Preparation method of high galangal polysaccharide and application thereof in preparation of anti-liver cancer drugs
Patent Information
- Application Number
- CN202410121414.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-29
- Publication Date
- 2026-08-21
- Estimated Expiration
- 2044-01-29
AI Technical Summary
[0005]目前有研究报道高良姜提取物改善胰岛素抵抗作用显著，但对于高良姜多糖应用于抗肝癌药物及其给药浓度未见详细报道
[0029]1、本发明首次发现通过本发明特定的制备方法获得的高良姜酸性多糖具有良好的抑制HepG2细胞株增殖的作用以及从发病机理方面，具有良好的预防和治疗肝癌的药用价值。
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Figure CN118027233B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of drug preparation and application technology, and relates to a method for preparing galangal polysaccharide and its application in the preparation of anti-liver cancer drugs, providing a scientific basis for the clinical application of galangal polysaccharide as an adjuvant therapy for liver cancer. Background Technology
[0002] Galangal is the dried rhizome of a plant belonging to the genus *Alpinia* in the ginger family (Zingiberaceae), mainly distributed in Guangdong, Guangxi, and Hainan provinces. Galangal has the effects of warming the stomach and dispelling cold, promoting digestion and relieving pain, and can alleviate stomach ailments. Current research on galangal both domestically and internationally mainly focuses on its chemical components such as flavonoids and aromatic oils, as well as its pharmacological functions such as antibacterial, antitumor, and lipid-lowering effects; however, research on galangal polysaccharides remains limited.
[0003] Liver cancer (HCC) is the fifth most common malignant tumor worldwide, and can be divided into two main categories: primary and secondary. Primary liver cancer originates from the epithelial or mesenchymal tissue of the liver; the former is called primary liver cancer, which is a highly prevalent and extremely dangerous malignant tumor in my country; the latter is called sarcoma, which is less common compared to primary liver cancer. Secondary or metastatic liver cancer refers to malignant tumors originating from multiple organs throughout the body that invade the liver. It is commonly seen as a liver metastasis from malignant tumors of the stomach, bile ducts, pancreas, colorectal, ovary, uterus, lung, and breast. The etiology and exact molecular mechanisms of primary liver cancer are not fully understood. Currently, its pathogenesis is considered to be a complex, multifactorial, and multi-step process, influenced by both environmental and dietary factors. Due to its tolerance to chemotherapy and its extremely high recurrence and metastasis rates after surgery, it is the third leading cause of death among malignant tumors. In recent years, increasing research has demonstrated that HCC may originate from a subpopulation of stem cells known as tumor-initiating cells (TICs) or cancer stem cells. These cells express specific surface markers and possess the characteristics of self-renewal, differentiation, tumor formation, and chemotherapy resistance. Although sorafenib is currently used as a targeted therapy for HCC, its tumor-suppressive effect and survival rate for liver cancer patients are not very good.
[0004] The HepG2 human hepatoblastoma cell line was isolated and established in 1979 by Aden et al. and Morris et al. from a primary hepatoblastoma in a 15-year-old Caucasian boy in Argentina. This cell line is epithelial-like, adherent, and grows in clusters. It grows rapidly, with a passage cycle of 1-2 days. It has a low metastasis rate, poor tumorigenesis in nude mice, is AFP-positive, HBsAg-negative, and exhibits high differentiation. Its metabolic enzymes retain their biotransformation characteristics well, requiring no exogenous activation system. In drug-related studies, the metabolic enzymes remain stable and do not change with passage number. The biotransformation enzymes it contains are homologous to those in normal human hepatocytes; therefore, it is often used as an ideal cell line for in vitro hepatocyte metabolism or genotoxicity assays. Among them, HepG2.2.15 is a widely used cell line. It is a derivative of HepG2, obtained by transfecting HepG2 recipient cells with a recombinant plasmid containing two head-to-tail HBV DNA full genes. It can reproduce asexually in vitro and can stably secrete HBsAg, HBeAg and complete Dane particles for a long time, producing a large number of replication intermediates. It is a good model for screening anti-HBV drugs in vitro and is used as an in vitro research tool for the development of new anti-HBV drugs.
[0005] Current research reports that galangal extract significantly improves insulin resistance, but there are no detailed reports on the application of galangal polysaccharides in anti-liver cancer drugs and their dosage. Summary of the Invention
[0006] The purpose of this invention is to provide a method for preparing galangal polysaccharide, which obtains galangal acidic polysaccharide through a specific preparation process. The galangal acidic polysaccharide has a significant effect on improving insulin resistance.
[0007] To achieve the above objectives, the technical solution of the present invention is as follows: a method for preparing Alpinia galanga polysaccharide, comprising the following steps:
[0008] Step S1: After washing the galangal rhizomes, crush them and sieve them. Extract the galangal powder several times by reflux with hot water. Combine the extracts and concentrate them under reduced pressure. After precipitation, deproteinization by Sevag method, dialysis, freeze-drying and drying, crude polysaccharide AHP is obtained.
[0009] Step S2: After the crude polysaccharide is dissolved, it is separated and eluted sequentially with ultrapure water, 0.1M NaCl, 0.2M NaCl and 0.3M NaCl solutions, and the eluent is collected.
[0010] Step S3: Determine the sugar content of each eluent tube using the phenol-sulfuric acid method;
[0011] Step S4: Redistill phenol and prepare test solution with distilled water. Use glucose as standard and accurately prepare glucose standard stock solution with redistilled water. Prepare standard curves according to the glucose standard stock solution at concentrations of 0.0, 30.0, 60.0, 90.0, 120.0, 150.0, and 180.0 μg / ml, and dilute to 50 ml in volumetric flasks.
[0012] Step S5: Take each standard stock solution, add phenol test solution, mix evenly in a shaker, quickly add concentrated sulfuric acid, shake, place at room temperature, and measure the absorbance value.
[0013] Step S6: Plot the sulfuric acid-phenol standard curve with the absorbance of the standard sample minus the absorbance of distilled water on the ordinate and the concentration of glucose standard on the abscissa, and calculate the regression equation.
[0014] Step S7: Weigh the polysaccharide sample, make up to volume in a volumetric flask, measure the absorbance value, and substitute it into the standard curve equation to calculate the concentration of each sample.
[0015] Step S8: Plot the polysaccharide content-tube number curve, and collect the polysaccharides according to the curve; after concentration, dialysis and freeze-drying, the preliminarily purified Alpinia galanga polysaccharide is obtained.
[0016] Step S9: Using a Sephadex G-100 column with ultrapure water as the eluent, further purify the polysaccharide, determine the sugar content again, and plot the polysaccharide content-tube number curve.
[0017] Step S10: After concentration and freeze-drying, purified polysaccharide is obtained.
[0018] Furthermore, in step S1, the galangal is pulverized and then passed through a 60-mesh sieve.
[0019] Furthermore, in step S1, the hot water reflux temperature is 100℃, and the extraction is performed 3 times, each time for 1 hour.
[0020] Further, in step S1, the extracts are combined and concentrated under reduced pressure to 10%-20% of the original volume; 95% ethanol is added until the ethanol concentration is 60%, and precipitation is carried out at 4°C for 12 hours.
[0021] Further, in step S2, the eluent is collected using an automated fraction collector at a flow rate of 0.5 mL / min for 30 minutes per tube.
[0022] Another objective of this invention is to provide an application of galangal polysaccharide in the preparation of anti-liver cancer drugs, providing a scientific basis for the clinical application of galangal polysaccharide in the adjuvant treatment of liver cancer.
[0023] This invention relates to the application of galangal polysaccharide in the preparation of anti-liver cancer drugs, wherein the galangal polysaccharide obtained by this invention is used to prepare drugs for the treatment and / or prevention of liver cancer.
[0024] Furthermore, the dosage concentration of the galangal polysaccharide is 0.1-0.5 mg / ml.
[0025] Furthermore, the dosage concentration of the galangal polysaccharide is 0.1 mg / ml.
[0026] Furthermore, the dosage concentration of the galangal polysaccharide is 0.3 mg / ml.
[0027] Furthermore, the dosage concentration of the galangal polysaccharide is 0.5 mg / ml.
[0028] The preparation method of Alpinia galanga polysaccharide of the present invention and its application in the preparation of anti-liver cancer drugs have the following beneficial effects:
[0029] 1. This invention is the first to discover that the galangal acidic polysaccharide obtained by the specific preparation method of this invention has a good inhibitory effect on the proliferation of HepG2 cell line and has good medicinal value in the prevention and treatment of liver cancer from the perspective of pathogenesis.
[0030] 2. The galangal polysaccharide obtained in this invention is mainly composed of rhamnose, arabinose, galactose, xylose, and galacturonic acid, with molar percentages of 8.9%, 16.9%, 21.4%, 11.8%, and 35.4%, respectively. It contains small amounts of fucose (0.5%) and glucuronic acid (2.0%), but no mannose. Chemical composition and monosaccharide composition analysis results show that AHP-3a contains a high content of uronic acid, indicating that the galangal polysaccharide of this invention is an acidic polysaccharide.
[0031] 3. Through the experiments of this invention, it was found that the optimal drug concentrations of 0.1, 0.3, and 0.5 mg / ml of high-alpinia galanga polysaccharide showed the best inhibitory effect on the migration and invasion of HepG2 liver cancer cells. Attached Figure Description
[0032] Figure 1 Effects of Alpinia galanga polysaccharide on the viability of LX-2 and HepG2 cells (Note: **p<0.01 compared with the control group);
[0033] Figure 2 The effect of Alpinia galanga polysaccharide on the proliferation of HepG2 cells (Note: Compared with the control group, *P<0.05, ***P<0.001);
[0034] Figure 3The effect of Alpinia galanga polysaccharide on the migration of HepG2 cells (Note: Compared with the control group, *P<0.05, **p<0.01, ***P<0.001). Detailed Implementation
[0035] The features and advantages of the present invention will be further understood through the following detailed description. The provided embodiments are merely illustrative of the methods of the present invention and do not limit the remaining content disclosed herein in any way. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available.
[0036] Galangal (purchased from Danzhou, Hainan), DEAE-52 cellulose (Greenherbs), Sephadex G-100 (Cytiva), automatic fraction collector (BS-100A; Shanghai Huxi, China).
[0037] Example 1
[0038] The present invention provides a method for preparing Alpinia galanga polysaccharide, comprising the following steps:
[0039] (1) The rhizomes of Alpinia galanga purchased from Danzhou, Hainan were washed with clean water and dried to constant weight. They were then pulverized, passed through a 60-mesh sieve, and the sieved powder was dried and stored for later use. 1.0 kg of Alpinia galanga rhizomes were pulverized and extracted three times by reflux with hot water (100℃), 1 hour each time. The extracts were combined and concentrated under reduced pressure to 10% of the original volume. 95% ethanol was added until the ethanol concentration reached 60%, and precipitation was carried out at 4℃ for 12 hours. The precipitated polysaccharide was dissolved in deionized water, deproteinized using the Sevag method, dialyzed, frozen, and dried to obtain crude polysaccharide (AHP).
[0040] (2) The crude polysaccharide after deproteinization was dissolved in deionized water and separated on a DEAE-52 chromatographic column (2.6 cm × 30 cm). Elution was performed sequentially with ultrapure water, 0.1 M NaCl, 0.2 M NaCl, and 0.3 M NaCl solutions. The eluent was collected using an automated fraction collector for 30 minutes per tube (flow rate 0.5 mL / min).
[0041] (3) The sugar content of each eluent tube was determined by the phenol-sulfuric acid method. The redistilled phenol was prepared into a 50 g / L test solution with distilled water. Using glucose as the standard, a 1.5 mg / ml glucose (Glu) standard stock solution was accurately prepared with redistilled water. The standard curve was prepared according to the Glu standard solution at concentrations of 0.0, 30.0, 60.0, 90.0, 120.0, 150.0, and 180.0 (μg / ml), and diluted to 50 ml in volumetric flasks.
[0042] (4) Take 0.2 ml of each standard solution and add 50 g / L phenol test solution. After mixing evenly with a shaker, quickly add 2.0 ml of concentrated sulfuric acid, shake, and place at room temperature for 30 min. Then measure the absorbance value at 490 nm using a spectrophotometer.
[0043] (5) Plot the sulfuric acid-phenol standard curve with the absorbance of standard sample minus the absorbance of distilled water on the ordinate and the concentration of glucose standard sample on the abscissa, and calculate the regression equation.
[0044] (6) Sample content determination: Accurately weigh 10 mg of polysaccharide sample, dilute to 100 ml in a volumetric flask and then measure the content. The specific method is the same as that of glucose standard solution. After measuring the absorbance value, substitute it into the standard curve equation to calculate the concentration of each sample.
[0045] (7) Plot the polysaccharide content-tube number curve and collect the polysaccharides according to the curve. After concentration, dialysis and freeze-drying, the preliminarily purified Alpinia galanga polysaccharide is obtained.
[0046] (8) The polysaccharide was further purified using a Sephadex G-100 column (2.6 cm × 30 cm) with ultrapure water as the eluent. The sugar content was determined again, and a polysaccharide content-tube number curve was plotted. The purified AHP was obtained after concentration and freeze-drying.
[0047] The present invention relates to the application of galangal polysaccharide in the preparation of drugs for the treatment and / or prevention of liver cancer, wherein the dosage concentration of galangal polysaccharide is 0.1 mg / ml.
[0048] Example 2
[0049] The present invention provides a method for preparing Alpinia galanga polysaccharide, comprising the following steps:
[0050] (1) The rhizomes of Alpinia galanga purchased from Danzhou, Hainan were washed with clean water and dried to constant weight. The rhizomes were then pulverized, passed through a 60-mesh sieve, and the sieved powder was dried and stored for later use. 1.0 kg of Alpinia galanga rhizomes were pulverized and extracted three times by reflux with hot water (100℃), 1 hour each time. The extracts were combined and concentrated under reduced pressure to 15% of the original volume. 95% ethanol was added until the ethanol concentration reached 60%, and precipitation was carried out at 4℃ for 12 hours. The precipitated polysaccharide was dissolved in deionized water, deproteinized using the Sevag method, dialyzed, frozen, and dried to obtain crude polysaccharide (AHP).
[0051] (2) The crude polysaccharide after deproteinization was dissolved in deionized water and separated on a DEAE-52 chromatographic column (2.6 cm × 30 cm). Elution was performed sequentially with ultrapure water, 0.1 M NaCl, 0.2 M NaCl, and 0.3 M NaCl solutions. The eluent was collected using an automated fraction collector for 30 minutes per tube (flow rate 0.5 mL / min).
[0052] (3) The sugar content of each eluent tube was determined by the phenol-sulfuric acid method. The redistilled phenol was prepared into a 50 g / L test solution with distilled water. Using glucose as the standard, a 1.5 mg / ml glucose (Glu) standard stock solution was accurately prepared with redistilled water. The standard curve was prepared according to the Glu standard solution at concentrations of 0.0, 30.0, 60.0, 90.0, 120.0, 150.0, and 180.0 (μg / ml), and diluted to 50 ml in volumetric flasks.
[0053] (4) Take 0.2 ml of each standard solution and add 50 g / L phenol test solution. After mixing evenly with a shaker, quickly add 2.0 ml of concentrated sulfuric acid, shake, and place at room temperature for 30 min. Then measure the absorbance value at 490 nm using a spectrophotometer.
[0054] (5) Plot the sulfuric acid-phenol standard curve with the absorbance of standard sample minus the absorbance of distilled water on the ordinate and the concentration of glucose standard sample on the abscissa, and calculate the regression equation.
[0055] (6) Sample content determination: Accurately weigh 10 mg of polysaccharide sample, dilute to 100 ml in a volumetric flask and then measure the content. The specific method is the same as that of glucose standard solution. After measuring the absorbance value, substitute it into the standard curve equation to calculate the concentration of each sample.
[0056] (7) Plot the polysaccharide content-tube number curve and collect the polysaccharides according to the curve. After concentration, dialysis and freeze-drying, the preliminarily purified Alpinia galanga polysaccharide is obtained.
[0057] (8) The polysaccharide was further purified using a Sephadex G-100 column (2.6 cm × 30 cm) with ultrapure water as the eluent. The sugar content was determined again, and a polysaccharide content-tube number curve was plotted. The purified AHP was obtained after concentration and freeze-drying.
[0058] The present invention relates to the application of galangal polysaccharide in the preparation of drugs for the treatment and / or prevention of liver cancer, wherein the dosage concentration of galangal polysaccharide is 0.3 mg / ml.
[0059] Example 3
[0060] The present invention provides a method for preparing Alpinia galanga polysaccharide, comprising the following steps:
[0061] (1) The rhizomes of Alpinia galanga purchased from Danzhou, Hainan were washed with clean water and dried to constant weight. They were then pulverized, passed through a 60-mesh sieve, and the sieved powder was dried and stored for later use. 1.0 kg of Alpinia galanga rhizomes were pulverized and extracted three times by reflux with hot water (100℃), 1 hour each time. The extracts were combined and concentrated under reduced pressure to 20% of the original volume. 95% ethanol was added until the ethanol concentration reached 60%, and precipitation was carried out at 4℃ for 12 hours. The precipitated polysaccharide was dissolved in deionized water, deproteinized using the Sevag method, dialyzed, frozen, and dried to obtain crude polysaccharide (AHP).
[0062] (2) The crude polysaccharide after deproteinization was dissolved in deionized water and separated on a DEAE-52 chromatographic column (2.6 cm × 30 cm). Elution was performed sequentially with ultrapure water, 0.1 M NaCl, 0.2 M NaCl, and 0.3 M NaCl solutions. The eluent was collected using an automated fraction collector for 30 minutes per tube (flow rate 0.5 mL / min).
[0063] (3) The sugar content of each eluent tube was determined by the phenol-sulfuric acid method. The redistilled phenol was prepared into a 50 g / L test solution with distilled water. Using glucose as the standard, a 1.5 mg / ml glucose (Glu) standard stock solution was accurately prepared with redistilled water. The standard curve was prepared according to the Glu standard solution at concentrations of 0.0, 30.0, 60.0, 90.0, 120.0, 150.0, and 180.0 (μg / ml), and diluted to 50 ml in volumetric flasks.
[0064] (4) Take 0.2 ml of each standard solution and add 50 g / L phenol test solution. After mixing evenly with a shaker, quickly add 2.0 ml of concentrated sulfuric acid, shake, and place at room temperature for 30 min. Then measure the absorbance value at 490 nm using a spectrophotometer.
[0065] (5) Plot the sulfuric acid-phenol standard curve with the absorbance of standard sample minus the absorbance of distilled water on the ordinate and the concentration of glucose standard sample on the abscissa, and calculate the regression equation.
[0066] (6) Sample content determination: Accurately weigh 10 mg of polysaccharide sample, dilute to 100 ml in a volumetric flask and then measure the content. The specific method is the same as that of glucose standard solution. After measuring the absorbance value, substitute it into the standard curve equation to calculate the concentration of each sample.
[0067] (7) Plot the polysaccharide content-tube number curve and collect the polysaccharides according to the curve. After concentration, dialysis and freeze drying, the preliminarily purified Alpinia galanga polysaccharide is obtained.
[0068] (8) The polysaccharide was further purified using a Sephadex G-100 column (2.6 cm × 30 cm) with ultrapure water as the eluent. The sugar content was determined again, and a polysaccharide content-tube number curve was plotted. The purified AHP was obtained after concentration and freeze-drying.
[0069] The present invention relates to the application of galangal polysaccharide in the preparation of drugs for the treatment and / or prevention of liver cancer, wherein the dosage concentration of galangal polysaccharide is 0.5 mg / ml.
[0070] Experimental procedure:
[0071] I. Experimental Materials
[0072] 1. Materials, reagents and experimental instruments
[0073] 1.1 Materials
[0074] LX-2 normal human hepatocytes, HepG2 liver cancer cells, DMEM medium (Gibco), 10% fetal bovine serum (Gibco) and 1% penicillin / streptomycin (Biosharp), 0.25% trypsin (Biosharp), PBS buffer (Biosharp), CCK8 assay kit (APExBIO), EDU cell proliferation assay kit (Beyotime), QuickBlock TM Immunostaining blocking solution (Beyotime), 0.3% Triton X-100 permeabilization solution (Beyotime), 4% paraformaldehyde (Beyotime), Transwell chambers, and Matrigel were all purchased from Corning Incorporated, USA. 4% cell fixative and 0.1% crystal violet staining solution were purchased from Solarbio, Beijing; Alpinia galanga polysaccharide was also used.
[0075] 1.2 Instruments
[0076] Adjustable-range pipette, 0.22μm microporous filter membrane, cryopreservation tubes, constant temperature CO2 cell culture incubator, clean bench, centrifuge, constant temperature water bath, precision electronic balance, ELISA reader, fluorescence microscope.
[0077] 1.3 Preparation of Sample Solutions
[0078] Alpinia polysaccharide was prepared into a stock solution of 2000 μg / ml.
[0079] 1.4 Cell Culture
[0080] (1) Cell thawing: After removing the cells from the -80°C freezer, quickly place them in 37°C water and shake rapidly to thaw. After thawing, disinfect the surface of the cryovials with alcohol, transfer them to a laminar flow hood, aspirate the cryopreservation solution containing the cells and transfer it to a centrifuge tube, incubate at 1500 rpm for 5 minutes, discard the supernatant, add fresh culture medium, gently pipette to disperse the cells into single cells, and transfer to a 25 cm³ centrifuge tube. 2 Culture flask.
[0081] (2) Cell culture: Seed cells into new 25cm cells. 2 Add approximately 4 ml of DMEM culture medium containing 10% fetal bovine serum and 1% penicillin and streptomycin to the culture flask, and culture at 37°C and 5% CO2. Change the culture medium every two days. When the cell adhesion rate is approximately 80%-90% and the cells are in good condition, subsequent experiments can be performed.
[0082] (3) Cell passage: When the cells have reached approximately 85% confluence and adherence to the culture dish, perform routine passage, remove the old culture medium, wash twice with 1 ml PBS, add an appropriate amount of trypsin to ensure full contact between the cells and trypsin, and incubate for 2-3 minutes for digestion. Under a microscope, when the cell shape changes from irregular to round and the intercellular spaces widen, add fresh culture medium containing serum to stop digestion, gently pipette to form a single-cell suspension, and seed the suspension into new culture dishes at a ratio of 1:3. Transfer the suspension to an incubator for further culture, and change the culture medium every two days thereafter.
[0083] (4) Cell cryopreservation: Collect and centrifuge cells as above. Add about 1 mL of cell cryopreservation solution to the centrifuge tube, resuspend the cell pellet, transfer it to a cryopreservation tube, seal the cryopreservation tube with sealing film, and label the cell type, passage number and cryopreservation date. Store in a freezer at -80℃ for long-term storage.
[0084] 2. Experimental Methods
[0085] 2.1 Monosaccharide Composition Analysis
[0086] Alpinia galanga polysaccharide AHP-3a (5 mg) was placed in an ampoule and hydrolyzed with 3 mol / L TFA at 120 °C for 3 hours. The hydrolysis product was dried under nitrogen atmosphere and washed three times with methanol to remove residual TFA. Finally, the hydrolyzed sample was dissolved in ultrapure water. Monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid, glucuronic acid) and polysaccharide samples were detected using an ion chromatograph (ICS5000, Thermo Fisher, USA). The chromatographic system was as follows: Dionex Carbopac™ PA-20 analytical column and electrochemical detector; mobile phase: A (H2O), B (15 mM NaOH), C (15 mM NaOH and 100 mM NaOAc); flow rate: 0.3 mL / min; column temperature: 30 °C.
[0087] 2.2 Effects of the drug on the activity of LX-2 and HepG2 cells
[0088] Logarithmically growing cells were collected and prepared into a single-cell suspension. The cell concentration was adjusted, and cells were seeded at a density of 10,000 cells / well in 96-well plates, with 5 replicates per group. The edges were filled with sterile PBS. The plates were incubated at 37°C and 5% CO2. Drug intervention was administered the following day. After 12 hours, the liquid in the 96-well plates was aspirated, and 100 μL of CCK8 medium was added to each well. The plates were incubated at 37°C and 5% CO2 for 1 hour. After incubation, the absorbance was measured at 450 nm using a microplate reader.
[0089] 2.3 Effects of drugs on the proliferation of LX-2, HepG2, and Huh7 cells (EDU cell proliferation assay)
[0090] HCC cells were fed at a rate of 6 × 10 4 Cells were seeded at 1 ml per well in 24-well plates. Once the cells reached 80% cell growth, different drug concentrations were added to the treatment groups. After 24 hours of drug administration, EdU working solution was added to both the control and treatment groups, and the plates were incubated at 37°C for 2 hours. Cells were then fixed with 4% paraformaldehyde for 15 minutes and permeabilized with PBS containing 0.3% Triton X-100 for 15 minutes. 300 μl of Click Additive Solution was added and incubated for 30 minutes. The reaction buffer was discarded, and each well was washed twice with washing buffer for 5 minutes each time. The washing buffer was discarded, and 300 μl of Hoechst was added and incubated for 10 minutes. Hoechst was discarded, and each well was washed twice for 5 minutes each time. Cells were observed under a fluorescence microscope. The ratio of EdU-positive cells (red) to the total number of Hoechst-positive cells (blue) was calculated to indicate cell proliferation capacity.
[0091] 2.4 Effect of drugs on the migration ability of HepG2 cells (as determined by scratch assay)
[0092] Draw even lines on the bottom of the 6-well plate with a black marker, and arrange HepG2 cells at 6 × 10⁻⁶ wells. 4 Cells were seeded per well in 6-well plates. When cell confluence reached 90%, three vertical lines were drawn using a pipette tip. Free cells were washed away with PBS, and serum-free medium containing different concentrations of high-galangal polysaccharide (300, 400, 500 μg / ml) was added. Images were taken under a microscope at 0 and 24 hours. The migration rate for each group was calculated. The formula for calculating the migration rate is: Cell migration rate (wound healing rate) = (Initial scratch area - Scratch area at time t) / Initial scratch area × 100%.
[0093] 2.5 Statistical Analysis
[0094] Data were processed and analyzed using the statistical analysis software GraphPadPrism 9.0. Experimental data are expressed as mean ± standard deviation (Mean ± SD). T-tests were used to compare differences between groups, nonparametric tests were used to assess unequal variances, and one-way ANOVA was used to compare multiple groups. A p-value < 0.05 was considered statistically significant.
[0095] II. Experimental Results
[0096] 3.1 Chemical composition and monosaccharide composition of Alpinia galanga polysaccharides
[0097] Table 1. Chemical composition and monosaccharide composition of Alpinia galanga polysaccharide of the present invention. Chemical composition of AHP-3a.
[0098]
[0099] Table 1 shows the chemical composition of AHP-3a of this invention. The contents of neutral polysaccharide, uronic acid, and total phenols are 28.00%, 45.96%, and 8.16%, respectively. The measurement results of total protein content indicate that the polysaccharide does not contain protein. The monosaccharide composition analysis is shown in Table 1. AHP-3a is mainly composed of rhamnose, arabinose, galactose, xylose, and galacturonic acid, with molar percentages of 8.9%, 16.9%, 21.4%, 11.8%, and 35.4%, respectively. It contains small amounts of fucose (0.5%) and glucuronic acid (2.0%), but no mannose. The results of chemical composition and monosaccharide composition analysis both show that AHP-3a contains a high content of uronic acid, indicating that AHP-3a of this invention is an acidic polysaccharide. In addition, the chemical and monosaccharide analysis of AHP-3a differs significantly from previously reported Alpinia galanga polysaccharides, which may be related to the selected components. Previous studies have shown that galangal neutral polysaccharides do not contain uronic acid, total phenols, or protein. Analysis of the monosaccharide composition revealed that galangal neutral polysaccharides are 100% composed of glucose. This suggests that the structure of AHP-3a is significantly different from that of galangal neutral polysaccharides.
[0100] 3.2 CCK8 Experimental Results
[0101] Effects of Alpinia officinarum polysaccharide on the activity of LX-2 and HepG2 cells
[0102] like Figure 1 As shown, galangal polysaccharides at concentrations of 100, 200, 300, 400, and 500 μg / ml had no inhibitory effect on the growth of LX-2 cells, while concentrations at these concentrations significantly inhibited the growth of HepG2 cells. Therefore, concentrations of 100, 300, and 500 μg / ml were used as the dosage concentrations for subsequent polysaccharide experiments.
[0103] 3.3 EdU Experimental Results
[0104] Effects of Alpinia officinarum polysaccharide on HepG2 cell proliferation
[0105] like Figure 2 As shown, after EdU staining, fluorescence imaging revealed that the red fluorescence (positive) gradually decreased with increasing concentration of Alpinia galanga polysaccharide. Quantitative fluorescence analysis showed that Alpinia galanga polysaccharide dose-dependently inhibited the proliferation of HepG2 liver cancer cells.
[0106] 3.4 Scratch Test Results
[0107] Effects of Alpinia officinarum polysaccharide on HepG2 cell migration
[0108] like Figure 3 As shown, 0.1, 0.3, and 0.5 mg / ml of Alpinia officinarum polysaccharide inhibited the migration and invasion of HepG2 liver cancer cells.
[0109] III. Experimental Conclusions
[0110] The results in summary indicate that:
[0111] Alpinia polysaccharide at concentrations of 0.1, 0.3, and 0.5 mg / ml inhibited the proliferation and migration of HepG2 liver cancer cells in a dose-dependent manner.
[0112] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.
Claims
1. The application of galangal polysaccharide in the preparation of anti-liver cancer drugs, characterized in that: Galangal polysaccharides are used in the preparation of drugs for the treatment and / or prevention of liver cancer. The preparation method of galangal polysaccharides includes the following steps: Step S1: After washing the galangal rhizomes, crush them and sieve them. Extract the galangal powder several times by reflux with hot water. Combine the extracts and concentrate them under reduced pressure. After precipitation, deproteinization by Sevag method, dialysis, freeze-drying and drying, crude polysaccharide AHP is obtained. Step S2: After the crude polysaccharide is dissolved, it is separated and eluted sequentially with ultrapure water, 0.1M NaCl, 0.2M NaCl and 0.3M NaCl solutions, and the eluent is collected. Step S3: Determine the sugar content of each eluent tube using the phenol-sulfuric acid method; Step S4: Redistill phenol and prepare test solution with distilled water. Use glucose as standard and accurately prepare glucose standard stock solution with redistilled water. Prepare standard curves according to the glucose standard stock solution at concentrations of 0.0, 30.0, 60.0, 90.0, 120.0, 150.0, and 180.0 μg / ml, and dilute to 50 ml in volumetric flasks. Step S5: Take each standard stock solution, add phenol test solution, mix evenly in a shaker, quickly add concentrated sulfuric acid, shake, place at room temperature, and measure the absorbance value. Step S6: Plot the sulfuric acid-phenol standard curve with the absorbance of the standard sample minus the absorbance of distilled water on the ordinate and the concentration of glucose standard on the abscissa, and calculate the regression equation. Step S7: Weigh the polysaccharide sample, make up to volume in a volumetric flask, measure the absorbance value, and substitute it into the standard curve equation to calculate the concentration of each sample. Step S8: Plot the polysaccharide content-tube number curve, and collect the polysaccharides according to the curve; after concentration, dialysis and freeze-drying, the preliminarily purified Alpinia galanga polysaccharide is obtained. Step S9: Using a Sephadex G-100 column with ultrapure water as the eluent, further purify the polysaccharide, determine the sugar content again, and plot the polysaccharide content-tube number curve. Step S10: After concentration and freeze-drying, purified polysaccharide is obtained.
2. The application of galangal polysaccharide according to claim 1 in the preparation of anti-liver cancer drugs, characterized in that: In step S1, the galangal is crushed and then passed through a 60-mesh sieve.
3. The application of galangal polysaccharide according to claim 1 in the preparation of anti-liver cancer drugs, characterized in that: In step S1, the hot water reflux temperature is 100℃, and the extraction is performed 3 times, each time for 1 hour.
4. The application of galangal polysaccharide according to claim 1 in the preparation of anti-liver cancer drugs, characterized in that: In step S1, the extracts are combined and concentrated under reduced pressure to 10%-20% of the original volume; Add 95% ethanol to a concentration of 60%, and allow to precipitate at 4°C for 12 hours.
5. The application of galangal polysaccharide according to claim 1 in the preparation of anti-liver cancer drugs, characterized in that: In step S2, the eluent is collected using an automated fraction collector at a flow rate of 0.5 mL / min for 30 minutes per tube.
6. The application of galangal polysaccharide according to claim 1 in the preparation of anti-liver cancer drugs, characterized in that: The dosage of the galangal polysaccharide is 0.1-0.5 mg / ml.
7. The application of galangal polysaccharide according to claim 1 in the preparation of anti-liver cancer drugs, characterized in that: The dosage concentration of the galangal polysaccharide was 0.1 mg / ml.
8. The application of galangal polysaccharide according to claim 1 in the preparation of anti-liver cancer drugs, characterized in that: The dosage concentration of the galangal polysaccharide was 0.3 mg / ml.
9. The application of galangal polysaccharide according to claim 1 in the preparation of anti-liver cancer drugs, characterized in that: The dosage concentration of the galangal polysaccharide was 0.5 mg / ml.