Medicine compound for treating colitis inflammatory diseases and preparation method of medicine compound

Abstract

The invention discloses a medicine compound for treating inflammatory diseases of colitis. The medicine compound is formed by compounding acetylated gastrodia elata gum and oxsalazine, the mass ratio of the acetylated gastrodia elata gum to the oxsalazine is (50-150): 20; the acetylated gastrodia elata gum is prepared by reacting a colloidal white solid obtained by water extraction and alcohol precipitation of gastrodia elata with acetic anhydride. According to the medicine compound, the gastrodia elata colloid polysaccharide with specific molecular weight is acetylated and subjected to porous treatment and then compounded with the oxsalazine according to a specific ratio, so that the medicine compound can be continuously released at a colon focus part, has colon targeting and slow release effects, can remarkably reduce oxidative stress of colon cells, and can be used for treating the colon focus part. The effect is better than that of any single gastrodia elata colloid polysaccharide and oxalazine, the synergistic effect is generated in the aspect of protecting colon cells, and the gastrodia elata colloid polysaccharide and oxalazine composition has actual popularization and application value.

Description

Technical Field

[0001] The present invention specifically relates to a drug complex for treating colonic inflammatory diseases and a preparation method thereof. Background Art

[0002] Colonic inflammatory diseases are a group of diseases characterized by inflammation of the colonic mucosa or the entire layer, mainly including ulcerative colitis, Crohn's disease, infectious colitis, etc. Ulcerative colitis is a chronic non-specific colonic inflammation, and severe cases develop ulcers. The lesions mainly involve the colonic mucosa and submucosa. The scope usually starts from the distal colon and can develop retrograde to the proximal colon, even involving the entire colon and the terminal ileum, showing a continuous distribution. The clinical manifestations mainly include diarrhea, abdominal pain, and mucus bloody stools. Crohn's disease can involve various parts of the gastrointestinal tract, mainly the terminal ileum and its adjacent colon. It is a chronic granulomatous inflammation, mostly showing segmental and asymmetric distribution. The onset is mostly hidden and gradual, and the course of the disease is chronic, with alternating active and remission periods of varying lengths and a tendency to relapse throughout life. The clinical manifestations vary depending on the lesion site, disease stage, and complications. The overall quality of life score of patients with colonic inflammatory diseases is usually at a relatively low level, and regular drug treatment is required to reduce the negative impact of the disease on daily life.

[0003] The traditional Chinese medicine Gastrodia elata Bl. is also known as Red Arrow, Wind-Stabilizing Grass, and Solitary Shaking Fungus. It belongs to the perennial herbaceous plants of the orchid family with fungal nutrition type. The dried rhizome of Gastrodia elata is sweet in taste and neutral in nature, and belongs to the liver meridian. It is one of the precious traditional Chinese medicines in China and has the effects of calming endogenous wind and stopping convulsions, suppressing hyperactive liver-yang, and expelling wind and dredging collaterals. Gastrodia elata polysaccharide is one of the main active components in Gastrodia elata. It not only has a high content but also low toxicity and side effects, and is one of the hotspots in current drug research and development. There are research reports that Gastrodia elata polysaccharide has anti-inflammatory properties and can effectively relieve the symptoms of inflammatory bowel disease (Di Xu, Therapeutic efficacy and underlying mechanisms of Gastrodia elata polysaccharides on dextran sulfate sodium-induced inflammatory bowel disease in mice: Modulation of the gut microbiota and improvement of metabolic disorders).

[0004] Olsalazine is a chemical drug formed by connecting two molecules of 5-aminosalicylic acid (5-ASA) through an azo bond. It is not absorbed in the stomach and small intestine. After reaching the colon, it is decomposed by intestinal bacteria into two molecules of 5-ASA, thereby exerting an anti-inflammatory effect. 5-ASA can inhibit the synthesis of prostaglandins and leukotrienes, reduce the permeability of the intestinal wall cell membrane, and relieve intestinal mucosal edema. It is mainly used for the treatment of acute and chronic ulcerative colitis, Crohn's disease, and long-term maintenance treatment during the remission period of these diseases.

[0005] There is currently no report on the combination of gastrodia polysaccharide and olsalazine for the treatment of colonic inflammatory diseases. Summary of the Invention

[0006] To solve the above problems, the present invention provides a drug complex for the treatment of colonic inflammatory diseases, which is composed of acetylated gastrodia glue and olsalazine;

[0007] The mass ratio of the acetylated gastrodia glue to olsalazine is 50-150:20;

[0008] The acetylated gastrodia glue is a colloidal white solid obtained by water extraction and alcohol precipitation of gastrodia, and is formed by reacting with acetic anhydride.

[0009] Further, the mass ratio of the acetylated gastrodia glue to olsalazine is 100:20.

[0010] Further, the acetylated gastrodia glue has a porous structure.

[0011] The present invention also provides a preparation method of the aforementioned drug complex, including the following steps:

[0012] 1) Take gastrodia, soak it in water for extraction, concentrate the extract, add ethanol, and collect the colloidal white solid to obtain gastrodia glial polysaccharide;

[0013] 2) Take the gastrodia glial polysaccharide obtained in step 1), dissolve it in water, add acetic anhydride dropwise to the dissolved solution for reaction, dialyze the liquid after the reaction is complete, and dry it to obtain acetylated gastrodia glue;

[0014] 3) Dissolve the acetylated gastrodia glue in water, freeze-thaw it to obtain acetylated gastrodia glue with a porous structure;

[0015] 4) Take the acetylated gastrodia glue with a porous structure obtained in step 3), grind it into powder, stir it with n-hexane, and at the same time dropwise add an aqueous solution of olsalazine. After the dropping is completed, continue to stir for 6-8 h, and remove n-hexane to obtain the gastrodia glue olsalazine complex.

[0016] Further, the amount of water added in step 1) is 15 times the amount of Gastrodia elata; the Gastrodia elata is in the form of Gastrodia elata pieces with a diameter less than 1 cm; the soaking time is 4 to 5 hours, the extraction is carried out by water bath extraction, the number of water bath extraction times is 3 times, the temperature for each time is 70 to 90 °C, and the time is 2 to 4 hours; the concentration is carried out by vacuum concentration at a temperature of 40 to 60 °C until the relative density reaches 1.05; ethanol is added to the solution until the ethanol concentration reaches 70%.

[0017] Further, in step 2), the dissolution solution is added with acetic anhydride at 40 to 60 °C and 800 to 1200 r / min; during the addition of acetic anhydride, the pH value of the solution is maintained at 9; during dialysis, the cut-off molecular weight of the dialysis bag is 2.5 kD, and the dialysis time is 3 to 5 days; the drying is freeze-drying; the mass-volume ratio of the Gastrodia elata glial polysaccharide, water and acetic anhydride is 2 to 6 g: 8 to 12 ml: 2 to 6 ml.

[0018] Further, in step 3), the mass-volume ratio of the acetylated Gastrodia elata gum to water is 0.5 g: 2 to 8 mL; the number of freeze-thaw cycles is 5 to 8 times, the freezing temperature for each time is -20 °C, the freezing time is 4 h, the thawing temperature is 25 °C, and thawing is carried out until the ice crystals completely melt.

[0019] Further, in step 4), the mass-volume ratio of the acetylated Gastrodia elata gum with porous structure, n-hexane and olsalazine aqueous solution is 50 to 150 mg: 30 ml: 0.5 ml; the concentration of the olsalazine aqueous solution is 30 to 50 mg / ml; the stirring speed of the n-hexane is 500 to 1000 r / min.

[0020] The present invention also provides the use of the aforementioned drug complex in the preparation of a drug for treating colonic inflammatory diseases.

[0021] Further, the colonic inflammatory diseases include colitis and Crohn's disease; the colitis includes ulcerative colitis.

[0022] The drug complex of the present invention acetylates and pores a Gastrodia elata glial polysaccharide with a specific molecular weight and then complexes it with olsalazine in a specific ratio, which can be continuously released at the colonic lesion site, has colon-targeted and sustained-release effects, and can significantly reduce the oxidative stress of colon cells. The effect is better than that of either the Gastrodia elata glial polysaccharide or olsalazine used alone, and a synergistic effect is produced in protecting colon cells, having practical popularization and application value.

[0023] Obviously, based on the above content of the present invention, according to the common general knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0024] The following is a further detailed description of the above content of the present invention in the form of specific embodiments. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Description of the Drawings

[0025] Figure 1 Preparation process of olsalazine gastrodia glue

[0026] Figure 2 Gastrodia elata Blume herbs after processing

[0027] Figure 3 Crude gastrodia polysaccharide (left) and separated gastrodia glutinous polysaccharide (right) after water extraction and alcohol precipitation

[0028] Figure 4 Standard curve of amylose for gastrodia glutinous polysaccharide fragments

[0029] Figure 5 Absolute molecular weight analysis chart

[0030] Figure 6 Molecular configuration diagram

[0031] Figure 7 1H NMR spectrum

[0032] Figure 8 Chain length distribution chart of gastrodia glutinous polysaccharide fragments

[0033] Figure 9 Acetylation reaction of gastrodia glue (left) and solubility of acetylated gastrodia glue (right)

[0034] Figure 10 Repeated freeze-thaw of acetylated gastrodia glue

[0035] Figure 11 Preparation of OLSA@Ac-GGEPs (left) and finished product powder (right)

[0036] Figure 12 NMR characterization

[0037] Figure 13 Before repeated freeze-thaw (left) and after repeated freeze-thaw (right)

[0038] Figure 14 Full-band absorption spectrum of olsalazine

[0039] Figure 15 Standard curve of olsalazine

[0040] Figure 16 Full-band spectrum of mesalazine

[0041] Figure 17Standard curve of olsalazine in gastric juice;

[0042] Figure 18 Standard curve of olsalazine in gastric juice and duodenal juice;

[0043] Figure 19 Standard curve of olsalazine in gastric juice, duodenal juice and small intestinal juice;

[0044] Figure 20 Standard curve of olsalazine in gastric juice, duodenal juice, small intestinal juice and colon juice;

[0045] Figure 21 Standard curve of mesalazine in gastric juice;

[0046] Figure 22 Standard curve of mesalazine in gastric juice and duodenal juice;

[0047] Figure 23 Standard curve of mesalazine in gastric juice, duodenal juice and small intestinal juice;

[0048] Figure 24 Standard curve of mesalazine in gastric juice, duodenal juice, small intestinal juice and colon juice;

[0049] Figure 25 Experimental device diagram (left), experimental diagram during release (right);

[0050] Figure 26 Timed sampling diagram;

[0051] Figure 27 In vitro release curve of olsalazine@gastrodia elata polysaccharide;

[0052] Figure 28 Effect of different drug treatments on the intracellular ROS expression level induced by DSS;

[0053] Figure 29 Comparative analysis of intracellular ROS relative fluorescence values;

[0054] Figure 30 Effect of different drug treatments on the mitochondrial polarization of cells induced by DSS;

[0055] Figure 31 Effect of different drug treatments on the ratio of red / green fluorescence of cell mitochondria. Detailed implementation mode

[0056] Example 1. Preparation of the drug-loaded gastrodia elata polysaccharide complex of the present invention

[0057] 1. Preparation of gastrodia elata glial polysaccharide

[0058] Weigh 2 kg of Gastrodia elata Blume pieces, crush them into small pieces with a diameter less than 1 cm, soak them in 15 times the amount (v / w) of water for 4 - 5 hours, then extract them in a water bath at 80 °C for 3 hours. Filter and separate the extract while it is hot with a 300 - mesh filter cloth. Repeat the extraction of the filter residue twice in the same way. Combine the three extracts, concentrate them under reduced pressure at 50 °C to a relative density of 1.05. After cooling to room temperature, add ethanol until the ethanol concentration reaches 70%, and collect the colloidal white solid to obtain Gastrodia elata Blume glial polysaccharide (GGEPs);

[0059] 2. Preparation of acetylated Gastrodia elata Blume glue

[0060] Weigh 4 g of Gastrodia elata Blume glial polysaccharide and 10 ml of water, dissolve them by ultrasonic treatment, stir at 50 °C and 1000 r / min, and simultaneously add 4 ml of acetic anhydride dropwise. During the dropping process, adjust the pH value with sodium hydroxide solution to keep the pH value of the mixed solution at 9. After the dropping is completed, react for 4 hours, cool to room temperature, put the reaction solution into a 2.5 kD dialysis bag, and dialyze in running water for 3 - 5 days. Freeze - dry the dialyzed reaction solution to obtain acetylated Gastrodia elata Blume glue (Ac - GGEPs);

[0061] 3. Preparation of acetylated Gastrodia elata Blume glue with porous structure

[0062] Weigh 0.5 g of acetylated Gastrodia elata Blume glue, dissolve it in 5 mL of ultrapure water to form a 10% aqueous solution. After equilibrating at room temperature for 0.5 h, freeze it at - 20 °C for 4 h, then thaw it at 25 °C until the internal ice crystals completely melt. Repeat the freeze - thaw process 6 times in the same way to obtain acetylated Gastrodia elata Blume glue with porous structure;

[0063] 4. Preparation of Gastrodia elata Blume glue olsalazine complex

[0064] Weigh 100 mg of acetylated Gastrodia elata Blume glue with porous structure, grind it into powder, add 30 ml of n - hexane, stir at 800 r / min, and simultaneously add 0.5 ml of a 40 mg / ml olsalazine aqueous solution dropwise. After the dropping is completed, continue to stir for 6 - 8 h, and remove the n - hexane to obtain Gastrodia elata Blume glue olsalazine complex OLSA@Ac - GGEPs.

[0065] The beneficial effects of the present invention are illustrated below through test examples.

[0066] Test Example 1 Research on drug - loaded Gastrodia elata Blume polysaccharide complex

[0067] 1. Research route and research content

[0068] The research route map is shown in Figure 1 。

[0069] 1.1 Processing and preparation of Gastrodia elata Blume

[0070] Gastrodia elata is a precious traditional Chinese medicine, and its dried tuber is the main part used in medicine. Gastrodia elata has the effects of calming endogenous wind and stopping convulsions, suppressing hyperactive liver-yang, expelling wind and dredging collaterals, etc., and is commonly used to treat symptoms such as headache and dizziness, limb numbness, infantile convulsions, etc. Its medicinal properties are mild, and it can be used alone or in combination with other medicinal materials to enhance the curative effect. Gastrodia elata requires a special growth environment and mostly grows in humid and cool environments. Therefore, its output is limited and it is particularly precious. The processing and preparation of Gastrodia elata is a delicate and multi-step process aimed at improving its shelf life, medicinal efficacy and clinical applicability. Gastrodia elata needs to be dug during the appropriate time period, carefully washed to remove soil and impurities, and then classified according to its size. Gastrodia elata needs to be softened by soaking or steaming to make the internal structure of Gastrodia elata soft and facilitate subsequent slicing operations. The sliced Gastrodia elata needs to be dried by methods such as sun drying or drying to reach an appropriate dryness level, which helps to extend the shelf life of Gastrodia elata. In order to further enhance the medicinal efficacy of Gastrodia elata, sometimes special preparation methods such as frying, roasting or stir-frying with ginger juice are also required. The prepared dried Gastrodia elata needs to be stored in a cool and dry place to prevent it from getting damp or deteriorating, so as to ensure its quality and effect in clinical use. The Gastrodia elata slices used in this study were prepared according to the local standard of Chongqing, "Technical Regulations for Primary Processing of Gastrodia elata" (No.: DB50 / T1498-2023)( Figure 2 ).

[0071] 1.2 Extraction of Colloidal Polysaccharides from Gastrodia elata Slices

[0072] Weigh 2 kg of Gastrodia elata slices, use a hammer to beat or a chopper to cut them into small pieces less than 1 cm, place them in a 20 L large bucket, add water until the water surface covers the Gastrodia elata pieces (more than 15 times the volume), and soak for 4 - 5 hours. Extract in a water bath at 80 °C for 3 hours, filter and separate the extract while it is hot with a 300-mesh filter cloth, retain the filter residue and repeat the above process 2 more times, and combine the 3 extracts. Use a rotary evaporator to distill the extract under reduced pressure at 50 °C until it becomes slightly viscous (relative density about 1.05) to obtain a concentrated solution. Cool the concentrated solution to room temperature, add ethanol to it until the ethanol concentration reaches 70%, and at this time, two different types of polysaccharides, dark-colored massive solids and colloidal white solids, are observed to precipitate. The white colloidal solid is colloidal Gastrodia elata polysaccharide (Gastrodia elata glue, Gelatinous GEPs, GGEPs). There are certain differences in the density and volume of the two polysaccharides, and they can be stratified by floating and then standing still. At this time, absorb and separate Gastrodia elata glue from the suspension, filter to remove ethanol, redissolve with water and repeat the above alcohol precipitation process to remove residual small molecule substances such as gastrodin and parishin, and after filtering again, freeze-dry to obtain solid Gastrodia elata glue( Figure 3 ).

[0073] 1.3 Characterization of Gastrodia elata Colloidal Polysaccharide Fragments

[0074] The gummy polysaccharide fragments in Gastrodia elata were found to have amyloid gelatinization properties through preliminary tests. Therefore, physicochemical studies were carried out on the prepared gummy Gastrodia elata polysaccharide GGEPs using methods such as starch identification and determination. Through key indicators such as amylose content, starch polymerization degree, branching degree, molecular weight, viscosity, and gelatinization properties, accurate and stable data and reliable technical support were provided for related research such as the development of starch-based materials from the gummy substances of Gastrodia elata.

[0075] 1.3.1 Determination of resistant starch content

[0076] Resistant starch, also known as anti-enzyme-digested starch and indigestible starch, cannot be enzymatically digested in the small intestine but can undergo fermentation reactions with volatile fatty acids in the colon of the human gastrointestinal tract. To detect the content of resistant starch in the sample, mainly α-amylase and amyloglucosidase are used to remove non-resistant starch, the residue is dissolved with KOH, adjusted to neutral with acetate buffer, and the starch is hydrolyzed to glucose by amyloglucosidase. The GOPOD reagent buffer reacts with D-glucose to form quinoneimine dye, and its content is determined by colorimetry. After testing, no resistant starch was detected in the gummy polysaccharide of Gastrodia elata.

[0077] Table 1 Detection results of resistant starch content in the gummy polysaccharide of Gastrodia elata

[0078]

[0079] 1.3.2 Determination of total starch content

[0080] To detect the starch content in the sample, mainly heat-resistant α-amylase is used to hydrolyze starch into branched and unbranched maltodextrins, and then amyloglucosidase is used to quantitatively hydrolyze maltodextrin into D-glucose. The GOPOD reagent buffer reacts with D-glucose to form quinoneimine dye, and its content is determined by colorimetry. After testing, the total starch content in the gummy polysaccharide of Gastrodia elata is 49.95%.

[0081] Table 2 Detection results of total starch content in the gummy polysaccharide of Gastrodia elata

[0082]

[0083] 1.3.3 Determination of amylose content

[0084] The amylose content in the sample is mainly determined by taking advantage of the different affinities of amylose and amylopectin for iodine. By preparing standard solutions of amylose with different ratios and making a standard curve, the amylose content in the sample can be quantitatively determined ( Figure 4 ). After testing, no amylose was detected in the gummy polysaccharide sample of Gastrodia elata.

[0085] Table 3 Detection results of amylose content in the gummy polysaccharide of Gastrodia elata

[0086] Number Mass (mg) OD Value Content (%) 1 11.20 0.049 Not Detected 2 10.95 0.049 Not Detected 3 10.83 0.049 Not Detected

[0087] 1.3.4 Molecular weight detection

[0088] For the molecular weight detection of starch, the absolute molecular weight of the sample is mainly measured by coupling gel chromatography, laser light scattering and differential detector. Gel chromatography can elute the molecules in the solvent in sequence according to weight or size. Larger molecules can only enter the part of the gel pores with larger pore sizes, while smaller molecules can enter more gel particles. In this way, larger molecules move a shorter distance in the gel bed, and smaller molecules move a longer distance. Larger molecules are eluted first, and smaller molecules are eluted later to achieve the separation effect, so as to achieve the purpose of separation according to molecular size; furthermore, the differential detector is used to detect the concentration information of the sample according to its refractive intensity, the multi-angle laser light scattering instrument is used to detect the light scattering information of macromolecules, and the molecular weight corresponding to each component is calculated according to the Mark-Houwink Equation. The molecular weight of Gastrodia elata glial polysaccharide is Mw = 35581.273 kDa.

[0089] Table 4 Detection results of the molecular weight of Gastrodia elata glial polysaccharide

[0090]

[0091] The chromatographic data is processed using the software ASTRA6.1. Figures 5 - 6 Shown are the absolute molecular weight analysis chart and the molecular configuration analysis chart of the sample. The absolute molecular weight analysis chart uses time (min) as the abscissa and molar mass (g / mol) as the ordinate; the molecular configuration chart uses molar mass (g / mol) as the abscissa and root mean square radius (nm) as the ordinate.

[0092] Absolute molecular weight analysis chart ( Figure 5 ) The red line: multi-angle laser light scattering signal, reflecting the molecular size of the sample; blue line: differential signal, reflecting the sample concentration; black line: molecular weight fitted by the two signals. The blue line is the differential signal, representing the concentration of the sample; the red line is the light scattering signal, representing the molecular size of the sample. In the molecular configuration chart ( Figure 6 ) with molar mass as the abscissa and radius of gyration as the ordinate, its slope can be used as a reference for molecular configuration. Usually, slope = 1 indicates a rod-shaped molecule, slope = 0.5 - 0.6 indicates a random coil, and slope = 1 / 3 indicates a spherical shape. The slope of Gastrodia elata glial polysaccharide is 0.16, so it is speculated to be a dense sphere.

[0093] 1.3.5 Gelatinization temperature detection

[0094] Starch is a tandem complex formed by glucose through α-1,4 and α-1,6 glycosidic bonds and has a crystalline semi-crystalline structure. As the temperature rises, the higher-order structures such as the double helix structure and crystalline structure in starch are destroyed. For native starch, this process is called gelatinization; different starches have different structures, and their phase transition processes, the temperatures and energies required for phase transition also vary; in the research of starch and related polymer derivatives, by measuring the phase transition process of starch and its derivatives with a differential scanning calorimeter (DSC), the structure and physicochemical properties of starch can be analyzed. After testing, the gelatinization temperature of gastrodia glue has an onset temperature of 52.6 °C, a peak temperature of 69.5 °C, an end temperature of 94.3 °C, and a gelatinization enthalpy of 5.533 J / g.

[0095] Table 5 Detection results of the gelatinization temperature of gastrodia glutinous polysaccharide

[0096]

[0097] 1.3.6 Branching degree and chain length distribution

[0098] Based on the information such as the chemical shift provided by nuclear magnetic resonance of hydrogen, the position of protons on the carbon chain can be inferred. The data is analyzed using MestReNova software. According to the peak emergence time of the sample, the peak emergence range is selected to obtain the final result. The following formula is used for calculation:

[0099]

[0100] DB: Branching degree

[0101] A: Peak area of α-1,6 bond.

[0102] B: Peak area of α-1,4 bond

[0103] The nuclear magnetic resonance results ( Figure 7 ) show that the branching degree of gastrodia glutinous polysaccharide is 6.65%.

[0104] Chain length distribution ( Figure 8 ): Standards with different chain lengths have different retention times on the chromatogram. Qualitative analysis is carried out according to the retention time of the sample on the chromatographic column, and then the concentration is calculated from the standard curve based on the sample peak area. The test results show that the average degree of polymerization is 19.7.

[0105] 1.3.7 Summary of the characterization of gastrodia glutinous polysaccharide

[0106] Characterization shows that the gummiferous gastrodia elata polysaccharide GGEPs obtained by extraction and separation of gastrodia elata slices are natural polysaccharides with a certain degree of branching, starch-like properties, and high molecular weight. The interaction between the high molecular weight and branched chains of gummiferous gastrodia elata polysaccharide GGEPs can provide better stability when used as a carrier. Moreover, the gelatinization properties of gastrodia elata gum also indicate that its solubility in water is limited or the dissolution rate is slow, and it requires more time to metabolize in digestive juice. Therefore, it can be used as a natural intestinal targeting delivery material.

[0107] 1.4 Acetylation modification of gastrodia elata gum

[0108] Acetylation modification of polysaccharides is an important chemical operation. It changes the structure and properties of polysaccharides by introducing acetyl groups into polysaccharide molecules. After acetylation modification, the hydroxyl groups of polysaccharides are exposed outside, increasing their solubility in water and possibly enhancing biological activity. The number and substitution position of acetyl groups have a direct impact on the biological activity of polysaccharides. For example, when the O-3 position is substituted by an acetyl group, the anti-tumor activity increases; while when the O-5 position is substituted by an acetyl group, the anti-tumor activity is significantly weakened. Acetylation modification can also change the physicochemical properties such as the hydrophobicity and hardness of polysaccharides, so that they show better performance in aspects such as emulsification and foam stability. This modification method is widely used in the structural modification and functional optimization of polysaccharides. Acetylation modification of polysaccharides can also be used to improve the properties of polysaccharides, such as enhancing stability and biological activity. Acetylation modification also provides new ideas and methods for the development of new polysaccharide products.

[0109] Weigh 4 g of gummiferous gastrodia elata polysaccharide and 10 ml of water into a 200 ml conical flask, dissolve it by ultrasonic wave, heat it to 50 °C, add a magnetic stirrer and stir at a speed of 1000 r / min. Prepare 20 ml of 6 M sodium hydroxide solution for later use. Drop a small amount of acetic anhydride into the gastrodia elata gum solution, and at the same time add sodium hydroxide solution for neutralization. Use pH test paper to detect the pH value and make the pH value equal to 9. Continuously add acetic anhydride and sodium hydroxide until 4 ml of acetic anhydride is added. After reacting for 4 hours, cool it to room temperature, put the reaction solution into a 2.5 kD dialysis bag, and place it for 3 - 5 days under the condition of running water or water bath (changing water frequently). Freeze-dry the dialysis-completed reaction solution to obtain acetylated gastrodia elata gum (Ac-GGEPs). See the preparation process diagram in Figure 9 .

[0110] 1.5 Repeated freeze-thaw of acetylated gastrodia elata gum

[0111] Weigh 0.5 g of acetylated gastrodia glue (dry sample), add 5 mL of ultrapure water to make a 10% aqueous solution, place it in a petri dish of appropriate size, equilibrate at room temperature for 0.5 h, then put the petri dish into the freezer at -20 °C for 4 h, and thaw (or melt) it at a constant temperature of 25 °C until the internal ice crystals of the gastrodia glue completely melt. Complete 1 freeze-thaw process, that is, freeze-thaw once, and repeat the freeze-thaw process 6 times. After the freeze-thaw process is completed, lyophilize the sample to obtain a porous structure and store it at 4 °C for later use. See the preparation process diagram in Figure 10 。

[0112] 1.6 Preparation of olsalazine-loaded acetylated gastrodia glue complex (double-solvent method)

[0113] Weigh 100 mg of acetylated gastrodia glue after repeated freeze-thawing, grind it into small particle powder repeatedly, add it to a 50 mL centrifuge tube together with 30 mL of n-hexane, add a stir bar of appropriate size and stir at a speed of 800 r / min. Ultrasonically dissolve 20 mg of olsalazine (OLSA) in 0.5 mL of water, suck this olsalazine solution with a plastic dropper, and add it dropwise to n-hexane under stirring. Place the above mixture in a magnetic stirrer or ultrasonic crusher and stir overnight. After stirring, take out the stir bar, slowly pour out most of the n-hexane, and remove the small amount of n-hexane remaining on the product by drying in a vacuum drying oven at 40 °C for 2 h to obtain acetylated gastrodia glue loaded with olsalazine by the double-solvent method, OLSA@Ac-GGEPs. No olsalazine was detected in the n-hexane solution by ultraviolet detection, so the drug loading is considered to be approximately 20%. See the preparation process diagram in Figure 11 。

[0114] 1.7 Characterization of OLSA@Ac-GGEPs

[0115] 1.7.1 NMR characterization

[0116] Weigh 0.009 g of OLSA@Ac-GGEPs, dissolve it in 0.6 mL of deuterated DMSO to prepare a test solution with a concentration of 15 mg / mL. Use a 400 HZ nuclear magnetic resonance spectrometer (model: AVANCE500) to detect the 1 1H NMR spectrum of the test sample. Test the NMR spectrum of pure OLSA according to the above steps. It can be known from 1H NMR characterization (as Figure 12 ), the double-solvent method can be used to encapsulate olsalazine into acetylated gastrodia polysaccharide to successfully prepare the OLSA@Ac-GGEPs delivery system.

[0117] 1.7.2 Scanning electron microscopy

[0118] Weigh a small amount of OLSA@Ac-GGEPs samples and dry them in an oven at 40 °C. Fix them on the sample stage. Since OLSA@Ac-GGEPs is not conductive, sputter gold for 30 s, set the voltage to 15 kV, and observe the microstructure of the samples under a scanning electron microscope (model: Quattro S) and collect images. From the electron microscopy results ( Figure 13 ), it can be seen that OLSA@Ac-GGEPs after repeated freeze-thaw cycles can form relatively uniform pores, which is beneficial for subsequent drug encapsulation.

[0119] 1.8 Determination of the drug content in OLSA@Ac-GGEPs

[0120] Currently, for mild to moderate UC patients clinically, aminosalicylate drugs are first selected for treatment, such as mesalazine and olsalazine. Mesalazine, also known as 5-aminosalicylic acid (5-ASA), mainly inhibits the synthesis of leukotriene synthase, reduces the production of leukotriene, and inhibits the synthesis of prostaglandin and the formation of the inflammatory mediator leukotriene, thus significantly inhibiting the inflammation of the intestinal mucosa; Olsalazine is an isomer of 5-aminosalicylic acid, composed of two molecules of 5-ASA coupled together, and releases two molecules of 5-aminosalicylic acid in the colon to play a role, with very low toxicity and is suitable for the long-term treatment of ulcerative colitis. Olsalazine will also be partially converted into mesalazine in water. Therefore, the determination of the drug content in the prepared complex OLSA@Ac-GGEPs needs to include two kinds: olsalazine and mesalazine.

[0121] 1.8.1 Determination of the content of olsalazine

[0122] (1) Preparation of the standard solution

[0123] Accurately weigh 10 mg of olsalazine into a 50 ml volumetric flask, and make up the volume with pH 6.8 phosphate buffer solution to prepare a 0.2 mg / ml stock solution.

[0124] (2) Determination of the detection wavelength

[0125] The aqueous solution or phosphate buffer solution of sodium olsalazine has a maximum absorption in the ultraviolet region. Accurately measure 1.0 ml of the olsalazine stock solution into a 50 ml volumetric flask, dilute it to the mark with pH = 6.8 phosphate buffer solution, and use pH = 6.8 phosphate buffer solution as the reference. Scan in the wavelength range of 200 - 700 nm. Figure 14 Result: There is a maximum absorption at 360 nm. Therefore, 360 nm is determined as the detection wavelength.

[0126] (3) Establishment and determination of the standard curve

[0127] Precisely pipette 0, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of the stock solution into 10-ml volumetric flasks, and dilute to the mark with pH = 6.8 phosphate buffer solution. Using the pH = 6.8 phosphate buffer solution as the reference, measure the absorbance at a wavelength of 360 nm. Perform a linear regression with concentration C against absorbance A, and the regression equation is C = 72.925×A - 0.0448, R2 = 0.999( Figure 15 ). It shows that olsalazine has a good linear relationship in the range of 4 - 20 μg·ml. Obtain standard solutions with different concentration gradients and measure the ultraviolet absorption at a wavelength of 360 nm.

[0128] (4) Sample detection

[0129] Take 1.27 mg of the OLSA@Ac-GGEPs sample and transfer it to a 10-ml volumetric flask. Dilute to the mark with pH 6.8 phosphate buffer solution and sonicate for 10 min to prepare a 0.127 mg / ml solution. Measure the ultraviolet absorption at 360 nm, which is 1.544. Calculate the concentration of olsalazine to be 0.0216 mg / ml according to the standard curve, and the drug loading is 17.01%. The drug loading measured by spectrophotometry is less than the theoretical value. This is because olsalazine is encapsulated in the large molecular weight gastrodia elata polysaccharide phase and cannot be completely dissolved into the phosphate buffer solution in a short time due to diffusion limitation. Even if waiting for a long time for sufficient diffusion, olsalazine will be partially converted into mesalazine in water. Therefore, the actually measured drug loading of olsalazine is less than the true value by 20%.

[0130] 1.8.2 Determination of mesalazine content

[0131] (1) Preparation of standard solution

[0132] Precisely weigh 10 mg of mesalazine and transfer it to a 50-ml volumetric flask. Dilute to the mark with pH 6.8 phosphate buffer solution to prepare a 0.2 mg / ml stock solution.

[0133] (2) Determination of the detection wavelength

[0134] Mesalazine in aqueous solution or phosphate buffer solution has a maximum absorption in the ultraviolet region. Precisely pipette 1.0 ml of the mesalazine stock solution into a 50-ml volumetric flask and dilute to the mark with pH = 6.8 phosphate buffer solution, using the pH = 6.8 phosphate buffer solution as the reference. Perform a scan in the wavelength range of 200 - 700 nm. Figure 16 Result: There is a maximum absorption at 303 nm. Therefore, 303 nm is determined as the detection wavelength.

[0135] (3) Establishment and determination of the standard curve

[0136] Same as 1.8.1(3)

[0137] (4) Sample detection

[0138] Same as 1.8.1(4) OLSA@Ac-GGEPs initial sample, mesalazine was not detected.

[0139] 1.9 In vitro release experiment of OLSA@Ac-GGEPs

[0140] 1.9.1 Establishment of olsalazine standard curve

[0141] Precisely weigh 10 mg of olsalazine into a 50 ml volumetric flask, dilute to the mark with pH 6.8 phosphate buffer solution to prepare a stock solution of 0.2 mg / ml. Precisely pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0 ml of the stock solution into 10 ml volumetric flasks, and dilute to the mark with artificial gastric juice, duodenal juice, small intestinal juice and colonic juice respectively. Using the corresponding simulated solution as the reference, measure the absorbance at a wavelength of 360 nm, and perform a linear regression with concentration C against absorbance A. The fitting curves R in the four different media 2 Are all around 0.999, Figures 17 - 20 Indicating that olsalazine shows a good linear relationship in the range of 4 - 20 μg·ml.

[0142] 1.9.2 Establishment of mesalazine standard curve

[0143] Precisely weigh 10 mg of mesalazine into a 10 ml volumetric flask, dilute to the mark with pH 6.8 phosphate buffer solution to prepare a stock solution of 1.6 mg / ml. Precisely pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0 ml of the stock solution into 10 ml volumetric flasks, and dilute to the mark with artificial gastric juice, duodenal juice, small intestinal juice and colonic juice respectively. Using the corresponding simulated solution as the reference, measure the absorbance at a wavelength of 303 nm, and perform a linear regression with concentration C against absorbance A. The fitting curves R in the four different media 2 Are all around 0.99, Figures 21 - 24 Indicating that mesalazine shows a good linear relationship in the range of 20 - 100 μg·ml.

[0144] 3.9.3 Obtaining samples for in vitro release experiment

[0145] (1) Pretreatment of dialysis bag

[0146] Take an appropriate amount of dialysis bag, cut it into a suitable length, soak it in distilled water overnight to remove impurities and possible chemical substances. Tie a knot at one end of the dialysis bag to ensure good sealing.

[0147] (2) Drug loading

[0148] Accurately measure 10 mg of OLSA@GGEPs into 10 ml of distilled water to prepare a drug solution with a concentration of 1 mg / ml. Inject it into the dialysis bag through the untied end, then tie a knot at this end to seal the drug in the dialysis bag, and try to avoid residual air bubbles.

[0149] (3) Preparation of digestive fluid

[0150] The preparation of the following digestive fluids is carried out in accordance with the Chinese Pharmacopoeia.

[0151] Artificial gastric juice: Take 16.4 mL of dilute hydrochloric acid, add 800 mL of water and 10 g of pepsin, shake well, and then dilute with water to 1000 mL; among them, the dilute hydrochloric acid is obtained by diluting 234 mL of concentrated hydrochloric acid with water to 1000 mL to obtain 9.5% - 10.5% dilute hydrochloric acid.

[0152] Duodenal fluid: Weigh 6.8 g of sodium chloride, 0.4 g of potassium chloride, and 0.06 g of potassium dihydrogen phosphate, dissolve them in an appropriate amount of water and make up the volume to 1000 mL to obtain solution A; weigh 10.0 g of sodium bicarbonate, dissolve and make up the volume to 100 mL to obtain solution B; weigh 0.04 g of calcium chloride, dissolve and make up the volume to 100 mL to obtain solution C; take 850 mL of solution A, add 100 mL of solution B and 10 mL of solution C, mix and adjust the pH to 7.5 - 8.0; add 1.0 g of trypsin and 2.5 g of bile salts, stir to dissolve, transfer to a 1000 mL volumetric flask and make up the volume to obtain the solution.

[0153] Artificial small intestinal fluid: Take 6.8 g of potassium dihydrogen phosphate at pH 6.8, add 500 mL of water to dissolve it, and adjust the pH value to 6.8 with 0.1 mol / L

[0154] sodium hydroxide solution; take 10 g of pancreatin, add an appropriate amount of water to dissolve it, mix the two solutions, and then dilute with water to 1000 mL to obtain the solution;

[0155] Artificial colon fluid: At pH 7.8, 5.59 g of dipotassium hydrogen phosphate and 0.41 g of potassium dihydrogen phosphate are dissolved in 1000 mL of water to obtain the solution.

[0156] (4) Simulated gastric juice release stage (2 hours)

[0157] Add 30 ml of simulated gastric juice to a clean stoppered conical flask. Place the dialysis bag containing the drug into the conical flask to ensure that the dialysis bag is completely immersed in the gastric juice. Place the conical flask in a constant temperature water bath oscillator, set the temperature to 37 °C, the oscillation speed to 100 rpm, and avoid light. Within 2 hours, at time points of 0.5 hour, 1 hour, and 1.5 hours, take out 2 ml of gastric juice from the conical flask for drug concentration detection, and supplement 2 ml of fresh simulated gastric juice after each sampling.

[0158] (5) Transfer to the simulated duodenal fluid stage (2 hours)

[0159] After 2 hours, transfer all the gastric juice in the dialysis bag and the conical flask to a new conical flask containing 30 ml of simulated duodenal fluid. Place the new conical flask in a constant temperature water bath oscillator, maintain the temperature at 37 °C, the oscillation speed at 100 rpm, and avoid light. During these 2 hours, take out 2 ml of the mixed solution at 0.5 hour, 1 hour, and 1.5 hours respectively for drug concentration detection. After each sampling, supplement 2 ml of the solution obtained by mixing simulated duodenal fluid and simulated gastric juice in a volume ratio of 30:30 (to keep the total volume and composition ratio relatively stable).

[0160] (6) Transfer to the simulated intestinal fluid stage (4 hours)

[0161] After the 2-hour simulated duodenal fluid stage ends, transfer the mixed solution containing the dialysis bag to a conical flask containing 30 ml of simulated intestinal fluid. Continue the release experiment in a constant temperature water bath oscillator at 37 °C and 100 rpm. Take 2 ml of samples at time intervals of 0.5 hour, 1 hour, 2 hours, and 3 hours for drug concentration detection. After each sampling, supplement 2 ml of the solution obtained by mixing simulated intestinal fluid and the previous mixed solution in a volume ratio of 30:(30 + 30).

[0162] (7) Transfer to the simulated colon fluid stage (8 hours)

[0163] After the intestinal fluid stage is completed, transfer the dialysis bag and the mixed solution to a conical flask containing 30 ml of simulated colon fluid. Continue the release at 37 °C and 100 rpm. Take 2 ml of samples at time points of 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, and 7 hours to detect the drug concentration. After each sampling, supplement 2 ml of the solution obtained by mixing simulated colon fluid and the previous mixed solution in a volume ratio of 30:(30 + 30 + 30).

[0164] The process diagram of the release experiment is shown in Figure 25 .

[0165] 1.9.4 Sample determination and result analysis

[0166] Draw a curve of concentration versus time based on the measured data. Measure the ultraviolet absorption of the samples taken at each time point at a wavelength of 360 nm with an ultraviolet spectrophotometer (Shimadzu UV2600i), calculate the concentration according to the corresponding standard curve of the simulated release medium, and according to the formula: cumulative release rate (%) = (V 0 ×C t +V×∑C i )×100% / m, where: V 0 is the total volume of the release medium. C tis the concentration of the drug in the release medium at the last sampling time point (μg·mL⁻¹); V is the volume of each sampling; ΣC i is the sum of the concentrations of the drug in the release medium at all sampling times from the first sampling time point to the last sampling time point; m is the total amount of the drug in the pharmaceutical preparation (mg).

[0167] This formula is used to calculate the proportion of the drug released from the preparation within a given time. By regularly sampling ( Figure 26 ) during the experiment and measuring the concentration of the drug in the samples, the cumulative release rate ( Figure 27 ) can be calculated, thereby evaluating the release performance of the drug.

[0168] From the continuous release tests of olsalazine, mesalazine, and OLSA@Ac-GGEPs in the simulated medium, it can be seen that after olsalazine and mesalazine monomers enter the simulated digestive environment, they are rapidly released in the stomach, duodenum, and small intestine. The release rate of olsalazine reaches 80% after entering the small intestine and is completely released 2 hours after entering the colon. Mesalazine is completely released before entering the colon and cannot reach the target organ.

[0169] From the in vitro release profile of olsalazine from OLSA@Ac-GGEPs in the simulated digestive tract environment, the release amount of olsalazine@gastrodia polysaccharide complex in gastric juice is about 0.5% in 2 hours. It can be seen that gastrodia glutinous polysaccharide wraps the drug molecules, significantly reducing the release of the drug in gastric juice; when the complex enters the intestinal juice, the release accelerates and the release amount reaches about 40%; when the complex reaches the colon, the drug is continuously released and gradually tends to balance in about 20 hours.

[0170] In addition, under the action of specific azoreductase in the colon, olsalazine can break the azo bond and be reduced to two molecules of mesalazine, playing anti-inflammatory and other therapeutic roles in the form of mesalazine in the colon. Therefore, the drug concentration of mesalazine is synchronously collected during the drug release process. From the in vitro release profile of mesalazine from OLSA@Ac-GGEPs in the simulated digestive tract environment, the release trend of mesalazine is consistent with that of olsalazine, indicating that olsalazine is decomposed into mesalazine after being released from the OLSA@Ac-GGEPs complex, and the release amount of mesalazine detected in the colon environment reaches about 70%.

[0171] Based on the above results, it is confirmed that gastrodia glutinous polysaccharide as a drug carrier can effectively protect the drug from premature release in the upper and middle segments of the digestive tract and can continuously release at the colon lesion site, having colon targeting and sustained release effects.

[0172] 1.10 Protective effect of OLSA@Ac-GGEPs on colon cells

[0173] 1.10.1 Detection of ROS by DCFH-DA probe method

[0174] Dextran sodium sulfate (DSS) is a water-soluble sulfated polysaccharide. Due to its negative charge, it is toxic to intestinal epithelial cells, can damage its barrier integrity and increase colonic epithelial permeability, inducing intestinal injury. When the antioxidant capacity of cells decreases and is insufficient to clear the excessive accumulation of lipid ROS, oxidative stress occurs in cells. To evaluate this, the effects of OLSA@Ac-GGEPs on the ROS levels in DSS-induced Caco-2 cells were tested, with Ac-GGEPs, OLSA, and mesalazine (MSZ) as controls. The specific steps are as follows:

[0175] (1) Stimulation

[0176] In a 6-well plate, after 2.5×10 5 cells were seeded in the wells and the adherent cells grew for 12 h, cell culture media containing 2.5% DSS, and 10 μg / mL OLSA@Ac-GGEPs, 10 μg / mL Ac-GGEPs, 10 μg / mL OLSA, and 10 μg / mL MSZ were added to the cell wells respectively. A blank treatment group was set to add normal culture media, and the cells were continued to be cultured in the incubator for 24 h. Then, probe staining was performed to detect the changes in intracellular ROS.

[0177] (2) Staining

[0178] A. Dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to make the final concentration 10 μmol / L. Remove the cell culture medium and add an appropriate volume of the diluted DCFH-DA.

[0179] B. Add 1 mL of the diluted DCFH-DA to one well of a 12-well plate. Incubate in a 37 °C cell incubator for 20 minutes.

[0180] C. Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells.

[0181] D. Observe and take pictures of live cells directly under a fluorescence microscope (200×). Green light: excitation light is set at 490 nm and emission light is set at 530 nm.

[0182] The results are shown in Figures 28 - 29The results showed that the fluorescence intensity of the DSS group was significantly higher than that of the blank group, indicating that 2.5% DSS induced oxidative stress in Caco-2 cells. After treatment with 10 μg / mL OLSA@Ac-GGEPs, 10 μg / mL Ac-GGEPs, 10 μg / mL OLSA, and 10 μg / mL MSZ, the fluorescence intensity decreased significantly. Moreover, the intracellular ROS level in the OLSA@Ac-GGEPs group decreased more significantly than that in the Ac-GGEPs, OLSA, and MSZ groups, indicating that OLSA@Ac-GGEPs treatment could reduce the intracellular ROS level, alleviate DSS-induced oxidative stress, and produce a synergistic effect.

[0183] 1.10.2 Detection of changes in mitochondrial membrane potential of cells

[0184] JC-1 is a commonly used fluorescent probe for detecting changes in mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix and presents green fluorescence. Therefore, the effect of OLSA@Ac-GGEPs on the mitochondrial membrane potential of DSS-induced Caco-2 cells was tested by the transition of JC-1 from red fluorescence to green fluorescence, with Ac-GGEPs, OLSA, and MSZ as controls. The specific steps are as follows:

[0185] (1) Stimulation

[0186] In a 6-well plate, after 12 h of adherent cell growth in the cell inoculation wells with 2.5×10 5 cells, cell culture media containing 2.5% DSS, and 10 μg / mL OLSA@Ac-GGEPs, 10 μg / mL Ac-GGEPs, 10 μg / mL OLSA, and 10 μg / mL MSZ were added to the cell wells respectively, and a blank treatment group was set to add normal culture media. The cells were continued to be cultured in the incubator for 24 h, and then probe staining was performed to detect the changes in the mitochondrial membrane potential of the cells.

[0187] (2) Staining

[0188] A. Take an appropriate amount of JC-1 (200×) and dilute JC-1 according to the ratio of adding 8 mL of ultrapure water to every 50 μl of JC-1 (200×). Vortex vigorously to fully dissolve and mix JC-1. Then add 2 mL of JC-1 staining buffer (5×) and mix well to obtain the JC-1 staining working solution.

[0189] B. Add 1 ml of cell culture medium.

[0190] C. Add 1 mL of JC-1 staining working solution and mix well. Incubate at 37 °C in a cell culture incubator for 20 minutes.

[0191] D. During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1×) by adding 4 mL of distilled water to every 1 mL of JC-1 staining buffer (5×), and place it on ice bath.

[0192] E. After the incubation at 37 °C, aspirate the supernatant and wash twice with JC-1 staining buffer (1×). Add 2 mL of cell culture medium, which may contain serum and phenol red.

[0193] F. Observe and take pictures of live cells directly under a fluorescence microscope (200×). For green light: set the excitation light at 490 nm and the emission light at 530 nm; for red light: set the excitation light at 525 nm and the emission light at 590 nm.

[0194] The results are shown in Figures 30 - 31 . It can be seen from the results that compared with the blank control group, the cell membrane potential in the DSS group decreased significantly, indicating that DSS induced mitochondrial polarization in cells; compared with the DSS group, after treatment with 10 μg / mL OLSA@Ac-GGEPs, 10 μg / mL Ac-GGEPs, 10 μg / mL OLSA, and 10 μg / mL MSZ, the ratio of red / green fluorescence in cells decreased significantly, indicating that the above drug treatments could significantly inhibit the decrease in cell mitochondrial membrane potential caused by DSS stimulation, and the effect of OLSA@Ac-GGEPs was more significant.

[0195] 2. Conclusion

[0196] In this study, glial polysaccharide was extracted and separated from the crude polysaccharide of Gastrodia elata, a traditional Chinese medicine, and used to load small molecule drugs of the aminosalicylate class to form the preparation method of the OLSA@Ac-GGEPs complex: extract the crude polysaccharide of Gastrodia elata from Gastrodia elata blocks by hot water alcohol precipitation method; float and wash to separate the glial part, freeze precipitate and dry it into powder; prepare the Gastrodia elata gum complex OLSA@Ac-GGEPs loaded with olsalazine through chemical modification, repeated freezing and thawing, and double solvent method; through in vitro release experiments, the colon-targeting and sustained-release effects of OLSA@Ac-GGEPs were confirmed; through the DSS-induced oxidative stress experiment on Caco-2 cells, it was confirmed that OLSA@Ac-GGEPs had a synergistic effect on the protection of colon cells.

[0197] In summary, the drug-loaded gastrodia glue complex of the present invention is an integrated traditional Chinese and Western medicine that targets the delivery of olsalazine to the colon and synergistically treats with gastrodian polysaccharide itself, aiming to treat ulcerative colitis that is clinically prevalent. The innovative therapy of the present invention improves the bioavailability of olsalazine, reduces adverse reactions. At the same time, the combination of olsalazine and gastrodia glue produces a synergistic effect in protecting colon cells, demonstrating the potential of natural polysaccharides as drug delivery carriers, and providing new methods and new insights for the treatment strategy of ulcerative colitis.

Claims

1. A drug complex for treating inflammatory diseases of the colon, characterized in that: It is a compound of acetylated Gastrodia elata gum and olsalazine; The mass ratio of the acetylated gastrodia gum to olsalazine is 50-150:20; The acetylated Gastrodia elata gum is prepared by reacting the colloidal white solid obtained by water extraction and alcohol precipitation of Gastrodia elata with acetic anhydride.

2. The drug complex according to claim 1, characterized in that: The mass ratio of the acetylated gastrodia gum to olsalazine is 100:

20.

3. The drug complex according to claim 1 or 2, characterized in that: The acetylated gastrodia gum has a porous structure.

4. A method for preparing the drug complex according to any one of claims 1 to 3, characterized in that: The steps include: 1) taking Gastrodia elata, soaking it in water for extraction, concentrating the extract and adding ethanol, collecting the colloidal white solid to obtain Gastrodia elata colloid polysaccharide; 2) taking the Gastrodia elata polysaccharide obtained in step 1), adding water to dissolve it, adding acetic anhydride dropwise to the dissolved solution for reaction, dialyzing the liquid after the reaction is complete, and drying to obtain acetylated Gastrodia elata gum; 3) dissolving the acetylated Gastrodia elata gum in water, and freezing and thawing the acetylated Gastrodia elata gum with a porous structure; 4) The porous acetylated Gastrodia elata gum obtained in step 3) is ground into powder, and n-hexane is added and stirred, and an olsalazine aqueous solution is added dropwise. After the addition is completed, stirring is continued for 6 to 8 hours, and the n-hexane is removed to obtain a Gastrodia elata gum olsalazine complex.

5. The preparation method according to claim 4, characterized in that: Step 1) The amount of water added is 15 times the amount of Gastrodia elata; the Gastrodia elata is a Gastrodia elata block with a diameter of less than 1 cm; the soaking time is 4 to 5 hours, the extraction is a water bath extraction, the number of water bath extractions is 3 times, each temperature is 70 to 90° C., and the time is 2 to 4 hours; the concentration is reduced pressure concentration, the temperature is 40 to 60° C., and the concentration is concentrated to a relative density of 1.05; the ethanol is added to the solution until the ethanol concentration reaches 70%.

6. The preparation method according to claim 4, characterized in that: Step 2) acetic anhydride is added dropwise to the dissolved solution at 40-60°C and 800-1200r / min; the pH value of the solution is maintained at 9 during the addition of acetic anhydride; the molecular weight cutoff of the dialysis bag during the dialysis is 2.5kD, and the dialysis time is 3-5 days; the drying is freeze-drying; the mass volume ratio of the Gastrodia elata colloid polysaccharide, water and acetic anhydride is 2-6g:8-12ml:2-6ml.

7. The preparation method according to claim 4, characterized in that: Step 3) The mass volume ratio of the acetylated gastrodia gum to water is 0.5 g: 2-8 mL; the number of freeze-thaw cycles is 5-8 times, each freezing temperature is -20°C, the freezing time is 4 hours, the thawing temperature is 25°C, and the thawing is performed until the ice crystals are completely melted.

8. The preparation method according to claim 4, characterized in that: Step 4) The mass volume ratio of the porous structured acetylated gastrodia gum, n-hexane and olsalazine aqueous solution is 50-150 mg:30 ml:0.5 ml; the concentration of the olsalazine aqueous solution is 30-50 mg / ml; and the stirring speed of the n-hexane is 500-1000 r / min.

9. Use of the drug complex according to any one of claims 1 to 3 in the preparation of a drug for treating colon inflammatory diseases.

10. The use according to claim 9, characterized in that: The colon inflammatory diseases include colitis and Crohn's disease; the colitis includes ulcerative colitis.

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