CN109265575B - 4O-methylglucuronic acid xylan obtained from Artemisia desertorum seed gum and its application in inhibiting liver tumor - Google Patents
4O-methylglucuronic acid xylan obtained from Artemisia desertorum seed gum and its application in inhibiting liver tumor Download PDFInfo
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- CN109265575B CN109265575B CN201811020812.7A CN201811020812A CN109265575B CN 109265575 B CN109265575 B CN 109265575B CN 201811020812 A CN201811020812 A CN 201811020812A CN 109265575 B CN109265575 B CN 109265575B
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- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- 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
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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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
-
- 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
Abstract
The invention obtains a sand sagebrush seed gum polysaccharide with good water solubility by processing the sand sagebrush seed gum (ASKG) with poor solubility, and confirms that the sand sagebrush seed gum polysaccharide is 4-oxymethyl glucuronic acid xylan: the main chain is xylose structural unit (1 → 4) -beta-Xylf, and the 2-position of every two xylose structural units (1 → 4) -beta-Xylf is connected with the end of 4-O-Me-a-GlcAp through 1, 2-O-glycosidic bond. Through intensive research, the artemisia desertorum seed gum polysaccharide can induce the apoptosis of HepG2 human liver cancer cells, so that the application of the artemisia desertorum seed gum polysaccharide in the aspect of inhibiting liver tumors is determined.
Description
Technical Field
The invention relates to extraction, separation and degradation of artemisia desertorum seed gum polysaccharide and application thereof.
Background
Artemisia desertorum, semi-shrub-like plant, is mostly distributed in northwest arid desert area, researches show that Artemisia desertorum seed has many edible nutrient substances such as Artemisia desertorum seed oil, Artemisia desertorum seed protein and Artemisia desertorum seed gum, etc., and has been widely used in food additive and feed industry, etc[1,2]. The polysaccharide is one of the important components of Artemisia desertorum seed, and is divided into water-soluble Artemisia desertorum seed polysaccharide and water-insoluble Artemisia desertorum seed gum.
The research on water-soluble Artemisia desertorum seed polysaccharide is extensive, and the product is beautiful[3]Extracting and purifying to obtain glucomannan component, analyzing its structure, and making Qingbin Guo[4]The xylan component is obtained by an ammonium persulfate salt precipitation method and subjected to structure analysis. Researches show that the water-soluble artemisia seed polysaccharide has the functions of immunoregulation, antioxidation, regulation of glycolipid metabolism and reduction of cancer cell activityMultiple biological activities[5,6]。
The artemisia seed gum (ASKG) is waxy colloid polysaccharide attached to the surface of artemisia seed, has large molecular weight, high water retaining property, high thickening property, high stability and other features, and may be used widely in food, medicine and other industry[7]. In addition, the artemisia seed gum as a natural plant gum polysaccharide has various biological activities peculiar to cellulose substances, such as blood sugar and blood fat reduction, cardiovascular and cerebrovascular disease prevention and the like. As a result, the Chinese medicine has already used the sand sagebrush seed gum as medicine for curing diabetes, Zhang, etc[8]Researches on the blood sugar reducing effect and mechanism of artemisia desertorum seed gum polysaccharide prove that the artemisia desertorum seed gum can obviously reduce the fasting blood sugar content of type II diabetic rats, increase the glucokinase activity in livers and promote the conversion of hepatic glycogen. The artemisia seed glue can also obviously increase the activity of SOD enzyme in the liver and serum of a diabetic rat, and remove active oxygen free radicals in the liver by improving the activity of antioxidant enzyme of an organism, so that the liver and islet cells are protected, the secretion of insulin and the synthesis of glycogen of the liver are promoted, and the effect of regulating blood sugar is achieved.
However, the sand sagebrush seed gum has poor solubility and strong intermolecular force, so that certain difficulty is caused in the activity exertion, the current research mostly focuses on the extraction preparation and direct application, and relatively few reports of structure-activity relationship exist. The method for improving the solubility of artemisia seed gum polysaccharide through carboxymethylation or sulfation modification is researched[9]These methods can destroy the original structure of the polysaccharide or modify the original structure, introduce foreign groups, have certain influence on the structural characterization of the polysaccharide, and the exertion of the activity of the polysaccharide cannot determine whether the polysaccharide is generated by the polysaccharide per se. In addition, the solubility of the artemisia desertorum seed gum is improved by alkaline treatment, but the obtained water-soluble artemisia desertorum seed gum polysaccharide has a high molecular weight (more than 500kD) and is difficult to enter cells to exert biological activity, so that the activity is not considered to be studied.
Reference documents:
[1] china academy of sciences plant institute, China higher plant atlas first volume [ J ].1990
[2] The excellent characteristics and development and utilization of Zhaoyang-black sand sagebrush (J) in Chinese forestry, 2011, (17) 45-47
[3] Study on properties, structure and immunological activity of Hodgy Artemisia ordosifolia seed gum polysaccharide [ D ]; northwest university, 2009
[4]Guo Q.Physicochemical,Structural and Conformational Properties ofHeteropolysaccharides from Seeds of Artemisia Sphaerocephala Krasch[J].Polysaccharides Structure Conformation Artemisia Sphaerocephala Krasch,2013,(10):40-42
[5] Research on in vitro hypoglycemic activity of Royal Arisaema pallidum polysaccharide and derivatives thereof [ D ]; gansu agricultural university, 2011
[6]Ren D,Lin D,Alim A,et al.Chemical characterization of a novelpolysaccharide ASKP-1 from Artemisia sphaerocephala Krasch seed and itsmacrophage activation via MAPK,PI3k/Akt and NF-κB signaling pathways inRAW264.7cells[J].Food&Function,2017,8(3):1299
[7] Research on in vitro hypoglycemic activity of Royal Arisaema pallidum polysaccharide and derivatives thereof [ D ]; gansu agricultural university, 2011
[8]Zhang,J.,Huang,Y.,Hou,T.,&Wang,Y.(2006).Hypoglycaemic effect ofArtemisia sphaerocephala Krasch seed polysaccharide in alloxan-induceddiabetic rats.Swiss Medical Weekly,136(33-34),529
[9]Wang,J.,Wang,Y.,Xu,L.,Wu,Q.,Wang,Q.,Kong,W.,Liang,J.,Yao,J.,&Zhang,J.(2017).Synthesis and structural features of phosphorylated Artemisiasphaerocephala polysaccharide.Carbohydrate Polymers,19-26.
Disclosure of Invention
The invention obtains artemisia desertorum seed gum polysaccharide (AGP-III-C) with good water solubility by processing artemisia desertorum seed gum (ASKG) with poor solubility, confirms that the artemisia desertorum seed gum polysaccharide is 4-oxymethyl glucuronate xylan, and further researches prove that the artemisia desertorum seed gum polysaccharide (AGP-III-C) can induce the apoptosis of HepG2 human liver cancer cells, thereby determining the application of the artemisia desertorum seed gum polysaccharide in the aspect of inhibiting liver tumors.
The concrete results are as follows:
the artemisia seed gum polysaccharide is characterized in that xylose structural units (1 → 4) -beta-Xylf are taken as a main chain, and 2 sites of every two xylose structural units (1 → 4) -beta-Xylf are connected with the tail end of 4-O-Me-a-GlcAp through 1, 2-O-glycosidic bonds, and the method comprises the following steps:
the Artemisia desertorum seed gum polysaccharide preferably has a molecular weight of 53kDa or its approximate value (e.g., 52 kDa-54 kDa).
The application of the artemisia desertorum seed gum polysaccharide in preparing the medicine for inhibiting the liver tumor can induce the apoptosis of HepG2 human liver cancer cells. In particular to the types of medicine which can directly reach the focus, such as injection, controlled release preparation (controlled release tablet, controlled release capsule, etc.), inhalant, etc.
A pharmaceutical composition, which comprises the artemisia desertorum seed gum polysaccharide and a pharmaceutically acceptable carrier.
Food comprising the above artemisia desertorum seed gum polysaccharide. For example: dairy products, candies, beverages, and the like.
The invention has the following beneficial effects:
the invention can be degraded by weak alkali treatment, separation and purification and oxidation (H) without destroying or changing the polysaccharide structure of the artemisia desertorum seed gum2O2-Vc oxidative degradation system), and deeply discussing the activity of the obtained small molecular weight component, and determining that the small molecular weight 4 oxymethyl glucuronate xylan component AGP-III-C can induce HepG2 human liver cancer cell apoptosis.
Drawings
FIG. 1 is a flow chart of the extraction, separation, purification and degradation of Artemisia desertorum seed gum.
FIG. 2 shows the elution pattern of AGP through a DEAE-cellulose column.
FIG. 3 is a graph showing the effect of Vc concentration on the molecular weight of an AGP-III degradation component.
FIG. 4 sugar content and uronic acid content of fractions before and after degradation.
FIG. 5 is a gas chromatogram of AGP-III and the degraded components AGP-III-A to AGP-III-D. Monosaccharide standards: 1. rhamnose (Rha)2. fucose (Fuc)3. arabinose (Ara)4. xylose (Xyl)5. mannose (Man)6. glucose (Glc)7. galactose (Gal)8. glucuronic acid (GlcA)9. galacturonic acid (GalA) a.4-O-methyl-glucuronic acid.
FIG. 6 Effect of AGP-III and degradation components on HepG2 cell viability. The result of the A experiment is expressed as the average value plus or minus standard deviation (n is more than or equal to 6); p <0.05, P <0.01, and B plots are two-by-two comparisons of experimental results, with different letters indicating a significant difference in P < 0.05.
FIG. 7 is a statistical chart showing the ratio of cycles of AGP-III-C treated HepG2 cells by flow cytometry. Experimental results are expressed as mean ± standard deviation (n ═ 3); p <0.05, p <0.01, compared to control.
FIG. 8 the effect of AGP-III-C on HepG2 cell morphology.
FIG. 9 Effect of AGP-III-C on mitochondrial membrane potential and ROS levels in HepG2 cells. HepG2 cells were treated with different concentrations of AGP-III-C (0, 1, 5, 25, 50, 100. mu.g/mL) and 5-FU (20. mu.g/mL) for 24h, stained with JC-1 and ROS, and visualized with a fluorescence microscope (. times.200). Qualitatively detecting JC-1 by using the multifunctional microplate reader (figure 9A) and qualitatively detecting ROS (figure 9B), wherein the experimental results are expressed as the average value +/-standard deviation (n is 3); p <0.05, p <0.01, compared to control.
FIG. 10 Effect of AGP-III-C on the MAPK signaling pathway in HepG2 cells. After the HepG2 cells are treated for 24h by different concentrations of AGP-III-C (0, 1, 5, 25, 50, 100 mu g/mL) and 5-FU (20 mu g/mL), the expression level of related proteins in the MAPK signal path is increased. Experimental results are expressed as mean ± standard deviation (n ═ 5); p <0.05, p <0.01, compared to control.
FIG. 11 is a GC-MS spectrum and mass spectrum fragment peaks of AGP-III-C and AGP-III-C-R.
FIG. 12 of AGP-III-C1H NMRAnd13c NMR chart. In the figure, A is 4-O-Me-alpha-GlcA, B is (1 → 4) -beta-Xyl; c (1 → 4) -beta-Xyl-2-O- (4-O-Me-alpha-GlcA.
FIG. 13 COSY map of AGP-III-C. A. B, C represent three structural units of AGP-III-C, respectively, wherein A:4-O-Me- α -GlcA; b: (1 → 4) - β -Xyl; c: (1 → 4) - β -Xyl-2-O- (4-O-Me-. alpha. -GlcA).
FIG. 14 AGP-III-C TCOSY map. A. B, C represent three structural units of AGP-III-C, respectively, wherein A:4-O-Me- α -GlcA; b: (1 → 4) - β -Xyl; c: (1 → 4) - β -Xyl-2-O- (4-O-Me-. alpha. -GlcA).
FIG. 15 HSQC map of AGP-III-C. A. B, C represent three structural units of AGP-III-C respectively, wherein, A:4-O-Me- α -GlcA; b: (1 → 4) - β -Xyl; c: (1 → 4) - β -Xyl-2-O- (4-O-Me-. alpha. -GlcA).
FIG. 16A TNNOESY map of AGP-III-C.
FIG. 17 HMBC map of AGP-III-C.
FIG. 18 depicts the structure of AGP-III-C.
Detailed Description
The invention treats sand sagebrush seed gum with weak base, separates and purifies the sand sagebrush seed gum, and then adopts H2O2The Vc oxidation degradation system degrades one of the separated and purified components AGP-III, and changes the concentration of Vc to obtain artemisia seed gum polysaccharide AGP-III-A, AGP-III-B, AGP-III-C, AGP-III-D with four small molecular weight sections; and the influence of artemisia desertorum seed gum polysaccharide on the apoptosis of human liver cancer cells HepG2 is deeply researched by measuring cell viability experiments, cell proliferation capacity, cell cycle, apoptosis state observation, mitochondrial membrane function change and related protein expression in MAPKs (important signal pathways) in apoptosis pathways.
As shown in fig. 1, the process of extracting, separating, purifying and degrading artemisia desertorum seed gum in this embodiment is as follows:
(1) extracting artemisia desertorum seed gum: weighing a certain amount of artemisia desertorum seeds, soaking in water with the volume 40 times of the artemisia desertorum seeds for 1 hour at room temperature, and extracting for 5 hours at the constant temperature of 80 ℃. Cooling to room temperature after reaction, crushing and centrifuging the adhesive gum block with a tissue triturator, separating out a semitransparent gelatinous substance, and discarding the supernatant water-soluble artemisia seed polysaccharide part and the artemisia seed shell residue. Freeze drying the semi-transparent colloidal material, namely artemisia desertorum seed gum ASKG;
(2) preparing artemisia desertorum seed gum polysaccharide: accurately weighing the sample ASKG obtained in the step (1), preparing into colloidal suspension with the material-liquid ratio of 1%, stirring for 1h at room temperature until the mixture is completely mixed, and adding 1mol/L NaOH (containing 0.1mol/L NaBH)4) Heating at 70 deg.C and magnetic stirring, extracting for 4 hr, adjusting pH of the extract to 7 with acetic acid, 10000r/min, centrifuging for 10min to remove insoluble substances, concentrating the supernatant, dialyzing, and freeze drying to obtain Artemisia desertorum seed gum polysaccharide ASKGP with good solubility, AGP for short;
(3) separating and purifying artemisia desertorum seed gum polysaccharide: using DEAE-cellulose column (25 cm. times.5 cm, HCO)3-Model) the AGP obtained in the step (2) is separated and purified, after the column is activated, 1g of AGP is weighed and added into 20mL of distilled water, and after the mixture is fully and uniformly mixed, the mixture is centrifuged to take the supernatant, and the supernatant is uniformly loaded. Distilled water, 0.2mol/L and 0.5mol/L NaHCO are used in sequence3Eluting the solution, detecting protein absorption A280 and sugar absorption A490 with separate tubes (phenol-sulfuric acid method), making elution curves, collecting single peak to obtain four components, concentrating, dialyzing, and freeze-drying to obtain polysaccharide samples with AGP-I, AGP-II, AGP-III, and AGP-IV (figure 2) yields of 0.10%, 0.034%, 0.27%, and 0.17%, respectively, wherein the AGP-III yield is the highest;
(4) degradation of AGP-III: since AGP-III has molecular weight of more than 500kDa and hardly enters cells to exert biological activity, H is used2O2the-Vc system degrades the component AGP-III. To obtain a low molecular weight polysaccharide fraction, suitable degradation concentrations were first investigated according to the conditions shown in Table 1. The specific process is as follows: accurately weighing 6 portions of quantitative AGP-III, preparing suspensions with the material-liquid ratio of 0.5%, placing the suspensions in No. 1-6 vials with stoppers, and maintaining the conditions shown in Table 12O2The concentration of Vc was changed at 5mmol/L (0.5mmol/L, 1.5mmol/L, 3mmol/L, 5mmol/L, 7mmol/L, 10 mmol/L).
TABLE 1 selection of degradation conditions
Sealing and reacting in a water bath kettle at 40 ℃ for 2 h. Terminating the reaction after 2H, concentrating the degraded product by rotary evaporator, and dialyzing with dialysis bag with molecular cut-off of 3500 at 4 deg.C for three days to remove H2O2And VC. And finally, respectively collecting degradation samples through freeze drying. The molecular weight of the degraded sample is then determined. The chromatographic columns are TSK-gel G4000 PWXL and TSK-gel G3000 PWXL, and H is performed at 40 deg.C while the polysaccharide concentration is kept at 0.5%2O2Has a concentration of 5mmol constant, VCThe degradation results are shown in FIG. 3, when the concentration of (B) is varied from 0.5mmol/L to 10 mmol/L. Found when H2O2The concentration is changed at a certain time VCThe molecular weight of AGP-III is obviously changed when C isH2O2/CVCWhen the molecular weight is more than or equal to 1, the molecular weight follows VCThe concentration is increased and gradually reduced, and the permeability of the solution is gradually increased; when C is presentH2O2/CVCWhen the content is 1, the degradation degree reaches the maximum; with VCThe degradation degree is reduced instead by the gradual increase of the concentration. The excess Vc at this time suppresses the reaction. Selecting four components AGP-III-A with molecular weight sections: 358.9kDa, AGP-III-B: 106.7kDa, AGP-III-C: 53.1kDa, AGP-III-D: a large number of preparations and subsequent studies were carried out at 23.6 kDa.
(5) The total sugar content of AGP-III and degradation components AGP-III-A, AGP-III-B, AGP-III-C, AGP-III-D is measured by adopting a phenol-sulfuric acid method. The total sugar content was 78.8%, 63.2%, 73.9%, 68.5%, 75.1%, respectively. The uronic acid content of the five components of AGP-III and AGP-III-A, AGP-III-B, AGP-III-C, AGP-III-D is determined by a carbazole-sulfuric acid method. The uronic acid content was 21.2%, 22.3%, 26.2%, 39.3%, 26.5%, respectively. Wherein the component AGP-III-C uronic acid content is significantly higher than the other components (FIG. 4). Monosaccharide composition analysis shows that AGP-III and four components AGP-III-A, AGP-III-B, AGP-III-C, AGP-III-D are all acidic xylan type components, and more active groups such as acidic sugar content is gradually increased with the increase of degradation degree, but partial branched sugar such as glucose and the like is lost (figure 5).
(6) The effect of MTT method on AGP-III and four degraded components AGP-III-A, AGP-III-B, AGP-III-C, AGP-III-D on the activity of HepG2 human hepatoma cells is shown in FIG. 6. When the concentration reaches 100 mug/mL, the reduction of the HepG2 cell viability is 83.81%, 67.8%, 65.1%, 59.6% and 64.7% which are respectively very significant compared with the blank group (P < 0.001). The results show that the five xylan components can reduce the activity of human liver cancer cells, the inhibition degree of the degraded components on the cell activity is greater than that of AGP-III, and the comparison result of every two shows that the inhibition degree of the components AGP-III-C on the activity of HepG2 cells is obviously stronger than that of other components. Therefore, the component AGP-III-C is selected for further activity study in the later period.
(7) To investigate whether AGP-III-C could inhibit the proliferation of HepG2 human hepatoma cells, this study selected a colony formation experiment to observe that HepG2 cells were treated with different concentrations (0, 1, 5, 25, 50, 100. mu.g/mL) of AGP-III-C and 5-FU (20. mu.g/mL) for times n > 6. Crystal violet is an alkaline staining solution that binds to nuclear DNA and stains bluish violet. The experimental results are as follows: the cells in the normal control group show blue-purple color, which indicates that the cells can normally grow within 7 days of culture, while the cells in the sample group become lighter in color with the increase of the sample concentration, which indicates that the number of the cells is small or insufficient. The experimental result shows that the HepG2 human liver cancer cell shows the proliferation inhibition phenomenon and shows concentration dependence after the sample is given. When the concentration reaches 50 mug/mL, the sample inhibits the proliferation of cells, the phenomenon of cell shedding and the like occurs, and the blue-violet color is basically not generated, and the degree is similar to that of the positive control 5-FU.
(8) The effect of AGP-III-C on the HepG2 cell cycle was examined by flow cytometry analysis. As shown in FIG. 7, when the AGP-III-C concentration is greater than 1. mu.g/mL, the relative percentage of G0/G1 gradually increases from 59.50% to 75.85% as the AGP-III-C concentration increases, as compared to the blank group. The relative percentage of the S phase and the G2-M phase is reduced, and the statistical significance is remarkable. It was shown that AGP-III-C induces apoptosis by blocking the HepG2 cell cycle at G0/G1.
(9) To investigate the effect of AGP-III-C on the morphology of HepG2 human hepatoma cells, it is shown in FIG. 8. The blank control group cells are uniform and bright, and show long spindle shapes, complete cell shapes and complete cell-cell contact. In the AGP-III-C treated cells, chromatin in cell nuclei contracts, shapes change, cells become round and small, contact between cells disappears, edges become rough, and the number of cells is reduced. And when the concentration is gradually increased, the change of the cell morphology is more obvious, and the cell gradually appears in a shedding state. The 5-FU treatment group was a positive control, and a change in cell morphology during apoptosis of cancer cells was observed. In order to further discuss whether the AGP-III-C induces the apoptosis of HepG2 human liver cancer cells, the research selects AO-EB double-fluorescence staining and DAPI fluorescence staining methods to observe the change of the apoptosis form of the HepG2 human liver cancer cells after the AGP-III-C treatment. AO can penetrate membrane intact cells to enter the nucleus and combine with double-stranded DNA to emit green fluorescence. EB can only penetrate the membrane damaged cells, and the embedded nuclear DNA emits orange-red fluorescence. As shown in fig. 8B, the cell membrane of the control group cells is intact and exhibits uniform green fluorescence, while the cell membrane permeability of the low concentration treatment group cells increases, EB gradually enters the cells and stains with AO to exhibit orange fluorescence, and with the increasing of the sample concentration, the amount of EB entering the cells gradually increases, which indicates that the integrity of the cell membrane decreases, so that the amount of red fluorescence increases. As can be seen from the figure, the red fluorescence is gradually increased along with the increase of the concentration of the AGP-III-C, which indicates that the AGP-III-C can induce the apoptosis of HepG2 human liver cancer cells. Similarly, the DAPI fluorescent dye can combine with nuclear DNA in normal cells to present uniform blue fluorescence, when the cells are in an apoptotic state, the integrity of cell membranes is damaged, and the DAPI dye entering the cells is gradually increased, so that the cells present bright blue fluorescence after observation. As shown in FIG. 8C, the cell membrane structure of the cells in the blank control culture medium was intact, and the DAPI fluorescent dye entered the cells was uniformly stained. The uniform blue fluorescence is presented, the cell morphology of the sample group is changed, the chromatin is condensed, the integrity of the cell membrane is reduced, the DAPI entering the cells is increased, the staining is not uniform, and the cells present bright blue fluorescence after observation. It shows that the AGP-III-C can induce HepG2 human liver cancer cell to die and is concentration-dependent.
(10) Effect of AGP-III-C on mitochondrial membrane function of HepG2 human hepatoma cells
Changes in mitochondrial membrane potential were measured using JC-1 specific fluorescent dye. JC-1 exists in two forms, and in normal cells, the multimeric form of JC-1 aggregates to give red fluorescence in the matrix of mitochondria, where mitochondrial membrane potentials are high. When the membrane potential of mitochondria is reduced and the permeability is changed, JC-1 can not form polymers and exists in a monomer form, and green fluorescence appears. The cells in the control group exhibited red fluorescence, indicating that JC-1 exists in a multimeric form and that mitochondria function normally. And as the concentration of AGP-III-C increases, the green fluorescence increases significantly, the red fluorescence decreases gradually, and the ratio of the green fluorescence to the red fluorescence increases significantly, as shown in FIG. 9A, which shows that AGP-III-C can induce the decrease of the mitochondrial membrane potential of the cell. The ROS levels in HepG cells after AGP-III-C treatment were further determined as shown in FIG. 9B. Compared with a blank control group, after 24 hours of treatment of 1 mu g/mL, 5 mu g/mL, 25 mu g/mL, 50 mu g/mL and 100 mu g/mL AGP-III-C, the ROS content of the HepG2 human hepatoma cells is respectively increased by 0.078 +/-0.0086%, 0.29 +/-0.0051%, 0.75 +/-0.056%, 1.18 +/-0.068% and 2.79 +/-0.011%. The results indicate that AGP-III-C can significantly (P <0.001) promote the increase of intracellular ROS level and is concentration-dependent.
Whether the MAPK signal channel participates in the AGP-III-C induced HepG2 human liver cancer cell apoptosis is investigated by detecting the expression level of the total protein and the phosphorylated protein of ERK1/2, JNK and p 38. As shown in FIG. 10, AGP-III-C can significantly increase the expression of p38, ERK and JNK phosphorylated proteins and is concentration-dependent. It is suggested that AGP-III-C is involved in regulating the process of AGP-III-C induced apoptosis by activating the MAPK pathway in HepG2 cells.
AGP-III-C was established above as the best active ingredient and was therefore structurally characterized. The structural information of AGP-III-C is obtained by GC-MS and NMR spectrum analysis by adopting a derivatization method.
Reduction of uronic acids in AGP-III-C
Since the acidic polysaccharide is prone to loss of uronic acid and the linked neutral sugar during acid hydrolysis, and during methylation derivatization, the sugar chain is prone to β -elimination under alkaline conditions in the reaction and thus cannot be detected. Therefore, it is necessary to reduce uronic acid first when the structure of acidic sugar is analyzed.
Reducing uronic acid by Taylor and Conrad, weighing about 10mg AGP-III-C sample, adding about 1.5mL distilled water, shaking uniformly, slowly adding 30mg CMC, keeping pH at about 4 with 0.01mol/L HCl, continuously stirring for 2h, and then continuously dropwise adding 1mL 100mg/mL NaBH into the reaction4Keeping the pH of the system at 7 in the reaction process, continuing the reaction for 1h, slowly adding diluted glacial acetic acid to adjust the pH to 5 after the reaction is finished, dialyzing for 3 days, and freeze-drying. Repeating the above steps 5 times until the uronic acid reduction is complete, and the obtained product is named as AGP-III-C-R.
Methylation analysis of AGP-III-C and AGP-III-C-R
Carrying out methylation analysis on AGP-III-C and AGP-III-C-R by adopting a needles method, dissolving about 10mg of AGP-III-C and AGP-III-C-R in 1.5mL of DMSO, carrying out ultrasonic dissolution for 1h, stirring overnight at 50 ℃ to fully dissolve polysaccharide, adding about 100mg of NaOH powder which is ground and dried in advance, and carrying out ultrasonic reaction for 1 h. Adding 2mL of methyl iodide at room temperature in a dark place, and carrying out ultrasonic reaction for 2 h. Water is added for quenching, and the reaction is ended. Dialyzed against water for 3 days and freeze-dried. Repeating the steps for 4 times until the methylation is complete, performing hydrolytic acetylation, respectively adding 3mL of formic acid into the completely methylated samples, performing depolymerization and hydrolysis at 100 ℃ for 3h, removing the formic acid after rotary evaporation to dryness, and then adding 2mol/L TFA and hydrolyzing at 121 ℃ for 2 h. After neutralization, the mixture is decompressed and dried, and 15 percent of NaBH is added4Reducing, adding glacial acetic acid dropwise for neutralization after 2h, and adding methanol into the sample after decompression and draining to remove borate by pumping for several times. Then, 1mL of pyridine and 1mL of acetic anhydride were added and reacted overnight. Vacuum pumping to dry, dissolving in CH2Cl2Distilled water was added and extracted several times. GC-MS analysis and G analysis of the prepared samplesAnd C, quantitative analysis.
The GC-MS programmed chromatographic column is rtx-5 MS column (30m multiplied by 0.25mm multiplied by 0.25 mu m), the programmed temperature rise condition is that the temperature is kept for 3min at 140 ℃, the temperature is raised to 250 ℃ at the speed of 2 ℃/min and is kept for 20min under the condition; helium is taken as carrier gas, and the flow rate is 0.6 mL/min; the mass spectrum ion source is EI+Source, temperature 210 ℃.
The GC program chromatographic column is rtx-5 ms column (30m multiplied by 0.25mm multiplied by 0.25 μm), the temperature programming condition is that the temperature is kept for 3min at 140 ℃, and the temperature is increased to 250 ℃ at the speed of 2 ℃/min and is kept for 20min under the condition; helium is taken as carrier gas, and the flow rate is 0.6 mL/min;
of AGP-III-C1H and13c analysis
Weighing about 50mg of AGP-III-C, dissolving in 1mL of heavy water, fully shaking for dissolving, centrifuging to obtain a supernatant, freeze-drying, and repeating the operation for 3 times. The sample was dissolved in 400. mu.L of heavy water and transferred to a nuclear magnetic tube for nuclear magnetic analysis.
NMR analysis at 30 ℃ on a Varian 600MHz NMR spectrometer1H and13and C, collecting a spectrum and a two-dimensional spectrum.
Results of the experiment
1 uronic acid reduction
After the uronic acid is reduced, a completely reduced sample AGP-III-C-R is obtained, GC analysis is carried out on the sample, the uronic acid disappears by comparing GC spectrums before and after reduction, and the content of the uronic acid measured by a colorimetry is 5.2%, which indicates that the uronic acid is basically completely reduced.
2 AGP-III-C and AGP-III-C-R complete methylation analysis
After complete methylation analysis of AGP-III-C and AGP-III-C-R, reduction and acetylation are carried out to obtain a partially methylated sugar alcohol acetyl ester derivatization sample. The pattern obtained after GC-MS analysis and the corresponding fragment peaks are shown in FIG. 11, the GC-MS pattern of the AGP-III-C derivative has 2 peaks, the GC-MS pattern of the AGP-III-C-R derivative has 3 peaks, and each peak is assigned according to the retention time of the chromatographic peak and the characteristic fragment of the mass spectrum peak, and the types of the sugar residues are deduced. Wherein 2,3-Me2-Xyl and 3-Me-Xyl areCommon peak, and new peak 2,3,4,6-Me is added in AGP-III-C-R4Glc, newly added peak from reduced 4-O-Me-GlcA and GlcA. Three sugar residues 2,3-Me in derivatized AGP-III-C-R2-Xyl, 3-Me-Xyl and 2,3,4,6-Me4-Glc represents three attachment modes, namely-4) Xylf (1-, -2,4) Xylf (4-, 4-O-Me-GlcAp (1-. methylation analysis results are shown in table 2 below:
TABLE 2 methylation analysis of GP-III-C and AGP-III-C-R
The relative molar ratio of each sugar residue was calculated from the peak area of the GC pattern and the response factor, three sugar residues in AGP-III-C-R-4) Xylf (1-, -2,4) Xylf (4-, 4-O-Me-GlcAp (1-molar ratio of 1:1.34: 1.23. wherein the branching glycosyl group-2, 4) Xylf (1-number is equal to the terminal glycosyl group number-GlcA, indicating complete methylation.
3 AGP-III-C1H and13c analysis
The glycosidic bond configuration of AGP-III-C, the linkage system, and other sugar chain structure information were analyzed by Nuclear Magnetic Resonance (NMR), and the results are shown in FIG. 12. And (3) assigning related chemical shifts by combining GC-MS (gas chromatography-Mass spectrometer) sugar residue assignment and nuclear magnetic spectrum analysis. Of AGP-III-C1In the H spectrum, there are three anomeric proton shifts, 5.15, 4.32, and 4.49, respectively, which are named A, B, C. The chemical shift of A is between 5.1 and 5.4, no split peak belongs to alpha-anomeric proton, and the signal is generated by 4-O-Me-GlcA. 4.49 and 4.32 have split peaks and chemical shifts of the protons of the sugar rings between 4.5 and 5.0ppm belong to the beta-type glycosidic linkages, representing the signals generated by the sugar residues-4) Xylf (1-and-2, 4) Xylf (4-), respectively.1Integrated peak area molar ratio of Xyl to GlcA in H spectrum B + C: and A is 1.53: 1.0. This is close to a backbone to side chain molar ratio of 1.90:1 in methylation.13Chemical shifts of anomeric carbon of sugar residues in C-spectrum are generally centered between 90-110, in AGP-III-C13Chemical shifts of the anomeric carbon of A, B, C in the C spectrum are respectively at 100.98, 104.51 and 104A signal peak at 01ppm, and additionally 62.83ppm indicated the presence of a methoxy group in AGP-III-C.
All of1H and13chemical shifts of the C signal were assigned by the homonuclear correlation spectrum (COSY), total correlation spectrum (TOCSY), TNNOESY spectrum and heteronuclear multiple bond correlation spectrum (HMBC), respectively.
Since the chemical shifts of the non-anomeric protons of the sugar rings generally overlap, analysis by means of a proton shift correlation map in each sugar residue is required. COSY and TOCSY spectra of AGP-III-C are shown in FIGS. 13 and 14. Direct coupling correlation peaks between adjacent protons can be found from the COSY spectra, such as sugar residue unit B anomeric signal H1(4.33ppm) associated with H2(3.15ppm), H2(3.15ppm) associated with H3(3.44ppm), H3(3.44ppm) associated with H4(3.64ppm), and H4(3.64ppm) associated with H5(4.02ppm), thereby assigning the chemical uniqueness of each proton in residue B. Residues a and C are also assigned in the same way. Additionally, the correlation peaks between anomeric protons and the rest of the protons in the sugar ring were assigned by TOCSY spectroscopy. Notably, in the TOCSY spectrum, a cross peak with a proton chemical shift of 3.34ppm was observed in the peak associated with the anomeric proton of residue a. This peak is attributed to the proton of the methoxy group,13the chemical shift in C was 62.51 ppm. In addition, a cross peak e: a was observed in HMBC spectra4C(84.90)/AH O‐Me(3.34) and f: A4H(3.34)/AC O‐Me(62.83) indicating that O-Me is attached at the O-4 position of residue A. This is consistent with the results of methylation analysis, demonstrating that the sugar residue A is the 4-O-methylglucuronic acid terminus. The chemical shift assignments for the remaining protons are shown in table 3.
The hydrocarbon-related signals can be detected by HSQC spectroscopy, as shown in FIG. 15, with sugar unit A strongly anomeric signals at 5.15ppm and 100.98 ppm; strong anomeric signals at 4.32ppm and 104.51ppm for saccharide unit B; sugar unit C strongly outranks at 4.49ppm and 104.01 ppm. Three main crosslinking residue units in AGP-III-C are further demonstrated. Meanwhile, the chemical shift of non-anomeric carbon can be assigned through HSQC, such as H4 in the sugar unit A is related to C4 signal at 84.90ppm, and H5 is related to C5 signal at 73.35 ppm. Other carbon chemical shifts can be assigned similarly.
The mode of linkage between glycosidic bonds of AGP-III-C was determined by TNNOESY and HMBC spectra, as shown in FIGS. 16 and 17. A cross peak b' was observed in the TNNOESY spectrum: B1H (4.49)/C4H (3.64) and C': c1H(4.33)/B4H(3.65), a cross peak a is observed in the HMBC spectrum: c1H(4.49)/B4C(78.81)；b：B1H(4.32)/C4C(79.46)；c：B4H(3.64)/C1C(104.10) signal correlation.
These results demonstrate that AGP-III-C is a main structural repeating unit of a sugar chain which is-4) Xylf (1-. And the cross peak a' in the TNNOESY spectrum: cross peak d in A1H (5.15)/C2H (3.28) and HMBC: C2H (3.27)/A1C(100.87) it was shown that the terminal-4-O-Me-GlcA is linked to the 2-position of the-2, 4) Xylf (1-sugar residue via a1, 2-O-glycosidic linkage. The structure of AGP-III-C was presumed to be shown in FIG. 18, based on the results of analysis of monosaccharide composition, methylation analysis and NMR spectrum.
TABLE 3 GP-III-C1H NMR and13chemical shift by C NMR
In order to clarify the primary structure of AGP-III-C, the kind of the single component, the type of the sugar residue, the configuration of the glycosidic bond, the connection sequence of the sugar residue and the like are analyzed and detected by analytical means such as monosaccharide composition analysis, infrared spectrometry, uronic acid reduction, methylation analysis, two-dimensional nuclear magnetic resonance spectroscopy and the like. In recent years, with the maturity of nuclear magnetic resonance spectroscopy, the analysis of the structure of the polysaccharide is more and more accurate, the sample does not need to be subjected to derivatization treatment, the structural information of the polysaccharide is directly and visually detected through detection, and the structural information of AGP-III-C can be accurately displayed in a nuclear magnetic spectrum. From AGP-III-C1The H map shows that the content of residue A4-O-Me-a-GlcAp is the highest, which indicates that the proportion of acid sugar is large, but the content of acid sugar does not exceed that of straight-chain xylanThe results are not much different from the methylation results in terms of mole numbers, and the connection mode between sugar residues is known through a TNNOESY spectrum and an HMBC spectrum, and the tail ends of the branched chains 4-O-Me-a-GlcAp are connected to two adjacent-4) Xylf (1-main chain structural units. By analyzing the structure of AGP-III-C, the 4-oxymethyl glucuronoxylan is found to be a typical type. Studies have reported that the degree of polymerization of xylan, the distribution position and ratio of uronic acid to xylose all affect the development of biological activity.
And (4) identification conclusion: the structural information of the purified AGP-III-C is as follows: the main chain is (1 → 4) -beta-Xylf, and the 2-position of every two xylose structural units is connected with the terminal of 4-O-Me-a-GlcAp through a1, 2-O-glycosidic bond.
Claims (5)
1. 4-O-methylglucuronic acid xylan obtained from artemisia desertorum seed gum is characterized in that: the molecular weight is 52 kDa-54 kDa; the main chain is xylose structural unit (1 → 4) -beta-Xylf, and the 2-position of every two xylose structural units (1 → 4) -beta-Xylf is connected with the terminal of 4-O-Me-a-GlcAp through 1, 2-O-glycosidic bond, as follows:
2. 4 oxymethyl glucuronate xylan according to claim 1 wherein: the molecular weight is 53 kDa.
3. Use of 4 oxymethyl glucuronoxylomannan as claimed in claim 1 or 2 in the manufacture of a medicament for the inhibition of liver tumors, which is capable of inducing apoptosis of HepG2 human liver cancer cells.
4. A pharmaceutical composition comprising the 4 oxymethyl glucuronate xylan according to claim 1 or 2 in combination with a pharmaceutically acceptable carrier.
5. A food product comprising the 4 oxymethyl glucuronoxylomannan of claim 1 or 2.
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