CN110522760B - Application of ascosan from Ascophyllum nodosum - Google Patents
Application of ascosan from Ascophyllum nodosum Download PDFInfo
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- CN110522760B CN110522760B CN201910665480.6A CN201910665480A CN110522760B CN 110522760 B CN110522760 B CN 110522760B CN 201910665480 A CN201910665480 A CN 201910665480A CN 110522760 B CN110522760 B CN 110522760B
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention relates to the field of medicines, in particular to a new application of ascophyllan, and especially relates to an application of ascophyllan in preparing anti-liver malignant tumor medicines. Tests show that the ascophyllin and the low molecular weight fragment thereof have obvious inhibition effects on adhesion, migration and infiltration of human liver cancer cells (HepG 2), and can be used for preparing anti-liver malignant tumor medicines, health products or foods.
Description
Technical Field
The invention relates to the technical field of medicines, and in particular relates to a new application of ascophyllan in the field of medicines.
Background
Hepatocellular carcinoma (HCC) is abbreviated as liver cancer, and is located at the 5 th site of the incidence rate and the 3 rd site of the death rate of malignant tumors all over the world. Liver cancer has become the third most common malignant tumor with mortality rate second to that of stomach cancer and esophagus cancer. Cancer is associated with genetic changes, and cells proliferate uncontrollably due to changes in DNA, which continually infiltrate surrounding normal tissues, leading to disease. Tumors can be treated by surgical resection as long as the growing tumor remains localized. Due to the infiltration and migration characteristics of liver cancer cells, a tiny disseminated focus may exist before an operation, so that the recurrence and metastasis rate after the liver cancer focus is removed by the operation is high, which is also an important reason for the failure of the operation treatment of most liver cancer patients. Therefore, inhibiting the metastasis of hepatoma cells is also one of the necessary strategies for treating liver cancer. The intercellular substance near the primary focus of malignant tumor generates new blood vessel, the cancer cell is separated from the primary focus, infiltrates and migrates into the surrounding tissue and blood vessel, enters the circulatory system and migrates to a distant position, invades the new position through the actions of adhesion, infiltration and migration, and grows to form the malignant tumor metastasis. Thus, adhesion, infiltration and migration are important processes for metastasis of malignant tumors. Screening and researching inhibitors for adhesion, infiltration and migration of liver cancer cells are one of important means for adjuvant therapy of liver cancer metastasis nuclear spread.
Ascophyllum nodosum (also called Chlamydomonas yanthera) is a large-scale marine brown alga and is one of the main raw materials for producing sodium alginate and mannitol in China; similar to other brown algae (such as kelp). Ascophyllum nodosum contains about 13% of ascophyllan (ascophyllan) and 8% of fucosan (fucoidan) in the total sugar content of the algal body. Researches report that the ascophyllum nodosum has the functions of resisting oxidation, inducing immune cell activation and reducing blood sugar, but no report is provided about the research and the activity of low molecular weight fragments of the ascophyllum nodosum, particularly the application of the ascophyllum nodosum in resisting human liver cancer tumor metastasis.
Disclosure of Invention
In view of this, the invention provides a new use of ascophyllan, in particular to a use of ascophyllan in preparing anti-liver malignant tumor products.
The invention detects the influence of the ascophyllum nodosum and the low molecular weight fragment thereof on the cell activity of human liver cancer cells (HepG 2) through experiments. The results show that the fucoidan low molecular weight fragment does not affect the cell viability of human hepatoma cells (HepG 2) in the determined concentration range (0-200. Mu.g/mL) and is non-toxic. In the concentration range of 0-50 mug/mL, the zosteran has no obvious influence on the cell activity of the liver cancer cell (HepG 2); when the concentration is 100-200 mug/mL, the influence of the ascophyllum nodosum on the cell viability of the liver cancer cell (HepG 2) is obvious, the cell viability is gradually reduced along with the increase of the ascophyllum nodosum concentration, and when the concentration reaches 200 mug/mL, the ascophyllum nodosum reduces the cell viability of the human liver cancer cell (HepG 2) by 10 percent and is slightly toxic.
The invention takes human liver cancer cells (HepG 2) as tested cells, utilizes a cell adhesion test to investigate the adhesion effect of the ascophyllan on tumor cells, and results show that the ascophyllan and low molecular weight fragments thereof can obviously reduce the adhesion capability of the cancer cells when the ascophyllan and the low molecular weight fragments thereof are used for treating the cancer cells. The results show that the ascophyllan and the low molecular weight segment thereof have the function of obviously inhibiting cancer cell adhesion.
The invention takes human liver cancer cells (HepG 2) as tested cells, and examines the effect of the ascophyllan and low molecular weight fragments thereof on the migration of tumor cells through a cell transfer test. The results show that the ascophyllan and the low molecular weight fragment thereof can obviously inhibit the migration of cancer cells. The paulownia fucoidan low molecular weight fragment treated by 100 mu g/mL for 12 hours can inhibit cell migration, and treated by 50 mu g/mL for 24 hours can obviously inhibit cell migration. After 50 mu g/mL of the ascophyllan is treated for 36 hours, the cell migration is obviously inhibited. Particularly, in the tested concentration range (50-100 mu g/mL), the low molecular weight fragment of the ascophyllan can inhibit the migration of cancer cells more obviously than the ascophyllan under the conditions of treating the cells for 12h, 24h and 36h, namely the activity of the low molecular weight fragment of the ascophyllan for inhibiting the migration of HepG2 cells is obviously higher than the ascophyllan.
The invention takes human liver cancer cells (HepG 2) as tested cells, and utilizes a Transwell infiltration test to investigate the influence of the ascophyllin and low molecular weight fragments thereof on tumor cell infiltration. The results show that 100. Mu.g/mL of ascophyllan inhibited cell infiltration by 26%; the 100. Mu.g/mL of ascophyllan low molecular weight fraction inhibited 42% of cell infiltration. The application shows that the ascophyllum nodosum and the low molecular weight fragment thereof have the effect of obviously inhibiting the infiltration of cancer cells, and the activity of the ascophyllum nodosum low molecular weight fragment in inhibiting the infiltration of HepG2 cells is obviously higher than that of ascophyllum nodosum.
Therefore, the invention provides the application of the ascophyllin preparing products for inhibiting the migration of human liver cancer cells.
From the experimental results, the ascophyllan and the low molecular weight fragment thereof provided by the invention have obvious inhibition effects on adhesion, migration and infiltration of human liver cancer cells (HepG 2).
In addition, the application of the ascosan according to the above embodiment of the present invention may further have the following additional technical features:
preferably, the application takes the ascophyllum nodosum or the ascophyllum nodosum low molecular weight fragment as one of the active ingredients or the only active ingredient to prepare the product for resisting the liver malignant tumor.
The low molecular weight fraction of ascophyllan refers to: and (3) carrying out enzymolysis treatment on the ascophyllan, and further separating to obtain a polysaccharide component with a molecular weight lower than that of the original ascophyllan.
Preferably, the application takes the ascophyllum nodosum or ascophyllum nodosum low molecular weight fragment as one of active ingredients or the only active ingredient for preparing the human liver cancer cell adhesion, migration and infiltration inhibitor.
Preferably, the anti-liver malignant tumor product is a medicament, a health product or a food for preventing or treating liver malignant tumor.
Preferably, the ascophyllan molecular weight distribution is: 180-460 kDa; the molecular weight distribution of the low molecular weight section of the ascophyllan is 2.0-7.0 kDa.
Preferably, the preparation method of the ascosan comprises the following steps:
weighing the phyllidium amabilis powder, adding distilled water according to the feed-liquid ratio of 1:20g/mL, extracting for 1-4 hours at the temperature of 80-100 ℃, stirring for multiple times in the extraction process, cooling, centrifuging, taking supernate, re-dissolving residues with distilled water, leaching for 1-4 hours again, collecting supernate, and combining the supernate obtained by two times of leaching;
adjusting the pH of the combined supernatant to 1.0-2.0 by using hydrochloric acid, standing the mixed supernatant in a chromatography cabinet at 4 ℃ overnight, centrifuging the mixed supernatant, collecting the supernatant, and adjusting the pH to be neutral by using sodium hydroxide;
mixing the neutral supernatant with anhydrous ethanol in equal volume, standing in a chromatography cabinet at 4 deg.C overnight, centrifuging, and collecting precipitate;
dissolving the precipitate with water, dialyzing and drying to obtain the ascosan.
The preparation method of the ascophyllan low molecular weight fragment comprises the following steps:
weighing 5g of ascophyllum nodosum, dissolving the ascophyllum nodosum in 1L of distilled water, adding alginate lyase, adjusting the pH to 5-7 by using hydrochloric acid/sodium hydroxide, then placing the mixture in a water bath kettle at the temperature of 30-50 ℃, and oscillating in a water bath for 1-4 hours;
after the water bath is finished, boiling in boiling water to inactivate the enzyme, centrifuging for 20min at 6000-10000 Xg, removing the precipitate, performing ultrafiltration by using an ultrafiltration separation device and an ultrafiltration membrane with the molecular weight of 10kDa cut-off, and separating to obtain a low molecular weight fraction solution of the ascophyllan with the molecular weight of less than 10 kDa;
and (3) passing the low molecular weight fraction solution of the ascophyllan through a Sephadex G75 gel chromatography column, collecting a sample at an elution peak, dialyzing by using a dialysis bag with a molecular weight cut-off of 1kDa, and freeze-drying to obtain the ascophyllan molecular weight fraction.
In a second aspect, the invention provides a medicament for the prevention or treatment of liver malignancies, comprising ascophyllan or an ascophyllan low molecular weight fragment, and a pharmaceutically acceptable excipient. Wherein, the medicine can be a medicine for inhibiting the metastasis of human hepatoma cells or a medicine for assisting in inhibiting the metastasis of human hepatoma cells. The medicine can be an injection preparation or an oral preparation. The oral agent can be tablet, capsule or granule. The injection preparation can be lyophilized powder for injection.
In a third aspect, the invention provides a food product for use in the prevention or treatment of liver malignancies, comprising ascophyllan or ascophyllan low molecular weight fragments, and a dietetically acceptable additive.
Drawings
FIG. 1 is a graph of the effect of different concentrations of ascophyllan and low molecular weight fragments thereof on the viability of human liver cancer cells (HepG 2) according to the invention;
FIG. 2 is a graph showing the effect of different concentrations of ascophyllan and low molecular weight fragments thereof on the adhesion ability of human hepatoma cells (HepG 2) according to the present invention;
FIG. 3 is a graph of the effect of different concentrations of ascophyllan on the migration ability of human liver cancer cells (HepG 2) according to the invention;
FIG. 4 is a graph showing the effect of fucoidan at various concentrations on the mobility inhibition of human hepatoma cells (HepG 2) according to the present invention;
FIG. 5 is a graph of the effect of different concentrations of ascophyllan low molecular weight fragments on the migration capacity of human hepatoma cells (HepG 2) according to the present invention;
FIG. 6 is the effect of fucoidan low molecular weight fragments of different concentrations of the present invention on the migration inhibition of human hepatoma cells (HepG 2);
FIG. 7 is a graph of the effect of 100. Mu.g/mL ascophyllin and ascophyllin low molecular weight fragments on the inhibition of human hepatoma cell (HepG 2) migration in accordance with the present invention;
FIG. 8 is a graph of the effect of 100. Mu.g/mL ascophyllan and low molecular weight fragments thereof on the amount of human hepatoma cells (HepG 2) infiltrated in accordance with the present invention;
FIG. 9 is a graph showing the effect of 100. Mu.g/mL ascophyllan and low molecular weight fragments thereof on the infiltration capacity of human hepatoma cells (HepG 2) according to the present invention.
Detailed Description
The technical solution of the present invention is explained below by specific concrete examples and drawings. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
In order to better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention can be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 preparation of Ascophyllin
Cleaning Ascophyllum nodosum with water, drying in a 60 ℃ oven, pulverizing with a pulverizer, and sieving with a 100-mesh sieve to obtain fine powder.
Weighing 100g of phyllidium soaking powder in a beaker, slowly adding 2000mL of distilled water (material-liquid ratio =1 = 20), stirring with a glass rod while adding water, uniformly stirring, placing in a 100 ℃ water bath kettle, and carrying out water bath at 80-100 ℃ for 1-4 h, wherein stirring is carried out for multiple times during the time to ensure that the phyllidium soaking powder is fully leached. After the leaching is finished, the leaching liquor of the Ascophyllum nodosum is taken out, cooled to room temperature and then centrifuged (5000 Xg, 30 min), and the supernatant is collected. Re-dissolving the residue with distilled water of a certain volume, uniformly stirring, leaching for 1-4 h at 80-100 ℃, taking out, cooling to room temperature, centrifuging (5000 Xg, 30 min), and collecting supernatant. The supernatants from the two leachings were combined and the pH of the supernatant was adjusted to 1.0-2.0,4 ℃ with 0.1M HCl solution and left overnight in a chromatography cabinet. The next day, it was centrifuged (5000 Xg, 30 min), the supernatant was collected and the pH of the supernatant was adjusted to neutral with 0.1M NaOH solution. Then absolute ethyl alcohol and the neutral supernatant liquid are mixed in equal volume, and after being stirred evenly, the mixture is placed in a chromatography cabinet at 4 ℃ for overnight. The next day, the pellet was centrifuged (5000 Xg, 30 min), the pellet was collected, dissolved in a small amount of water, and dialyzed against running water for 48h using a dialysis bag with a cut-off of 3500 Da. After dialysis, collecting the liquid in the dialysis bag, decompressing, rotary steaming to a certain volume, and carrying out vacuum freeze drying to obtain the ascophyllan for later use.
EXAMPLE 2 preparation of low molecular weight fragments of Ascophyllum nodosum
Accurately weighing 5g of Ascophyllum nodosum, dissolving in 1L of distilled water, and adding alginate lyase (the final concentration is 0.04-2.00U/mL). After being stirred evenly, the pH value is adjusted to 5 to 7 by hydrochloric acid/sodium hydroxide, and then the mixture is put into a water bath kettle at the temperature of 30 to 50 ℃ and is subjected to oscillating water bath for 1 to 4 hours. After the water bath is finished, boiling the water bath for 20min to inactivate the enzyme, and centrifuging for 20min at 6000-10000 Xg to remove the precipitate. And performing ultrafiltration separation by using an ultrafiltration separation device and an ultrafiltration membrane with the molecular weight cut-off of 10 kDa. And (3) passing the enzymolysis product of the ascophyllan with the molecular weight less than 10kDa separated by ultrafiltration through a Sephadex G75 (1.6 cm I.D.. Times.100 cm) (GE Healthcare BioSciences AB, uppsala and Sweden) gel chromatography column to obtain a single elution peak, collecting a sample at the elution peak, dialyzing for 2 days by using a dialysis bag with the molecular weight cut-off of 1KDa, and freeze-drying to obtain the ascophyllan low molecular weight fragment for later use.
EXAMPLE 3 culture of human hepatoma cells (HepG 2)
Fetal bovine blood containing 10% of human liver cancer cells (HepG 2)DMEM medium containing serum (FBS), 1 Xdouble antibody (100. Mu.g/mL streptomycin and 100 IU/mL penicillin) was subjected to 5.0% CO 2 The cells were subcultured at 37 ℃ in the cell culture chamber for use.
Example 4 in vitro cell assay
This example examines the effect of ascophyllan and ascophyllan low molecular weight fragments on the viability of human hepatoma cells (HepG 2).
Human liver cancer cells (HepG 2) (about 3X 10) 4 Individual cells/well) were seeded in 96-well plates; the fucoidan prepared in example 1 and the low molecular weight fragment of the fucoidan prepared in example 2 were added to a growth medium containing 10% FBS and 1 XPcillin-streptomycin double antibody solution, respectively, to a gradient of final concentration (0,6.25, 12.5, 25, 50, 100 and 200. Mu.g/mL), human hepatoma cells (HepG 2) were treated separately, 24h later, the supernatant per well was discarded, 50. Mu.L of 5mg/mL MTT solution was added to each well of a 96-well plate, and incubated at 37 ℃ for 30min. Then, 100. Mu.L of DMSO was added, and the 96-well plate was gently shaken to completely dissolve blue-violet formazan. Finally, the absorbance (absorbance OD) of each well at a wavelength of 570nm was measured by a microplate reader 570 ). The cell viability (viability%) was calculated as follows:
wherein the treatment groups are cells treated with different concentrations of the sample, the control group is 0 concentration treatment group without polysaccharide, and the blank is OD of DMSO 570 The value is obtained.
The result is shown in figure 1, when the concentration is 100-200 mug/mL, the low molecular weight fragment of the ascophyllum nodosum has no obvious influence on the cell viability of the human liver cancer cell (HepG 2), and the result shows that the low molecular weight fragment of the ascophyllum nodosum has no toxicity on the human liver cancer cell within the tested concentration range; in the concentration range of 0-50 mug/mL, the ascophyllum nodosum has no obvious influence on the cell activity of the liver cancer cell (HepG 2), when the concentration is 100-200 mug/mL, the ascophyllum nodosum has obvious influence on the cell activity of the liver cancer cell (HepG 2), the cell activity is gradually reduced along with the increase of the ascophyllum nodosum concentration, and when the concentration reaches 200 mug/mL, the ascophyllum nodosum reduces the activity of 10 percent of the human liver cancer cell (HepG 2) and is slightly toxic.
Example 5 Effect of Ascophyllin and Low molecular weight fragments thereof on adhesion to human liver cancer cells (HepG 2)
mu.L of rat tail collagen type I (Solambio C8061) was added to each well of a 48-well cell culture plate and spread flat, incubated for 1h, then the supernatant was aspirated, and dried at room temperature for 2h. The human liver cancer cells (HepG 2) of example 3 were prepared into a cell suspension (about 1X 10) using a growth medium 5 One cell/well), add to each well of a 48-well cell culture plate, immediately add growth media containing different concentrations of ascophyllum nodosum or ascophyllum nodosum low molecular weight fragments to final concentrations of 0, 50, 100 and 200 μ g/mL, respectively. After incubation for 1h at 37 ℃, the medium was aspirated, each well of the 48-well cell culture plate was washed 2 times with PBS, and the non-adherent cells were washed off, then 50 μ L of 5mg/mL MTT solution was added to each well of the 48-well plate and incubated at 37 ℃ for 30min. 100 μ L of DMSO was added and the 48-well plate was gently shaken to completely dissolve blue-violet formazan. Finally, the absorbance (absorbance OD) of each well at a wavelength of 570nm was measured by a microplate reader 570 ). The results are shown in FIG. 2.
As shown in fig. 2: compared with the concentration of 0 without adding the ascophyllum nodosum or the low molecular weight fragment thereof, the human liver cancer cells (HepG 2) are treated by the ascophyllum nodosum or the low molecular weight fragment thereof with the concentration of 50, 100 and 200 mu g/mL, and the adhesive capacity of the cancer cells can be obviously reduced. The adhesive capacity of the ascophyllan treated group is reduced by 12.6%, 46.4% and 73.4% respectively when the measured concentration is 50, 100 and 200 mu g/mL; the adhesion capacities of the treatment groups of the low molecular weight segments of the ascophyllum nodosum are respectively reduced by 25.6%, 36.6% and 59.8%, which shows that the ascophyllum nodosum and the low molecular weight segments thereof have the effect of obviously inhibiting the adhesion of cancer cells.
Example 6 Effect of Ascophyllin and Low molecular weight fragments on migration of human hepatoma cells (HepG 2)
Human liver cancer cell (HepG 2) 1 × 10 5 The cells/well density were seeded in 6-well plates and cultured for 18h. Preparation of a scratch of monolayer cells using a sterile 200 μ L pipette tip, followed by gentle washing 1 time with PBSTo remove scraped nonadherent cells. Human liver cancer cells (HepG 2) are treated for 0 to 36 hours by respectively using 0, 50 and 100 mu g/mL of the ascophyllin or the low molecular weight fragment thereof with different concentrations. Images were taken using bright field microscopy (40 x magnification) at 0, 12, 24 and 36h. The results are shown in fig. 3, 4, 5 and 6. The analysis was performed using ImageJ and the mobility inhibition was calculated according to the following formula:
wherein, T 0 For 0 hour scratch area, tx = T 12 ，T 24 Or T 36 (scratch area at 12, 24 or 36 hours, respectively). Tx 0 ＝T 12-0 ，T 24-0 Or T 36-0 (area of scratch at 12, 24 or 36 hours, respectively, at a concentration of 0. Mu.g/mL).
As shown in fig. 3 to 6: compared with the concentration of 0 without adding the ascophyllum nodosum or the low molecular weight fragment thereof, the human liver cancer cells (HepG 2) are treated by the ascophyllum nodosum or the ascophyllum nodosum low molecular weight fragment with the concentration of 50, 100 mu g/mL, and the cancer cell migration can be obviously inhibited. The ascophyllan low molecular weight fragment is treated for 12 hours by 100 mu g/mL to inhibit cell migration, and the migration inhibition rate is 8.9%; after treatment for 24h and 36h, cell migration is remarkably inhibited, and migration inhibition rates are 26.8% and 33.3%; the cell migration is obviously inhibited after the treatment of 50 mu g/mL for 36h, and the migration inhibition rate is 12.5%. After 50 mug/mL of ascosan is treated for 36 hours, cell migration is remarkably inhibited, and the migration inhibition rate is 14.2%; after the treatment of 100 mu g/mL for 36h, the cell migration is obviously inhibited, and the migration inhibition rate is 25.3%. As can be seen from fig. 7: particularly, in the tested concentration range (50-100 mu g/mL), the low molecular weight fragment of the Ascophyllum can inhibit the migration of cancer cells more obviously than the Ascophyllum under the conditions of treating the cells for 12h, 24h and 36h, namely the activity of the low molecular weight fragment of the Ascophyllum for inhibiting the migration of HepG2 cells is obviously higher than that of the Ascophyllum.
Example 7 Effect of Ascophyllin and Low molecular weight fragments on human hepatoma cell (HepG 2) infiltration
At the bottom of the inside of the transwell chamber100 μ L of matrigel (BD 356237) was spread on the outside bottom of the transwell chamber, human FN fibronectin (Solambio F8180) was coated on the outside of the transwell chamber, and the human liver cancer cells (HepG 2) of example 3 (about 1X 10) 5 Individual cells/well) were inoculated in transwell chambers and divided into experimental group 1, experimental group 2 and control group, experimental group 1 treated cells with 100 μ g/mL of the ascophyllan prepared in example 1, experimental group 2 treated cells with 100 μ g/mL of the ascophyllan low molecular weight fragment prepared in example 2, and control group treated cells with an equal volume of growth medium at 37 ℃,5% co 2 After incubation in an incubator for 24 hours, fixing the lower layer transfer cells by using 30% formaldehyde for the experimental group and the control group, then staining the cells with crystals to be purple, and taking a picture under a microscope, wherein the result is shown in figure 8; dissolving the stained cells with 33% acetic acid solution, and measuring the absorbance OD at 570nm with microplate reader 570 The results are shown in FIG. 9.
As shown in FIG. 8, treatment of human hepatoma cells (HepG 2) with 100. Mu.g/mL concentration of ascophyllan or low molecular weight fragments thereof significantly inhibited cancer cell infiltration compared to the control group without addition of ascophyllan or low molecular weight fragments thereof. As shown in fig. 8: the numbers of the infiltration cells of the experimental group 1 treated with the ascophyllan and the infiltration cells of the experimental group 2 treated with the ascophyllan low molecular weight fragment were less than those of the control group. As shown in fig. 9, after the crystal violet of the experimental group and the control group was dissolved out, the absorbance was measured to obtain the wetting ability; the 100 mug/mL of the cells of the experimental group 1 treated by the ascophyllin inhibited the infiltration of the cells by 26 percent compared with the control group; the 100 μ g/mL paulosan low molecular weight fraction treated cells of experimental group 2 inhibited 42% cell infiltration relative to the control group. The results show that the ascophyllum nodosum and the low molecular weight fragment thereof have the effect of obviously inhibiting the infiltration of cancer cells, and the capacity of inhibiting the infiltration of human liver cancer cells (HepG 2) of the ascophyllum nodosum low molecular weight fragment is obviously superior to that of ascophyllum nodosum.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (4)
1. The application of the ascophyllan as one of active ingredients or the only active ingredient in preparing the anti-liver cancer metastasis medicament is characterized in that the preparation method of the ascophyllan comprises the following steps:
weighing the phyllidium amabilis powder, adding distilled water according to the feed-liquid ratio of 1:20g/mL, extracting for 1-4 hours at the temperature of 80-100 ℃, stirring for multiple times in the extraction process, cooling, centrifuging, taking supernate, re-dissolving residues with distilled water, extracting for 1-4 hours again, collecting supernate, and combining the supernate obtained by two times of extraction;
adjusting the pH of the combined supernatant to 1.0 to 2.0 by using hydrochloric acid, placing the combined supernatant in a chromatography cabinet at 4 ℃ overnight, centrifuging, collecting the supernatant, and adjusting the pH to be neutral by using sodium hydroxide;
mixing the neutral supernatant with anhydrous ethanol in equal volume, standing in a chromatography cabinet at 4 deg.C overnight, centrifuging, and collecting precipitate;
dissolving the precipitate with water, dialyzing and drying to obtain the ascosan.
2. Use according to claim 1, characterized in that: the molecular weight distribution of the ascosan from the Ascophyllum nodosum is as follows: 180-460 kDa.
3. The application of the ascophyllan low molecular weight fragment as one of active ingredients or the only active ingredient in preparing the anti-liver cancer metastasis medicaments is characterized in that the preparation method of the ascophyllan low molecular weight fragment comprises the following steps:
weighing 5g of ascophyllum nodosum, dissolving the ascophyllum nodosum in 1L of distilled water, adding alginate lyase, adjusting the pH to 5~7 by using hydrochloric acid/sodium hydroxide, then placing the mixture in a water bath kettle at the temperature of 30 to 50 ℃, and oscillating the mixture in a water bath for 1 to 4 hours;
after the water bath is finished, boiling the inactivated enzyme with boiling water, 6000-10000 ″, and thengCentrifuging for 20min, removing precipitate, ultrafiltering with ultrafiltration separation device and ultrafiltration membrane with 10kDa molecular weight cut-off, and separating to obtain low molecular weight fraction solution of fucoidan with molecular weight less than 10 kDa;
and (3) passing the solution of the low molecular weight fraction of the ascophyllum nodosum through a Sephadex G75 gel chromatographic column, collecting a sample at an elution peak, dialyzing by using a dialysis bag with a molecular weight cut-off of 1kDa, and freeze-drying to obtain the low molecular weight fraction of the ascophyllum nodosum.
4. Use according to claim 3, characterized in that: the molecular weight distribution of the low molecular weight section of the ascophyllum nodosum is 2.0-7.0 kDa.
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