CN112010991A - A ferrified radix astragali polysaccharide for inhibiting human gastric cancer cell, and its preparation method and application - Google Patents
A ferrified radix astragali polysaccharide for inhibiting human gastric cancer cell, and its preparation method and application Download PDFInfo
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Abstract
The invention discloses an astragalus polysaccharide for inhibiting human gastric cancer cells and a preparation method and application thereof, and relates to the field of an astragalus polysaccharide ferration method and application thereof. The invention aims to solve the technical problems of lack of safety and iron content of the existing medicines for treating gastric cancer. Hydrogen bond linkage between molecules of the ferrified astragalus polysaccharide; the preparation method comprises the following steps: adding astragalus polysaccharide and trisodium citrate into single distilled water, heating in a water bath, and adding a ferric trichloride solution and a sodium hydroxide solution to obtain a suspension; centrifuging to remove the lower layer precipitate, adding anhydrous ethanol into the supernatant to completely precipitate, centrifuging, collecting precipitate, washing, vacuum drying to obtain crude product of polysaccharide-iron complex, dialyzing with running water, washing twice, and vacuum drying. The synthetic method of the invention has simple and convenient operation and low price. The ferrified astragalus polysaccharide has obvious proliferation inhibition on gastric cancer cells. The prepared ferrated astragalus polysaccharide is used as a bioactive component for inhibiting human gastric cancer cells and is applied to preparing antitumor drugs.
Description
Technical Field
The invention relates to an ironing method of astragalus polysaccharide and the application field.
Background
Polysaccharides (polysaccharides) are abundant in nature and widely distributed, and are widely found in plants, animals, microorganisms, and fungi. Scientists begin to research polysaccharides since 1960, and find that polysaccharides have the characteristics of wide sources and strong pharmacological activity, and have low cytotoxicity and high safety, so that the polysaccharides become one of the research hotspots in the field of medicine. The Astragalus is a plant of Astragalus (Astragalus) of leguminosae, has mild property and slightly sweet taste, and has the effects of tonifying qi, consolidating exterior, resisting toxin, promoting granulation, reducing swelling and the like. The chemical components of radix astragali mainly comprise saponins, flavonoids and polysaccharides[38]. The Astragalus polysaccharides are isolated from dried Astragalus roots. Research shows that the crude extract of astragalus polysaccharide contains polysaccharide components, wherein the polysaccharide components comprise arabinose, galactose, glucose and the like. The Astragalus polysaccharides have antioxidant, anti-tumor, immunity regulating, digestion promoting and antiinflammatory effects. The astragalus polysaccharide has attracted much attention in recent years because of its wide source, low toxic and side effects, low cost and no drug resistance.
In recent years, domestic and foreign researches show that the traditional Chinese medicine and the active ingredients thereof have the characteristics of small toxic and side effects, high treatment efficiency and the like, and become the main direction of research on future antitumor drugs. The death and morbidity of malignant tumors in China show a continuously rising trend. According to recent statistics, the incidence of malignant tumors in China is at the front end of the world, and the death rate of each year of patients accounts for one third of the total death rate of the malignant tumors. Research shows that the polysaccharide medicine has wide pharmacological and biological activity, less toxic side effect, antitumor, blood sugar reducing, antiaging, antiviral, blood fat reducing, etc. Among patients with tumors, most people are afflicted with iron-deficiency anemia. The total incidence rate of anemia in cancer patients is about 30-90%, and the incidence rate of anemia in Chinese investigation on cancer patients is up to 60.83%. The low physical condition and the reduced quality of life that are common in cancer patients are caused by anemia and iron deficiency, and even severe patients increase the risk of death. Iron deficiency can block DNA synthesis and antibody production in lymphocytes, and can cause hypoxic diseases in brain and other organs, which can aggravate tumor and help cancer. And the gastric cancer SGC-7901 cell is easy to cause chronic atrophy of mucous membranes of tongue, esophagus, stomach and small intestine due to the deficiency of iron element, so that gastric acid is reduced. This causes a large amount of bacteria to gather and multiply in the stomach, and makes nitrate taken into the body and amines in the stomach synthesize strong carcinogen nitrosamine, which is easy to form gastric cancer. Therefore, how to find a safe and effective medicine for the gastric cancer patients and enhance the absorption of iron elements, and simultaneously, how to reduce the anemia of the patients is the key point of research.
Disclosure of Invention
The invention provides an iron-treated astragalus polysaccharide for inhibiting human gastric cancer cells and a preparation method and application thereof, aiming at solving the technical problems that the existing medicine for treating gastric cancer lacks safety and lacks iron content.
A ferrization astragalus polysaccharides for inhibiting human gastric cancer cells has a structural formula of
The ferriated astragalus polysaccharides form intermolecular hydrogen bonding links. The preparation method of the ferrated astragalus polysaccharide for inhibiting the human gastric cancer cells comprises the following steps:
firstly, adding astragalus polysaccharide and trisodium citrate into single distilled water, controlling the temperature to be 50-80 ℃ to carry out water bath heating, then controlling the pH value to be 5-9, stirring and adding a ferric trichloride solution and a sodium hydroxide solution, stopping adding the ferric chloride solution and the sodium hydroxide solution after precipitation occurs, and stirring for 1-2 hours under the condition of water bath to obtain a suspension;
secondly, cooling the suspension obtained in the first step in an ice water bath, centrifuging to remove lower-layer precipitates, collecting supernatant, adding absolute ethyl alcohol into the supernatant, fully stirring, standing for complete precipitation, centrifuging, removing the supernatant, collecting precipitates, washing with an ethanol solution, absolute ethyl alcohol and absolute ethyl ether in sequence, and drying in vacuum to obtain a polysaccharide-iron complex crude product, wherein the volume fraction of the ethanol solution is 95%;
and thirdly, putting the crude polysaccharide-iron complex obtained in the second step into a dialysis bag, dialyzing with running water, then sequentially washing twice with an ethanol solution, absolute ethyl alcohol and absolute ethyl ether respectively, wherein the volume fraction of the ethanol solution is 95%, and drying in vacuum to obtain the ferrified astragalus polysaccharide.
In step one, Astragalus polysaccharides (98%, batch: C29J9Y53671) Zhejiang Union Shuo Biotech Co., Ltd.
The ferrified astragalus polysaccharide is used as a bioactive component for inhibiting human gastric cancer cells and is applied to preparing antitumor drugs.
The invention has the beneficial effects that:
the invention adopts a ferric trichloride synthesis method to carry out iron-ization on astragalus polysaccharide. The synthesis method is simple and convenient to operate and low in price.
Meanwhile, the invention obtains the ferriferous astragalus polysaccharide through a series of detection to be dark reddish brown powder, which is odorless, tasteless and easy to dissolve in water. The pH is neutral, and the product is insoluble in methanol, ethanol, and diethyl ether. The stability of the ferrified astragalus polysaccharide is good, and when the pH value is more than 14, the phenomenon of iron ion hydrolysis does not occur; and the average iron content of the detected ferrated astragalus polysaccharide is 2.66 percent.
The experimental result shows that the ferrated astragalus polysaccharide has obvious proliferation inhibition on gastric cancer SGC-7901 cells and is in a dose-dependent relationship. Half Inhibitory Concentration (IC) of ferrated Astragalus polysaccharides on gastric cancer SGC-7901 cells50) 18. mu.g/mL-1. Median Inhibitory Concentration (IC) of Positive drugs50) 3.5. mu.g/mL-1。
The prepared ferrated astragalus polysaccharide is used as a bioactive component for inhibiting human gastric cancer cells and is applied to preparing antitumor drugs.
Drawings
FIG. 1 is a UV spectrum of the ferrated Astragalus polysaccharide prepared in example one;
FIG. 2 is an infrared spectrum of the ferrified astragalus polysaccharide prepared in example one;
FIG. 3 is a gas chromatogram of the ferrified astragalus polysaccharide prepared in example one;
FIG. 4 shows the preparation of the ferriated astragalus polysaccharides of example one1H-NMR spectrum;
FIG. 5 shows a first embodimentPreparation of ferriferous Astragalus polysaccharides I2-KI spectrogram;
FIG. 6 is a plot of Congo red experimental data for the ferrated Astragalus polysaccharides prepared in example one, wherein ● represents Congo red and A-solidup represents the ferrated Astragalus polysaccharides;
FIG. 7 is a differential thermal scan of the ferrated Astragalus polysaccharide prepared in one example;
FIG. 8 is an X-ray diffraction pattern of the ferriated astragalus polysaccharides prepared in example one;
FIG. 9 is a scanning electron micrograph (500) of the ferrated Astragalus polysaccharide prepared in example one;
FIG. 10 is a scanning electron micrograph (1000) of the ferrated Astragalus polysaccharide prepared in example one;
FIG. 11 is a scanning electron micrograph (2000) of the ferrated Astragalus polysaccharide prepared in example one;
FIG. 12 is an electrophoresis chart of detection of the expression level of gastric cancer cell CDK1 by using ferrated astragalus polysaccharide;
FIG. 13 is a graph showing data analysis of the expression level of CDK1 in gastric cancer cells by using ferrated astragalus polysaccharides;
FIG. 14 is the electrophoresis chart of detecting the expression level of gastric cancer cell CyclinB1 by using the ferrified astragalus polysaccharide;
FIG. 15 is a graph showing the data analysis of the expression level of the astragalus polysaccharides ferrated in gastric cancer cell CyclinB 1;
FIG. 16 is an electrophoretogram of detecting the expression level of CDC25c in gastric cancer cells by using ferrated astragalus polysaccharide;
FIG. 17 is a graph showing the data analysis of the expression level of CDC25c in gastric cancer cells by using the ferrated astragalus polysaccharides;
FIG. 18 is an electrophoresis chart of detecting the expression level of FoxM1 in gastric cancer cells by using ferrated astragalus polysaccharides;
FIG. 19 is a graph of data analysis of FoxM1 expression level of gastric cancer cells by using ferriferous astragalus polysaccharides.
Detailed Description
The technical solution of the present invention is not limited to the specific embodiments listed below, and includes any combination of the specific embodiments.
The first embodiment is as follows: the structural formula of the ferriferous astragalus polysaccharide for inhibiting the human gastric cancer cells is shown as follows
The second embodiment is as follows: the preparation method of the ferrated astragalus polysaccharide for inhibiting the human gastric cancer cells comprises the following steps:
firstly, adding astragalus polysaccharide and trisodium citrate into single distilled water, controlling the temperature to be 50-80 ℃ to carry out water bath heating, then controlling the pH value to be 5-9, stirring and adding a ferric trichloride solution and a sodium hydroxide solution, stopping adding the ferric chloride solution and the sodium hydroxide solution after precipitation occurs, and stirring for 1-2 hours under the condition of water bath to obtain a suspension;
secondly, cooling the suspension obtained in the first step in an ice water bath, centrifuging to remove lower-layer precipitates, collecting supernatant, adding absolute ethyl alcohol into the supernatant, fully stirring, standing for complete precipitation, centrifuging, removing the supernatant, collecting precipitates, washing with an ethanol solution, absolute ethyl alcohol and absolute ethyl ether in sequence, and drying in vacuum to obtain a polysaccharide-iron complex crude product, wherein the volume fraction of the ethanol solution is 95%;
and thirdly, putting the crude polysaccharide-iron complex obtained in the second step into a dialysis bag, dialyzing with running water, then sequentially washing twice with an ethanol solution, absolute ethyl alcohol and absolute ethyl ether respectively, wherein the volume fraction of the ethanol solution is 95%, and drying in vacuum to obtain the ferrified astragalus polysaccharide.
The third concrete implementation mode: the second embodiment is different from the first embodiment in that: in the first step, the mass ratio of the astragalus polysaccharide to the trisodium citrate is 2: (0.1-0.8). The rest is the same as the second embodiment.
The fourth concrete implementation mode: the second or third embodiment is different from the first or second embodiment in that: in the first step, the mass-volume ratio of the astragalus polysaccharide to the single distilled water is 2 g: (59-62) mL. The other is the same as the second or third embodiment.
The fifth concrete implementation mode: this embodiment is different from one of the second to fourth embodiments in that: the concentration of the ferric trichloride solution in the step one is2mol·L-1The concentration of the sodium hydroxide solution is 2 mol.L-1. The other is the same as one of the second to fourth embodiments.
The sixth specific implementation mode: the present embodiment is different from one of the second to fifth embodiments in that: in the second step, the rotating speed is controlled to be 3500 r.min-1Centrifuging for 15min, removing the lower layer precipitate, and collecting the supernatant. The other is the same as one of the second to fifth embodiments.
The seventh embodiment: the present embodiment is different from one of the second to sixth embodiments in that: and when absolute ethyl alcohol is added into the supernatant in the second step, the volume ratio of the absolute ethyl alcohol to the supernatant is 3: 1. the other is the same as one of the second to sixth embodiments.
The specific implementation mode is eight: the present embodiment is different from one of the second to seventh embodiments in that: in the second step, the rotating speed is controlled to be 3500 r.min-1Centrifuging for 15min, removing supernatant, and collecting precipitate. The rest is the same as one of the second to seventh embodiments.
The specific implementation method nine: the present embodiment differs from the second to eighth embodiments in that: and in the third step, the mixture is dialyzed for over 72 hours by running water. The rest is the same as the second to eighth embodiments.
The specific embodiment is as follows: the embodiment of the invention relates to an application of the astragalus polysaccharide for inhibiting human gastric cancer cells as a bioactive component for inhibiting human gastric cancer cells in preparing antitumor drugs.
The following examples were used to demonstrate the beneficial effects of the present invention:
the first embodiment is as follows:
the embodiment of the invention relates to a preparation method of ferrated astragalus polysaccharide for inhibiting human gastric cancer cells, which comprises the following steps:
firstly, 2g of astragalus polysaccharide and 0.5g of trisodium citrate are added into a round-bottom flask filled with 60mL of single distilled water, the temperature is controlled at 70 ℃ for heating in water bath, then the pH value is controlled at 8.0, and the mixture is stirred and dropwise added into the mixture with the concentration of 2 mol.L-1Ferric trichloride solution and sodium hydroxide solution with the mass concentration of 20 percent are added after precipitation occurs, and the ferric chloride solution and the sodium hydroxide solution are stoppedStirring the sodium solution for 1h under the condition of keeping a water bath to obtain a suspension;
secondly, the suspension obtained in the step one is put into an ice water bath for cooling, and the rotating speed is controlled to be 3500 r.min-1Centrifuging for 15min, removing lower layer precipitate, collecting supernatant, adding anhydrous ethanol with three times volume into supernatant, stirring, standing for precipitation, and controlling rotation speed at 3500 r.min-1Centrifuging for 15min, removing supernatant, collecting precipitate, sequentially washing with ethanol solution, anhydrous ethanol and anhydrous ether, discarding washing solution, and vacuum drying to obtain polysaccharide-iron complex crude product, wherein the volume fraction of ethanol solution is 95%;
and thirdly, putting the crude polysaccharide-iron complex obtained in the step two into a dialysis bag, dialyzing with running water for over 72 hours, then sequentially washing with an ethanol solution, absolute ethyl alcohol and absolute ethyl ether twice respectively, wherein the volume fraction of the ethanol solution is 95%, and drying in vacuum to obtain the ferrated astragalus polysaccharide.
The structural formula of the ferrified astragalus polysaccharide obtained in the example is
The ferriated astragalus polysaccharides form intermolecular hydrogen bonding links.
The molecular weight test result shows that the molecular weight of the ferriferous astragalus polysaccharide is 6.04 multiplied by 104。
By adopting a particle size detector, the average particle size of the ferrified astragalus polysaccharide can be measured to be 172.8 nm. The grain size of the astragalus polysaccharide after the iron treatment is obviously reduced compared with the astragalus polysaccharide. This is because astragalus polysaccharides are hydrolyzed during the process of ferration, which results in a reduction in the particle size of the ferrated polysaccharides. Meanwhile, the ferrated astragalus polysaccharide is easier to penetrate cells than astragalus polysaccharide and is easy to be absorbed and utilized by organisms.
The average iron content of the detected ferrified astragalus polysaccharide is 2.66 percent.
Through the stability test of the phenanthroline iron solution, the stability of the ferriated astragalus polysaccharide is good, the phenanthroline iron solution is stable, and the absorbance is basically unchanged within 3 hours.
The physical and chemical properties of the ferrified astragalus polysaccharide are detected, and the ferrified astragalus polysaccharide is dark reddish brown powder, is odorless and tasteless and is easy to dissolve in water. The pH is neutral, and the product is insoluble in methanol, ethanol, and diethyl ether. When the stability of the ferrified astragalus polysaccharide is good and the pH value is more than 14, the phenomenon of iron ion hydrolysis does not occur; meanwhile, the results of phenol-sulfuric acid reaction, Molish reaction and Fehling reagent reaction prove that the ferrified astragalus polysaccharide is a saccharide substance; the ninhydrin reaction does not appear blue, and the biuret reaction does not appear red, which proves that the ferrated astragalus polysaccharide does not contain amino acid, protein and other substances; the ferric trichloride does not react, and the blackish green color is formed, which indicates that no phenols or tannin substances exist in the iron-treated astragalus polysaccharide; i is2No color change appears in the KI reaction, and the fact that the ferrated astragalus polysaccharide does not contain starch substances is proved.
FIG. 1 is a UV spectrum of the Astragalus polysaccharides with iron content prepared in example I, wherein it can be seen that Astragalus polysaccharides with iron content has a characteristic polysaccharide peak at 201nm, which indicates that Astragalus polysaccharides with iron content is a polysaccharide substance; there was no absorption at both 260nm and 280nm, indicating the absence of nucleic acids and proteins in the ferrated astragalus polysaccharides.
FIG. 2 is an infrared spectrum of the astragalus polysaccharides prepared in the first example, wherein the infrared spectrum of the astragalus polysaccharides is 3399.83cm-1Has a strong broad absorption peak, which is mainly caused by-OH stretching vibration in the ferrated astragalus polysaccharide; 2926.4cm-1And 2857.3cm-1Two peaks are-CH2Absorption peak of stretching vibration; the absorption peak of the C-H bending vibration is 1389.63cm-1The three peaks are characteristic peaks of polysaccharide, and can prove that the ferrified astragalus polysaccharide is a polysaccharide substance.
1604.60cm-1And 1256.36cm-1Two absorption peaks, caused by-COOH stretching vibrations; 1066.26cm-1The absorption peak is caused by the stretching vibration of C-O-C on the pyran ring; 909.67cm-1The absorption peak shows that the ferrified astragalus polysaccharide is in a beta configuration; in conclusion, the iron-treated astragalus polysaccharide is the beta-pyranose.
FIG. 3 is a gas chromatogram of the astragalus polysaccharides with iron being prepared in example one, which shows that the monosaccharides constituting the astragalus polysaccharides with iron are glucose and sorbose. The reduction of monosaccharide types may be that the structure of astragalus polysaccharide is changed after the astragalus polysaccharide is subjected to iron treatment, and in the iron treatment process, O-H bonds of the polysaccharide are broken to form a compound with Fe, so that the original structure of the astragalus polysaccharide is changed.
FIG. 4 shows the preparation of the ferriated astragalus polysaccharides of example one1H-NMR spectrum, according to spectrum analysis: the shift value of the proton peak of the ferrated astragalus polysaccharide is mostly less than 5, and the chemical shift is more than 4.95 and is alpha-configuration sugar residue, and the chemical shift is less than 4.95 and is beta-configuration sugar residue. Therefore, it can be presumed that the ferriated astragalus polysaccharides have the β configuration. Suggesting that the ferrified astragalus polysaccharide is pyranose with beta-type glycosidic bonds. However, because polysaccharides are macromolecular compounds and their structures are very complex, the information given by hydrogen spectroscopy is not very sufficient.
FIG. 5 shows the preparation of example I of the ferrified Astragalus polysaccharides2KI spectrogram, as can be seen, the ferrated astragalus polysaccharide and I2After the KI solution is mixed, the maximum absorption peak does not exist at 565nm, which indicates that the sidechain and the branched chain exist in the ferrated astragalus polysaccharide.
FIG. 6 is a graph of Congo red experimental data of the iron-treated Astragalus polysaccharides prepared in example one, wherein ● represents Congo red, and A-solidup represents iron-treated Astragalus polysaccharides, and it can be seen from the graph that the maximum absorption wavelength of the complex of iron-treated Astragalus polysaccharides and Congo red is 0-0.05 mol.L NaOH concentration-1When the concentration of NaOH reaches 0.05 mol.L, the concentration is obviously improved-1The maximum absorption wavelength is maximized. When the concentration of NaOH is between 0.1 and 0.25 mol.L-1Within the range, the maximum absorption wavelength of the ferriferous astragalus polysaccharide and Congo red complex is relatively stable, and the concentration of NaOH is more than 0.25 mol.L-1Then, the maximum absorption wavelength of the ferrified astragalus polysaccharide and congo red complex is rapidly reduced, which indicates that the ferrified astragalus polysaccharide has a triple helix structure.
FIG. 7 is a differential thermal scanning chart of the prepared Astragalus polysaccharides having an endothermic peak in the range of 60-115 deg.C, and it can be seen that the decomposition occurs at 82 deg.C, which shows that the Astragalus polysaccharides having an endothermic peak in the range of 60-115 deg.C has a phase change after absorbing enough heat. The graph can show that the ferriferous astragalus polysaccharide has an endothermic peak in DSC, and the temperature of the strongest endotherm is 82 ℃, which is the result of strong endotherm generated when the ferriferous astragalus polysaccharide needs to absorb heat to the outside during thermal decomposition.
FIG. 8 is an X-ray diffraction pattern of the ferrated Astragalus polysaccharides prepared in example one, which shows that the ferrated Astragalus polysaccharides have no distinct peak in the 2 θ range shown in the figure, have very small peaks only around 30 °, indicating that only very small amount of crystals are present, and no diffraction peaks in other ranges, indicating that the main form of the ferrated Astragalus polysaccharides is amorphous and less crystalline. It can be seen that the incorporation of Fe causes the formation of very small crystals of the ferrated Astragalus polysaccharides, but the bulk morphology of the ferrated Astragalus polysaccharides is substantially identical to APS and is amorphous, since the amount of Fe introduced is not sufficient to change the overall morphology of the polysaccharides. This reflects the poor molecular regularity and the very complex structural composition of polysaccharides.
FIG. 9 is a scanning electron micrograph (magnification 500) of the astragalus polysaccharides obtained in the first example, FIG. 10 is a scanning electron micrograph (magnification 1000) of the astragalus polysaccharides obtained in the first example, and FIG. 11 is a scanning electron micrograph (magnification 2000) of the astragalus polysaccharides obtained in the first example, and it can be seen that the astragalus polysaccharides have a structure mainly in the form of a sheet, a rod or a rod.
The proliferation inhibition effect of the ferrified astragalus polysaccharide on SGC-7901 cells is as follows: the method takes human gastric cancer cell SGC-7901 as a research object, and adopts an MTT method to detect the proliferation inhibition effect of the ferriferous astragalus polysaccharide on the human gastric cancer cell SGC-7901.
Subject: human gastric carcinoma cell SGC-7901, supplied by Harbin university of commerce, college of medicine.
The specific operation is as follows:
cell passage of gastric cancer cells in logarithmic phase of growth in a clean bench, preparing cell suspension after trypsinization, sucking a small amount of cell suspension, dripping the cell suspension on a cell counting plate, counting with a microscope, and regulating the cell number to 4 × 104each.mL-1. The single cell suspension was inoculated into a 96-well plate, and PBS buffer was added to the outermost round of the 96-well plate, after overnight in an incubator. Taken out the next dayRespectively adding 128, 64, 32, 16, 8 μ g/mL-1The ferrated astragalus polysaccharide has 5 parallel holes in each concentration, the blank control group is added with no ferrated astragalus polysaccharide but with culture medium of the same volume, and the positive control group is added with culture medium of the same volume of 3.5 mu g/mL-1HCPT of (5%) at 37 ℃ and CO of 5%2And continuing culturing in the incubator. Taking out after 72h, and adding prepared 0.5 mg/mL into each hole-1MTT 100. mu.L, 5% CO at 37 ℃2And continuing culturing in the incubator. After 4 hours, remove, aspirate MTT from the well and add 150. mu.L of DMSO solution per well. After shaking for ten minutes, the optical density (OD value) was measured by a microplate reader, and the measurement wavelength was 570 nm. Calculating the inhibition rate of the iron astragalus polysaccharide on gastric cancer SGC-7901 cells, and calculating the median Inhibitory Concentration (IC) by using SPSS software50)。
And SPSS software is adopted for data processing. The results of each group are expressed in (x +/-s), the multi-sample mean comparison adopts one-factor variance analysis, and P <0.05 has statistical significance.
Percent cytostatic rate (average OD value of blank control group-average OD value of administration group) × 100%/average OD value of blank control group
The MTT test result shows that 128 mug. multidot.mL-1、64μg·mL-1、32μg·mL-1、16μg·mL-1、8μg·mL-1The growth inhibition rate of the ferrated astragalus polysaccharide solution on gastric cancer SGC-7901 cells for 72h is shown in the following table 1. Half inhibition rate (IC) of stomach cancer SGC-7901 cell by ferriferous astragalus polysaccharide50) 18. mu.g/mL-1。
TABLE 1 Effect of ferriferous Astragalus polysaccharides on SGC-7901 cell proliferation ((
n＝5)
Comparison with blank control: p <0.05, P <0.01
MTT experimental results show that the ferrified astragalus polysaccharide has effect on gastric cancer SGC-7901 cellsSignificantly inhibited proliferation and was dose dependent. The positive control group hydroxycamptothecin has proliferation inhibiting effect on gastric cancer SGC-7901 cells. Half Inhibitory Concentration (IC) of ferrated Astragalus polysaccharides on gastric cancer SGC-7901 cells50) 18. mu.g/mL-1. Median Inhibitory Concentration (IC) of Positive drugs50) 3.5. mu.g/mL-1。
The cells in the six-hole plate are observed under an inverted microscope, and the cells in the negative control group are clearly seen to be full in shape, uniform in distribution and basically grow adherent.
The gastric cancer SGC7901 cell subjected to the dry prognosis of the ferrated astragalus polysaccharide is detected by a flow cytometer to find an obvious apoptosis peak, and the result shows that the synthesis of DNA is inhibited. After 72 hours, the apoptosis rates of the three groups of astragalus polysaccharide, namely the low, medium and high doses, are respectively (7.501 +/-0.217)%, (10.296 +/-0.178)%, (15.207 +/-0.355)% (shown in table 2) through parallel measurement of the three groups, and analysis shows that the apoptosis rate is in a rising trend along with the increase of the administration dose of the ferrated astragalus polysaccharide when the measured apoptosis peak is higher, and the apoptosis rates are all significantly different (P is less than 0.01). The apoptosis rate of the positive control group is measured to be (4.906 +/-0.168)%, and the positive control group has significant difference (P <0.01) compared with the blank control group.
TABLE 2 apoptosis rate of ferrated astragalus polysaccharides on gastric cancer cells (x + -s, n ═ 3)
Comparison with blank control: p <0.05, P <0.01
FIG. 12 is an electrophoresis chart of detection of the expression level of gastric cancer cell CDK1 by using ferrated astragalus polysaccharide; FIG. 13 is a graph showing data analysis of the expression level of CDK1 in gastric cancer cells by using ferrated astragalus polysaccharides; FIG. 14 is the electrophoresis chart of detecting the expression level of gastric cancer cell CyclinB1 by using the ferrified astragalus polysaccharide; FIG. 15 is a graph showing the data analysis of the expression level of the astragalus polysaccharides ferrated in gastric cancer cell CyclinB 1; FIG. 16 is an electrophoretogram of detecting the expression level of CDC25c in gastric cancer cells by using ferrated astragalus polysaccharide; FIG. 17 is a graph showing the data analysis of the expression level of CDC25c in gastric cancer cells by using the ferrated astragalus polysaccharides; FIG. 18 is an electrophoresis chart of detecting the expression level of FoxM1 in gastric cancer cells by using ferrated astragalus polysaccharides; FIG. 19 is a graph showing the data analysis of FoxM1 expression level of gastric cancer cells by using the iron-treated Astragalus polysaccharides; the expression level of the protein of the ferrated astragalus polysaccharide on gastric cancer cells is shown above. After a protein immunoblotting technology Western Blot method is adopted and analyzed by a gel imaging system, after gastric cancer SGC-7901 cells acted by the ferrified astragalus polysaccharides are obtained, the protein expression levels of CDK1, CyclinB1, CDC25c and p21 in the cells are obviously influenced, the protein expression levels of CDK1, CyclinB1 and CDC25c are obviously reduced after the ferrified astragalus polysaccharides act on the gastric cancer SGC-7901 cells for 48 hours, the protein expression level of p21 is obviously increased, on one hand, the formation of CDK1/CyclinB1 complexes is inhibited, on the other hand, the activity of the CDK1/CyclinB1 complexes is reduced by reducing the expression of CDC25c and increasing the expression of p21, and the cells are prevented from entering an M phase, so that the cycle blocking effect is achieved on the gastric cancer SGC-7901 cells.
Claims (10)
1. A ferrified astragalus polysaccharide for inhibiting human gastric cancer cells is characterized in that the structural formula of the ferrified astragalus polysaccharide is shown in the specification
2. A method for preparing ferrated astragalus polysaccharide for inhibiting human gastric cancer cells is characterized by comprising the following steps:
firstly, adding astragalus polysaccharide and trisodium citrate into single distilled water, controlling the temperature to be 50-80 ℃ to carry out water bath heating, then controlling the pH value to be 5-9, stirring and adding a ferric trichloride solution and a sodium hydroxide solution, stopping adding the ferric chloride solution and the sodium hydroxide solution after precipitation occurs, and stirring for 1-2 hours under the condition of water bath to obtain a suspension;
secondly, cooling the suspension obtained in the first step in an ice water bath, centrifuging to remove lower-layer precipitates, collecting supernatant, adding absolute ethyl alcohol into the supernatant, fully stirring, standing for complete precipitation, centrifuging, removing the supernatant, collecting precipitates, washing with an ethanol solution, absolute ethyl alcohol and absolute ethyl ether in sequence, and drying in vacuum to obtain a polysaccharide-iron complex crude product, wherein the volume fraction of the ethanol solution is 95%;
and thirdly, putting the crude polysaccharide-iron complex obtained in the second step into a dialysis bag, dialyzing with running water, then sequentially washing twice with an ethanol solution, absolute ethyl alcohol and absolute ethyl ether respectively, wherein the volume fraction of the ethanol solution is 95%, and drying in vacuum to obtain the ferrified astragalus polysaccharide.
3. The method for preparing the astragalus polysaccharide ferrated for inhibiting the human gastric cancer cells as claimed in claim 2, wherein the mass ratio of the astragalus polysaccharide to the trisodium citrate in the step one is 2: (0.1-0.8).
4. The method for preparing the astragalus polysaccharide ferruginous to inhibit human gastric cancer cells according to claim 2, wherein the mass-to-volume ratio of the astragalus polysaccharide to the single distilled water in the first step is 2 g: (59-62) mL.
5. The method according to claim 2, wherein the concentration of ferric chloride solution in step one is 2 mol.L-1The concentration of the sodium hydroxide solution is 2 mol.L-1。
6. The method for preparing the astragalus polysaccharide with iron content for inhibiting human gastric cancer cells as claimed in claim 2, wherein the rotation speed is controlled to 3500 r-min in the second step-1Centrifuging for 15min, removing the lower layer precipitate, and collecting the supernatant.
7. The method according to claim 2, wherein when absolute ethanol is added to the supernatant in the second step, the volume ratio of absolute ethanol to supernatant is 3: 1.
8. the astragalus membranaceus with iron property for inhibiting human gastric cancer cells according to claim 2The preparation method of the polysaccharide is characterized in that the rotating speed is controlled to 3500 r.min in the second step-1Centrifuging for 15min, removing supernatant, and collecting precipitate.
9. The method for preparing the ferrated astragalus polysaccharide for inhibiting human gastric cancer cells according to claim 2, wherein the dialysis is performed in running water for more than 72 hours in the third step.
10. The use of the astragalus polysaccharide for inhibiting human gastric cancer cells as claimed in claim 1, wherein the astragalus polysaccharide is used as a bioactive component for inhibiting human gastric cancer cells in the preparation of antitumor drugs.
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| CN105924535A (en) * | 2016-04-27 | 2016-09-07 | 广西农业职业技术学院 | Preparation method of Astragalus polysaccharide-iron |
| CN107714720A (en) * | 2017-10-30 | 2018-02-23 | 兰州大学 | A kind of preparation method for the Radix Angelicae Sinensis astragalus polyose iron complexes for treating hypoferric anemia |
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2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105924535A (en) * | 2016-04-27 | 2016-09-07 | 广西农业职业技术学院 | Preparation method of Astragalus polysaccharide-iron |
| CN107714720A (en) * | 2017-10-30 | 2018-02-23 | 兰州大学 | A kind of preparation method for the Radix Angelicae Sinensis astragalus polyose iron complexes for treating hypoferric anemia |
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| LI RUI等: "Optimization of extraction technology of Astragalus polysaccharides by response surface methodology and its effect on CD40", 《CARBOHYDRATE POLYMERS》 * |
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