CN114276471B - Application of caulerpa lentillifera polysaccharide - Google Patents
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Abstract
The invention relates to the technical field of marine organisms, in particular to a preparation method and application of long-stem grape caulerpa polysaccharide. The caulerpa lentillifera polysaccharide extracted by the method shows the effect of inhibiting the growth of tumors in vitro.
Description
Technical Field
The invention relates to the technical field of marine organisms, in particular to a preparation method and application of caulerpa lentillifera polysaccharide.
Background
Caulerpa lentillifera (Caulerpa lentillifera) belongs to the order of Lupeophyta (Chlorophyta) of the order of Lupeophytidae (Bryophyllales) and belongs to the genus Caulerpa (Caulerpa) of the family Caulerpaceae, and is distributed in tropical and subtropical regions such as south China sea, southeast Asia, okinawa Japan, taiwan province, oceania, and the like. The coccoid of Caulerpa lentillifera is glittering and translucent and shaped like grapes, so it is often called Hai grape; the seaweed is rich in nutrition, delicious in taste and has a health-care function, is called as a longevity vegetable, is deeply favored by vast coastal residents, and is an economic seaweed with research and development prospects.
According to related researches, the caulerpa lentillifera is rich in moisture content which accounts for 97% of the wet weight, the dietary fiber accounts for 24-26% of the dry weight, the protein accounts for 16% of the dry weight, the fat only accounts for 0.03%, and the caulerpa lentillifera is cholesterol-free and is a high-protein low-fat health food. Also contains a large amount of amino acids, minerals, unsaturated fatty acids, and trace elements such as calcium, phosphorus, magnesium, iodine, and copper. Other related researches also find that the caulerpa lentillifera has the pharmacological effects of resisting bacteria, treating diabetes, reducing blood pressure, dispelling wind-damp, beautifying, protecting health and the like.
In recent years, malignant tumors have become one of the main causes of harm to human health. Due to aging, industrialization, and urbanization of the population, and changes in lifestyle, the number of new cases of malignant tumors worldwide is increasing year by year, and the mortality rate is also increasing. At present, the prevention and control situation of malignant tumors at home and abroad is severe, and great challenges are faced. Common treatment means for cancer mainly comprise operations, radiotherapy, chemotherapy and combined treatment, can prolong the survival time of a patient to a certain extent and improve the life quality of the patient, but radiotherapy, chemotherapy and surgical treatment can cause many intolerable side effects of a human body, such as symptoms seriously damaging the life quality of the patient, such as nausea, vomiting, hair loss, blood cell reduction and the like. Therefore, the search of antitumor active ingredients with low side effects from natural plants becomes a research hotspot in the field of overcoming malignant tumors in recent years, marine plants have characteristics which some terrestrial plants do not have due to unique living environments, the marine plants are widely distributed and have large quantity, and the method for obtaining the antitumor active ingredients from the marine plants is a novel idea with development prospects.
Disclosure of Invention
The invention aims to solve the technical problem of the prior art and provides a new application of the Caulerpa lentillifera polysaccharide.
In order to realize the purpose of the invention, the following technical means are specifically adopted:
use of Botrytis longipedicularis polysaccharide in preparation of medicine for inhibiting Lewis cell, A549 cell or H1975 cell.
Application of Caulerpa lentillifera polysaccharide in preparing antitumor drugs is provided.
Preferably, the tumor is non-small cell lung cancer.
Preferably, the tumor is lung adenocarcinoma.
Advantageous effects
(1) The invention provides an optimized scheme of a process for extracting the caulerpa lentillifera polysaccharide by a water extraction and alcohol precipitation method, so that the extraction efficiency of the caulerpa lentillifera polysaccharide is greatly improved, and the high-purity caulerpa lentillifera polysaccharide can be obtained.
(2) The caulerpa lentillifera polysaccharide extracted by the method shows the effect of inhibiting the growth of non-small cell lung cancer cells and lung adenocarcinoma cells in vitro for the first time.
Drawings
FIG. 1 shows the standard curve of sulfuric acid-anthrone obtained by the present invention
FIG. 2 is a graph of a single-factor experiment showing the effect of different extraction times on the extraction rate of Caulerpa lentillifera polysaccharide
FIG. 3 is a graph of a single factor experiment showing the effect of different feed solutions on the extraction rate of Caulerpa lentillifera polysaccharide
FIG. 4 is a graph of a single-factor experiment showing the effect of different extraction times on the extraction rate of Caulerpa lentillifera polysaccharide
FIG. 5 is a graph of single factor experiment of the effect of different extraction temperatures on the extraction rate of Caulerpa lentillifera polysaccharide
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The method for measuring the content of the polysaccharide in the vitis amurensis dunn of the invention adopts a sulfuric acid-anthrone method to measure
1. Operation method for measuring polysaccharide content by sulfuric acid-anthrone method
A. Preparation of a standard solution:
(1) Anthrone reagent: accurately weighing 0.1g of anthrone, adding 80% concentrated sulfuric acid to dissolve, transferring to a brown volumetric flask, adding 80% concentrated sulfuric acid to a constant volume of 100ml, and shaking up; it is ready for use (not longer than 2 hours).
(2) Dextran standard solution: placing the glucan in a phosphorus pentoxide dryer, precisely weighing 0.1g after 12h, and making the volume of the glucan to be 100mL by using distilled water to prepare a glucan standard solution of 1.0 mg/mL.
B. Drawing a standard curve: 0mL, 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL and 1.00mL of dextran standard solution were precisely pipetted into 10mL stoppered test tubes, and 1.0mL was made up with distilled water to prepare 3 groups per concentration. 4.0mL of the anthrone-sulfuric acid solution was added, and the reaction solutions were mixed by using a vortex shaker, and then the tube was placed in a boiling water bath and reacted for 10min. Cooling to room temperature in cold water bath for 10min, and immediately measuring the maximum required wavelength at 626nm of an ultraviolet-visible spectrophotometer. A standard curve was prepared using dextran mass as abscissa and absorbance as ordinate, and is shown in FIG. 1.
C. And (3) measuring the content of the sample, namely measuring the absorbance of the polysaccharide sample of the caulerpa lentillifera at 626nm by taking 1.0mL of sample solution according to the method, measuring for 3 times in parallel, and calculating the content of the polysaccharide.
2. The invention relates to a method for extracting caulerpa lentillifera polysaccharide by water extraction and alcohol precipitation
(1) Selecting fresh seaweed as a raw material, removing impurities such as weeds, silt and the like in the seaweed, drying the seaweed for 36-72 hours at 50 ℃, taking out the dried seaweed, crushing the seaweed, and sieving the seaweed with a 60-mesh sieve to obtain seaweed powder;
(2) The single factor experiment is a method of considering that when a plurality of influencing factors exist in the experiment, the level of only one of the influencing factors is changed, other influencing factors are fixed at a proper level, and after the optimal level range of the influencing factor is obtained, other influencing factors are considered step by step. According to the method, the extraction times, the material-liquid ratio (g: ml), the extraction time (h) and the extraction temperature (DEG C) are taken as experimental factors, and the influence of each factor on the extraction rate of the botrytis macrorrhiza polysaccharide is researched.
(3) Different times of extraction
1) Weighing 1.0g of caulerpa longipedicla powder, placing the caulerpa longipedicla powder in a round bottom flask, adding 40ml of distilled water, carrying out reflux extraction for 1 time at 80 ℃, extracting for 2 hours, carrying out rotary evaporation and concentration on the obtained extracting solution to 10ml, adding 95% ethanol with 4 times of volume, carrying out ethanol precipitation for 12 hours at 4 ℃, centrifuging to obtain precipitates, dissolving the precipitates to prepare 100ml of crude polysaccharide aqueous solution, preparing 2.5ml of the crude polysaccharide aqueous solution to prepare 25ml of solution to be detected, and determining the polysaccharide extraction rate by a sulfuric acid-anthrone method on 1ml of solution to be detected, wherein the extraction rate is 2.944%.
2) Weighing 1.0g of caulerpa longipedicla powder, placing the powder in a round-bottom flask, adding 40ml of distilled water, carrying out reflux extraction for 2 times at 80 ℃, extracting for 2 hours, carrying out rotary evaporation and concentration on the obtained extracting solution to 10ml, adding 95% ethanol with 4 times of volume, carrying out ethanol precipitation for 12 hours at 4 ℃, centrifuging to obtain precipitate, dissolving the precipitate to prepare 100ml of crude polysaccharide aqueous solution, preparing 2.5ml of the crude polysaccharide aqueous solution into 25ml of to-be-detected solution, and determining the polysaccharide extraction rate by using a sulfuric acid-anthrone method on 1ml of to-be-detected solution, wherein the extraction rate is 3.420%.
3) Weighing 1.0g of caulerpa longipedicla powder, placing the powder in a round-bottom flask, adding 40ml of distilled water, carrying out reflux extraction at 80 ℃ for 3 times, extracting for 2 hours, carrying out rotary evaporation and concentration on the obtained extracting solution to 10ml, adding 95% ethanol with 4 times of volume, carrying out ethanol precipitation at 4 ℃ for 12 hours, centrifuging to obtain precipitate, dissolving the precipitate to prepare 100ml of crude polysaccharide aqueous solution, preparing 2.5ml of the crude polysaccharide aqueous solution to prepare 25ml of to-be-detected solution, and determining the polysaccharide extraction rate of 1ml of to-be-detected solution by using a sulfuric acid-anthrone method, wherein the extraction rate is 3.705%.
The effect of the different extraction times is shown in fig. 2.
According to the extraction rate and the trend of the polysaccharide of the caulerpa lentillifera curds, the time cost and the yield are considered, and the extraction times are selected to be 2 times to carry out the next single-factor experiment.
(4) Different material-liquid ratios
1) Weighing 1.0g of caulerpa longipedicla powder, placing the powder in a round bottom flask, adding 30ml of distilled water, carrying out reflux extraction for 2 times at 80 ℃, extracting for 2 hours, carrying out rotary evaporation and concentration on the obtained extracting solution to 10ml, adding 95% ethanol with 4 times of volume, carrying out ethanol precipitation for 12 hours at 4 ℃, centrifuging to obtain precipitate, dissolving the precipitate to prepare 100ml of crude polysaccharide aqueous solution, preparing 2.5ml of the crude polysaccharide aqueous solution to prepare 25ml of solution to be detected, and determining the polysaccharide extraction rate by using a sulfuric acid-anthrone method on 1ml of solution to be detected, wherein the extraction rate is 4.395%.
2) Weighing 1.0g of caulerpa longipedicla powder, placing the powder in a round-bottom flask, adding 50ml of distilled water, carrying out reflux extraction for 2 times at 80 ℃, extracting for 2 hours, carrying out rotary evaporation and concentration on the obtained extracting solution to 10ml, adding 95% ethanol with 4 times of volume, carrying out ethanol precipitation for 12 hours at 4 ℃, centrifuging to obtain precipitate, dissolving the precipitate to prepare 100ml of crude polysaccharide aqueous solution, preparing 2.5ml of the crude polysaccharide aqueous solution into 25ml of to-be-detected solution, and determining the polysaccharide extraction rate of 1ml of to-be-detected solution by using a sulfuric acid-anthrone method, wherein the extraction rate is 4.891%.
3) Weighing 1.0g of caulerpa longipedicla powder, placing the caulerpa longipedicla powder in a round bottom flask, adding 70ml of distilled water, carrying out reflux extraction for 2 times at 80 ℃, extracting for 2 hours, carrying out rotary evaporation and concentration on the obtained extracting solution to 10ml, adding 95% ethanol with 4 times of volume, carrying out ethanol precipitation for 12 hours at 4 ℃, centrifuging to obtain precipitates, dissolving the precipitates to prepare 100ml of crude polysaccharide aqueous solution, preparing 2.5ml of the crude polysaccharide aqueous solution to prepare 25ml of solution to be detected, and determining the polysaccharide extraction rate by a sulfuric acid-anthrone method on 1ml of solution to be detected, wherein the extraction rate is 4.133%.
The extraction effect of different extraction liquid-to-feed ratios is shown in fig. 3.
According to the extraction rate and the trend of the polysaccharide of the caulerpa lentillifera, a feed-liquid ratio of 1.
(5) Different extraction time
1) Weighing 1.0g of caulerpa longipedicla powder, placing the powder in a round-bottom flask, adding 50ml of distilled water, carrying out reflux extraction for 2 times at 80 ℃, extracting for 1h, carrying out rotary evaporation and concentration on the obtained extracting solution to 10ml, adding 95% ethanol with 4 times of volume, carrying out ethanol precipitation for 12h at 4 ℃, centrifuging to obtain precipitate, dissolving the precipitate to prepare 100ml of crude polysaccharide aqueous solution, preparing 2.5ml of the crude polysaccharide aqueous solution into 25ml of to-be-detected solution, and determining the polysaccharide extraction rate of 1ml of to-be-detected solution by a sulfuric acid-anthrone method, wherein the extraction rate is 4.210%.
2) Weighing 1.0g of caulerpa longipedicla powder, placing the caulerpa longipedicla powder in a round bottom flask, adding 50ml of distilled water, carrying out reflux extraction for 2 times at 80 ℃, extracting for 1.5h, carrying out rotary evaporation and concentration on the obtained extracting solution to 10ml, adding 95% ethanol with 4 times of volume, carrying out ethanol precipitation for 12h at 4 ℃, centrifuging to obtain precipitate, dissolving the precipitate to prepare 100ml of crude polysaccharide aqueous solution, preparing 2.5ml of the crude polysaccharide aqueous solution to prepare 25ml of solution to be detected, and determining the polysaccharide extraction rate by using a sulfuric acid-anthrone method on 1ml of solution to be detected, wherein the extraction rate is 4.440%.
3) Weighing 1.0g of caulerpa longipedicla powder, placing the caulerpa longipedicla powder in a round bottom flask, adding 50ml of distilled water, carrying out reflux extraction for 2 times at 80 ℃, extracting for 2 hours, carrying out rotary evaporation and concentration on the obtained extracting solution to 10ml, adding 95% ethanol with 4 times of volume, carrying out ethanol precipitation for 12 hours at 4 ℃, centrifuging to obtain precipitates, dissolving the precipitates to prepare 100ml of crude polysaccharide aqueous solution, preparing 2.5ml of the crude polysaccharide aqueous solution to prepare 25ml of solution to be detected, and determining the polysaccharide extraction rate by a sulfuric acid-anthrone method on 1ml of solution to be detected, wherein the extraction rate is 4.891%.
The extraction effect at different extraction times is shown in fig. 4.
According to the extraction rate and the extraction trend of the polysaccharide of the caulerpa lentillifera, the extraction time of 2 hours is selected for carrying out the next single-factor experiment.
(6) Different extraction temperatures
1) Weighing 1.0g of caulerpa longipedicla powder, placing the caulerpa longipedicla powder in a round bottom flask, adding 50ml of distilled water, carrying out reflux extraction for 2 times at 70 ℃, extracting for 2 hours, carrying out rotary evaporation and concentration on the obtained extracting solution to 10ml, adding 95% ethanol with 4 times of volume, carrying out ethanol precipitation for 12 hours at 4 ℃, centrifuging to obtain precipitates, dissolving the precipitates to prepare 100ml of crude polysaccharide aqueous solution, preparing 2.5ml of the crude polysaccharide aqueous solution to prepare 25ml of solution to be detected, and determining the polysaccharide extraction rate by a sulfuric acid-anthrone method on 1ml of solution to be detected, wherein the extraction rate is 4.450%.
2) Weighing 1.0g of caulerpa longipedicla powder, placing the powder in a round-bottom flask, adding 50ml of distilled water, carrying out reflux extraction for 2 times at 80 ℃, extracting for 2 hours, carrying out rotary evaporation and concentration on the obtained extracting solution to 10ml, adding 95% ethanol with 4 times of volume, carrying out ethanol precipitation for 12 hours at 4 ℃, centrifuging to obtain precipitate, dissolving the precipitate to prepare 100ml of crude polysaccharide aqueous solution, preparing 2.5ml of the crude polysaccharide aqueous solution into 25ml of to-be-detected solution, and determining the polysaccharide extraction rate of 1ml of to-be-detected solution by using a sulfuric acid-anthrone method, wherein the extraction rate is 4.891%.
3) Weighing 1.0g of caulerpa longipedicla powder, placing the powder in a round-bottom flask, adding 50ml of distilled water, carrying out reflux extraction for 2 times at 90 ℃, extracting for 2 hours, carrying out rotary evaporation and concentration on the obtained extracting solution to 10ml, adding 95% ethanol with 4 times of volume, carrying out ethanol precipitation for 12 hours at 4 ℃, centrifuging to obtain precipitate, dissolving the precipitate to prepare 100ml of crude polysaccharide aqueous solution, preparing 2.5ml of the crude polysaccharide aqueous solution into 25ml of to-be-detected solution, and determining the polysaccharide extraction rate by using a sulfuric acid-anthrone method on 1ml of to-be-detected solution, wherein the extraction rate is 4.888%.
The extraction effect at different extraction times is shown in fig. 5.
According to the extraction rate and the trend of the polysaccharide of the caulerpa lentillifera, the extraction temperature of 80 ℃ is selected for carrying out the next single-factor experiment.
(7) According to the content of the caulerpa lentillifera polysaccharide under different condition levels, the single-factor condition with the largest influence is selected, and on the basis, the orthogonal experiment with 4-factor 3 level is adopted to further optimize the experiment condition, which is shown in table 1. The experimental data are subjected to variance analysis by using an orthogonal design assistant, the optimal process parameters for extracting the caulerpa lentillifera polysaccharide are predicted, and the specific factor levels and results are shown in tables 2-3.
The orthogonal experiment method is a design method for researching multi-factor and multi-level, selects part of representative horizontal combinations from the comprehensive experiment according to Galois theory to carry out the experiment, and analyzes the result to find out the optimal horizontal combination. Under the condition that a plurality of inspected factors are fixed, the influence of each interactive item on the extraction rate of the caulerpa lentillifera polysaccharide is inspected, and the optimal process condition is obtained by utilizing orthogonal design assistant analysis.
(8) And (3) verifying a regression model: the optimal process conditions for simulating the extraction of the polysaccharide from the caulerpa lentillifera by analyzing by orthogonal design helper software are as follows: extracting for 2 times at 80 ℃ for 2h in a feed-liquid ratio of 1. In order to verify the accuracy of the model, 3 groups of parallel verification experiments are carried out according to the optimal process conditions predicted by software, the extraction rates of the three groups of the parallel verification experiments are 4.562%, 5.233% and 4.878%, and the fact that the orthogonal experiment method optimizes the water extraction and alcohol precipitation method to extract the caulerpa lentillifera polysaccharide is proved to be feasible.
TABLE 1 orthogonal Experimental design factors and levels
Table 2 orthogonal design experiments and results
TABLE 3 ANOVA TABLE
| Factors of the design | Sum of squares of deviations | Degree of freedom | Ratio of F | Critical value of F |
| Number of times of extraction | 1.663 | 2 | 3.085 | 4.460 |
| Extraction temperature/. Degree.C | 0.355 | 2 | 0.659 | 4.460 |
| Ratio of material to liquid | 0.033 | 2 | 0.061 | 4.460 |
| Extraction time/h | 0.105 | 2 | 0.195 | 4.460 |
| Error of the measurement | 2.16 | 8 |
3. Experimental scheme for preparing Caulerpa lentillifera polysaccharide under optimized conditions
(1) Selecting algae: selecting fresh caulerpa lentillifera as a raw material, removing impurities such as weeds, silt and the like, and cleaning for later use;
(2) Drying and powdering: drying the caulerpa lentillifera selected in the step (1) at 50 ℃ for 36-72 hours, taking out the dried seaweed, crushing, and sieving with a 60-mesh sieve to obtain caulerpa lentillifera powder;
(3) Mixing the material and the liquid: weighing crushed and sieved caulerpa lentillifera powder, adding distilled water, and properly and uniformly stirring to obtain a material liquid mixture, wherein the material liquid ratio g/ml is 1;
(4) Water extraction of crude polysaccharide: extracting the crude polysaccharide from the feed liquid mixture obtained in the step (3) at the extraction temperature of 80 ℃ for 2 hours for 2 times, mixing the extracting solutions, and performing rotary evaporation to obtain a crude polysaccharide extracting solution a of 1ml/g dry seaweed;
(5) Alcohol precipitation of crude polysaccharide: uniformly mixing the crude polysaccharide extracting solution a obtained in the step (4) with 95% ethanol according to the volume ratio of 1;
(6) Removing protein by Sevage method: adding a sevage reagent (chloroform: n-butanol =4: 1) with the volume of 1/3 times of the crude polysaccharide extracting solution b obtained in the step (5), oscillating at room temperature for 20min, performing high-speed centrifugation at the rotating speed of 5000r/min for 10min after the reaction is finished, and taking a supernatant; repeating the above steps for about 5-8 times until the solution is not precipitated, and concentrating by rotary evaporation to obtain crude polysaccharide extractive solution c of 1ml/g dry Sargassum;
(7) And (3) dialysis: dialyzing the polysaccharide extract c obtained in the step (6) at 4 ℃ for 48h in a dialysis bag with the molecular weight of 2500Da to remove an organic solvent to obtain a crude polysaccharide extract d;
(8) Freeze-drying and storing: and (4) freeze-drying the crude polysaccharide extracting solution d prepared in the step (7) to obtain a seaweed polysaccharide extract, and storing the obtained freeze-dried powder at the temperature of-20 ℃.
4. Antitumor experiment of Caulerpa lentillifera polysaccharide extracted by the method
(1) Experiment for detecting influence of Caulerpa lentillifera polysaccharide on Lewis cell activity by MTT method
1) And (3) cell culture: selecting Lewis cells as target tumor cells, adding fresh medium containing 10% fetal bovine serum, setting the temperature at 37 deg.C and the content of CO at 5% 2 And saturated humidity incubator, changing the culture medium every other day until the cells are confluent to the bottom of the flask, digesting and passaging with trypsin containing 0.25% EDTA, selecting cells in logarithmic growth phase for experiment.
2) Preparing a liquid medicine: docetaxel (DTX) was dissolved in 0.1% DMSO to prepare a 1mg/L solution; dissolving Caulerpa lentillifera polysaccharide in 0.1% DMSO to obtain medicinal solutions with concentrations of 20mg/L, 40mg/L, 60mg/L, and 80mg/L, respectively, and sterilizing with 0.22 μm microporous membrane.
3) MTT method: lewis cells in logarithmic growth phase were diluted to 4X 10 4 one/mL, 6 groups of 6 wells were inoculated into 96-well cell culture plates, each group was plated with 6 duplicate wells at 200. Mu.L/wellThe cells are suspended and cultured for 24h to adhere to the wall. The 6 groups are respectively four long-stem grape caulerpan polysaccharide groups (namely an adding medicine group) with different concentrations, a positive control group and a blank control group, the long-stem grape caulerpan polysaccharide groups with different concentrations are respectively added with 20mg/L, 40mg/L, 60mg/L and 80mg/L of long-stem grape caulerpan polysaccharide, and the positive control group is a 1mg/L DTX control group; the blank control group was medium only, and 6 groups were exposed to 5% CO 2 The cells were incubated at a constant temperature (37 ℃) for 48 hours with 20. Mu.L of MTT solution (5 mg/ml) per well, and after further incubation for 4 hours, the old culture medium was discarded. Adding 150 mu L of DMSO into each hole, oscillating for 10min at room temperature, and measuring the OD value at a single wavelength of 490nm on a microplate reader. Calculating the Inhibition Rate (IR) of the Caulerpa lentillifera polysaccharide to Lewis cells according to the OD value, wherein the calculation formula is as follows: inhibition (IR) = (blank control OD average-dosing OD average)/blank control OD value × 100%. The results are shown in Table 4.
TABLE 4MTT assay for inhibition of Lewis cellular Activity by Caulerpa lentinan
(2) Experiment for detecting influence of botryococcus longipediculus polysaccharide on A549 cell activity by MTT (methanol to transfer) method
1) Cell culture: selecting A549 cells as target tumor cells, adding fresh culture medium containing 10% fetal calf serum, placing at 37 deg.C, and 5% CO 2 And changing culture solution every other day in an incubator with saturated humidity until the bottom of the bottle is full of cells, digesting the cells by using trypsin containing 0.25% EDTA and carrying out passage, and selecting the cells in logarithmic phase for experiment.
2) Preparing a liquid medicine: docetaxel (DTX) was dissolved in 0.1% DMSO to prepare a 1mg/L solution; dissolving Caulerpa lentillifera polysaccharide in 0.1% DMSO to obtain medicinal solutions with concentrations of 20mg/L, 40mg/L, 60mg/L, and 80mg/L, respectively, and sterilizing with 0.22 μm microporous membrane.
3) MTT method: a549 cells in logarithmic growth phase were diluted to 4X 10 4 And (4) inoculating the cells per mL into a 96-well cell culture plate in 6 groups, wherein each group is provided with 6 multiple wells, 200 mu L of cell suspension is added into each well, and the cells are cultured for 24h to adhere to the wall. The 6 groups are respectively four long-stem grape caulerpan polysaccharide groups (namely an adding medicine group) with different concentrations, a positive control group and a blank control group, the long-stem grape caulerpan polysaccharide groups with different concentrations are respectively added with 20mg/L, 40mg/L, 60mg/L and 80mg/L of long-stem grape caulerpan polysaccharide, and the positive control group is a 1mg/L DTX control group; the blank control group was medium only. Place 6 groups in 5% CO 2 The cells were incubated at a constant temperature (37 ℃) for 48 hours with 20. Mu.L of MTT solution (5 mg/ml) per well, and after further incubation for 4 hours, the old culture medium was discarded. Add DMSO 150. Mu.L to each well, shake for 10min at room temperature, and measure OD value at single wavelength 490nm on a microplate reader. Calculating the Inhibition Ratio (IR) of the botryococcus longissimus polysaccharide to A549 cells according to the OD value, wherein the calculation formula is as follows: inhibition (IR) = (blank control OD average-dosing OD average)/blank control OD value × 100%. The results are shown in Table 5.
TABLE 5MTT assay for inhibition of A549 cell activity by botrytis longipedicularis polysaccharides
| Group of | Dosage (mg/L) | Inhibition ratio (%) |
| DTX |
1 | 43.68 |
| Caulerpa lentillifera polysaccharide 1 (20 mg/L) | 20 | 19.78 |
| Caulerpa lentillifera polysaccharide 2 (40 mg/L) | 40 | 28.69 |
| Caulerpa lentillifera polysaccharide 3 (60 mg/L) | 60 | 35.77 |
| Caulerpa lentillifera polysaccharide 4 (80 mg/L) | 80 | 39.08 |
(3) Experiment for detecting influence of Caulerpa lentillifera polysaccharide on activity of H1975 cells by MTT method
1) Cell culture: selecting H1975 cells as target tumor cells, adding fresh culture medium containing 10% fetal calf serum, placing at 37 deg.C, and containing 5% CO 2 And a humidity-saturated incubator, the culture medium was changed every other day until the cells were confluent at the bottom of the flask, digested with trypsin containing 0.25% EDTA and passaged, and the cells in the logarithmic growth phase were selected for the experiment.
2) Preparing a liquid medicine: dissolving Docetaxel (DTX) in 0.1% DMSO to obtain 1mg/L medicinal solution; dissolving Caulerpa lentillifera polysaccharide in 0.1% DMSO to obtain medicinal solutions with concentrations of 20mg/L, 40mg/L, 60mg/L, and 80mg/L, and sterilizing with 0.22 μm microporous membrane.
3) MTT method: h1975 cells in logarithmic growth phase were diluted to 4X 10 4 And (4) inoculating the cells per mL in 6 groups into a 96-well cell culture plate, wherein each group is provided with 6 multiple wells, 200 mu L of cell suspension is added into each well, and the cells are cultured for 24h to adhere to the wall. The 6 groups respectively comprise four different concentrations of Caulerpa lentillifera polysaccharide groups (i.e. additive group), positive control group and blank control group, the different concentrations of Caulerpa lentillifera polysaccharide groups are respectively added with 20mg/L, 40mg/L, 60mg/L and 80mg/L Caulerpa lentillifera polysaccharide, and the positive control group is 1mg/L DTX control group; the blank control group was medium only, and 6 groups were placed in 5% CO 2 The cells were incubated at a constant temperature (37 ℃) for 48 hours with 20. Mu.L of MTT solution (5 mg/ml) per well, and after further incubation for 4 hours, the old culture medium was discarded. Add DMSO 150. Mu.L to each well, shake for 10min at room temperature, and measure OD value at single wavelength 490nm on a microplate reader. Calculating the Inhibition Ratio (IR) of the caulerpa lentinan to H1975 cells according to the OD value, wherein the calculation formula is as follows: inhibition (IR) = (blank control OD average-dosing OD average)/blank control OD value × 100%. The results are shown in Table 6.
TABLE 6MTT assay for inhibition of H1975 cell Activity by Caulerpa lentillifera polysaccharides
| Group of | Dosage (mg/L) | Inhibition ratio (%) |
| DTX |
1 | 34.56 |
| Caulerpa lentillifera polysaccharide 1 (20 mg/L) | 20 | 15.43 |
| Caulerpa lentillifera polysaccharide 2 (40 mg/L) | 40 | 25.79 |
| Caulerpa lentillifera polysaccharide 3 (60 mg/L) | 60 | 40.37 |
| Caulerpa lentillifera polysaccharide 4 (80 mg/L) | 80 | 51.02 |
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (3)
1. Use of Botrytis longipedicularis polysaccharide in preparation of medicine for inhibiting Lewis cell, A549 cell or H1975 cell.
2. The application of the caulerpa lentillifera polysaccharide in preparing an anti-tumor medicament, wherein the tumor is non-small cell lung cancer.
3. The use of claim 2, wherein the tumor is lung adenocarcinoma.
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| CN110256595A (en) * | 2019-06-29 | 2019-09-20 | 深圳市蓝汀鼎执生物科技有限公司 | A kind of extracting method of sea grape polysaccharide |
| CN113274490A (en) * | 2021-05-13 | 2021-08-20 | 华南农业大学 | Preparation method and application of botryococcus longipediculus polysaccharide disease-resistant inducer |
| CN113817077A (en) * | 2021-10-08 | 2021-12-21 | 广西壮族自治区水产科学研究院 | Extraction method of caulerpa lentillifera polysaccharide |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110256595A (en) * | 2019-06-29 | 2019-09-20 | 深圳市蓝汀鼎执生物科技有限公司 | A kind of extracting method of sea grape polysaccharide |
| CN113274490A (en) * | 2021-05-13 | 2021-08-20 | 华南农业大学 | Preparation method and application of botryococcus longipediculus polysaccharide disease-resistant inducer |
| CN113817077A (en) * | 2021-10-08 | 2021-12-21 | 广西壮族自治区水产科学研究院 | Extraction method of caulerpa lentillifera polysaccharide |
Non-Patent Citations (3)
| Title |
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| anti-inflammatory activity and structure identification of a sulfated polysaccharide CLGP4 from caulerpa lentillifera;YuJiao Sun 等;《International journal of biological macromolecular》;20200330;第146卷;第931-938页 * |
| Caulerpa lentillifera polysaccharides enhance the immunostimulatory activity in immunosuppressed mice in correlation with modulating gut microbiota.;Sun Yujiao 等;《Food & Function》;20191231;第10卷(第7期);第4315-4329页 * |
| 海蒿子多糖提取、纯化及其药理活性研究进展;孔丽 等;《大连海洋大学学报》;20210922;第12页 * |
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