength of 625 nm using the sulfuric acid-anthrone method, and the absorbance was plotted against the tube number; The same sugar-containing components were collected and combined, and the collected components were dialyzed with double distilled water, with the water changed every 4 hours. The dialyzation lasted for 3 days, and then dialyzed with triple distilled water for 1 day. The dialyzed polysaccharide samples were concentrated under reduced pressure and freeze-dried.
5. The method for preparing oligosaccharides of Cynarodisiac according to claim 1 or 4, characterized in that: In S4, 60 mg of freeze-dried polysaccharide sample of Cynaroides sinensis was weighed, 1-2 mL of 0.2 M NH4HCO3 solution was added to dissolve, and the supernatant was taken for sample loading at 12000×g for 5 min, and the mobile phase was 0.2 M NH4HCO3 solution; after being collected by an automatic fraction collector, the distribution of Cynaroides sinensis polysaccharide was detected by absorbance at 490 nm using the phenol-sulfuric acid method, and the OD was calculated by the tube number. 490nm The values were plotted; the collected solutions corresponding to the main sugar peak of the eluted peaks were combined, concentrated and evaporated to dryness under reduced pressure at 50 °C to decompose NH4HCO3; the pure polysaccharide of Cynaroides sinensis was obtained after dissolving in double distilled water and freeze-drying.
6. A preparation method according to any one of claims 1 to 5 to obtain Cynaroides oligosaccharides with high antioxidant activity.
7. A Cynaroides oligosaccharide according to claim 6, characterized in that: When the concentration of S. cylindrica oligosaccharide was 5.0 mg / mL, the scavenging rates of DPPH, hydroxyl radicals and superoxide anions were 89.93%, 70.55% and 83.66%, respectively, and the total antioxidant capacity was 23.15 U / mg mass.
8. Use of the Cynaropoda oligosaccharide according to claim 6 in the preparation of antioxidant and anti-tumor products.