A pharmaceutical composition for treating colonic inflammatory disease and a method of preparing the same

Abstract

The application discloses a medicine compound for treating colon inflammatory diseases, which is compounded by acetylated Gastrodia elata gum and olsalazine; the mass ratio of the acetylated Gastrodia elata gum and the olsalazine is 50-150:20; the acetylated Gastrodia elata gum is a gelatinous white solid obtained by water extraction and alcohol precipitation of Gastrodia elata and reacted with acetic anhydride. The medicine compound can continuously release at a colon lesion site, has colon targeting and slow-release effects, can significantly reduce oxidation stress of colon cells, and has a synergistic effect in protecting colon cells, and has practical popularization and application value.

Description

TECHNICAL FIELD

[0001] The present application relates to a pharmaceutical composition for treating colonic inflammatory diseases and a preparation method thereof. BACKGROUND

[0002] Colonic inflammatory diseases are a group of diseases characterized by inflammation of the colonic mucosa or full-thickness, mainly including ulcerative colitis, Crohn's disease, infectious colitis, etc. Ulcerative colitis is a chronic nonspecific colonic inflammation, with ulceration in severe cases, mainly involving the colonic mucosa and submucosa, with a range often starting from the distal colon and progressing proximally, even involving the entire colon and terminal ileum, with continuous distribution. The main clinical manifestations are diarrhea, abdominal pain, and mucopurulent bloody stool. Crohn's disease can involve various parts of the gastrointestinal tract, mainly the terminal ileum and adjacent colon, and is a chronic granulomatous inflammation, which is often segmental and asymmetrically distributed. The onset is usually insidious and gradual, with chronic course, alternating between long and short active and remission periods, with a tendency to recur throughout life. The clinical manifestations vary with the lesion site, disease stage, and complications. The overall quality of life score of patients with colonic inflammatory diseases is usually at a low level, and standardized drug treatment is needed to reduce the negative impact of the disease on daily life.

[0003] Gastrodia elata Bl., also known as Chixian, Dingfengcao, and Dushaozhi, is a mycophytic perennial herbaceous plant of Orchidaceae. The dried tuber of Gastrodia elata Bl. is sweet in taste, flat in nature, and belongs to the liver, and is one of the precious traditional Chinese medicinal materials in China, with the functions of calming wind, stopping convulsion, and soothing liver yang. Gastrodia elata polysaccharide is the main active ingredient in Gastrodia elata, with high content and low toxicity, and is one of the current hotspots in drug research and development. Studies have reported 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 elatapolysaccharides 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 medicine which is made of two molecules of 5-aminosalicylic acid (5-ASA) linked by an azo bond. It is not absorbed in the stomach and small intestine, and is broken down by intestinal bacteria into two molecules of 5-ASA after reaching the colon, thereby playing an anti-inflammatory role. 5-ASA can inhibit the synthesis of prostaglandins and leukotrienes, reduce the permeability of the intestinal wall cell membrane, and reduce intestinal mucosal edema, and is mainly used for the treatment of acute and chronic ulcerative colitis, Crohn's disease, and long-term maintenance treatment in the remission period of these diseases.

[0005] There is no report on the combination of Gastrodine polysaccharide and Olsalazine for the treatment of colonic inflammatory diseases. SUMMARY

[0006] To solve the above problems, the present application provides a pharmaceutical compound for the treatment of colonic inflammatory diseases, which is made of acetylated Gastrodine gum and Olsalazine.

[0007] The mass ratio of the acetylated Gastrodine gum to Olsalazine is 50-150:20.

[0008] The acetylated Gastrodine gum is a gelatinous white solid obtained by water extraction and alcohol precipitation of Gastrodine, and is reacted with acetic anhydride.

[0009] Further, the mass ratio of the acetylated Gastrodine gum to Olsalazine is 100:20.

[0010] Further, the acetylated Gastrodine gum has a porous structure.

[0011] The present application also provides a preparation method of the aforementioned pharmaceutical compound, comprising the following steps:

[0012] 1) Take Gastrodine, soak and extract with water, concentrate the extract, add ethanol, collect the gelatinous white solid, and obtain Gastrodine gum polysaccharide;

[0013] 2) Take the Gastrodine gum polysaccharide obtained in step 1), dissolve with water, add acetic anhydride dropwise to the dissolved solution, dialyze the solution after the reaction is complete, and dry to obtain acetylated Gastrodine gum;

[0014] 3) Dissolve the acetylated Gastrodine gum with water, freeze-thaw to obtain acetylated Gastrodine gum with a porous structure;

[0015] 4) Take the acetylated Gastrodine gum with a porous structure obtained in step 3), grind into powder, stir with n-hexane, and dropwise add Olsalazine aqueous solution, continue stirring for 6-8 hours after the dropwise addition is completed, remove the n-hexane, and obtain the Gastrodine gum Olsalazine compound.

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

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

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

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

[0020] The application also provides a use of the aforementioned pharmaceutical compound in the preparation of a drug for treating colonic inflammatory diseases.

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

[0022] The pharmaceutical compound of the application, after the Gastrodia elata gum polysaccharide with a specific molecular weight is acetylated and porous, is compounded with oxalate in a specific ratio, can be continuously released at the colonic lesion site, has the effects of colonic targeting and sustained release, can significantly reduce the oxidative stress of colonic cells, has an effect superior to that of any single Gastrodia elata gum polysaccharide or oxalate, produces a synergistic effect in protecting colonic cells, and has practical popularization and application value.

[0023] Obviously, according to the above content of the application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical ideas of the application.

[0024] The above summary of the application will be further elaborated in the following detailed description in the form of examples. However, it should not be understood that the above summary of the subject matter of the application is limited to the following examples. Any technology achieved based on the above summary of the application falls within the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Preparation process of loading olsalazine with Gastrodia elata gum;

[0026] Figure 2 Gastrodia elata medicinal materials after processing;

[0027] Figure 3 Gastrodia elata crude polysaccharide after water extraction and alcohol precipitation (left) and Gastrodia elata gum polysaccharide after separation (right);

[0028] Figure 4 Amylose standard curve of Gastrodia elata gum polysaccharide fragments;

[0029] Figure 5 Absolute molecular weight analysis chart;

[0030] Figure 6 Molecular configuration chart;

[0031] Figure 7 Nuclear magnetic hydrogen spectrum

[0032] Figure 8 Chain length distribution chart of Gastrodia elata gum polysaccharide fragments;

[0033] Figure 9 Gastrodia elata gum acetylation reaction (left) and solubility of acetylated Gastrodia elata gum (right);

[0034] Figure 10 Repeated freezing and thawing of acetylated Gastrodia elata gum;

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

[0036] Figure 12 Nuclear magnetic characterization;

[0037] Figure 13 Before repeated freezing and thawing (left) and after repeated freezing and thawing (right);

[0038] Figure 14 Olsalazine full-waveband absorption spectrum;

[0039] Figure 15 Olsalazine standard curve;

[0040] Figure 16 Mesalamine full-waveband spectrum;

[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), release in experiment diagram (right);

[0050] Figure 26 Timing sampling diagram;

[0051] Figure 27 Osalazine / Gastrodine polysaccharide in vitro release curve diagram;

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

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

[0054] Figure 30 Effect of different drug treatments on DSS-induced cell mitochondrial polarization;

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

[0056] Example 1, preparation of drug-loaded gastrodine polysaccharide complex of the present application

[0057] 1. Preparation of gastrodine polysaccharide

[0058] Take 2 kg of Gastrodia elata pieces, crush into small pieces with diameter less than 1 cm, soak in 15 times (v / w) of water for 4-5 hours, then extract in water bath at 80°C for 3 hours, filter the extract while hot with 300 mesh filter cloth, repeat the extraction for 2 times with the same method for the residue, combine the 3 times of extract, concentrate under reduced pressure at 50°C to relative density 1.05, cool to room temperature, then add ethanol to reach 70% ethanol concentration, collect the gelatinous white solid, and obtain Gastrodia elata polysaccharide (GGEPs);

[0059] 2. Preparation of acetylated Gastrodia elata gel

[0060] Take 4 g of Gastrodia elata polysaccharide and 10 ml of water, dissolve by ultrasonic, then add 4 ml of acetic anhydride dropwise under stirring at 50°C and 1000 r / min, adjust the pH value with sodium hydroxide solution during the dropwise addition to keep the pH value of the mixed solution at 9, react for 4 hours after the dropwise addition is completed, cool to room temperature, load the reaction solution into a 2.5 kD dialysis bag, dialyze in water flow for 3-5 days, freeze-dry the reaction solution after dialysis to obtain acetylated Gastrodia elata gel (Ac-GGEPs);

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

[0062] Take 0.5 g of acetylated Gastrodia elata gel, dissolve into 10% aqueous solution with 5 mL of ultrapure water, equilibrate at room temperature for 0.5 h, then freeze at -20°C for 4 h, thaw at 25°C until the internal ice crystals completely melt, repeat the freeze-thaw process for 6 times in the same way to obtain acetylated Gastrodia elata gel with porous structure;

[0063] 4. Preparation of Gastrodia elata gel oxalate complex

[0064] Take 100 mg of acetylated Gastrodia elata gel with porous structure, grind into powder, add 30 ml of n-hexane, stir at 800 r / min, then add 0.5 ml of 40 mg / ml oxalate aqueous solution dropwise, continue to stir for 6-8 h after the dropwise addition is completed, remove the n-hexane to obtain Gastrodia elata gel oxalate complex OLSA@Ac-GGEPs.

[0065] The beneficial effects of the present application are illustrated by the following test examples.

[0066] Test Example 1 Study of drug-loaded Gastrodia elata polysaccharide complex

[0067] 1. Research route and research content

[0068] The research route chart is shown in Figure 1 .

[0069] 1.1 Processing of Gastrodia elata

[0070] Gastrodia elata is a precious traditional Chinese medicine, and its dried tubers are the main part for medicinal use. Gastrodia elata has the effects of relieving wind and convulsion, calming liver yang, and dispelling wind and dredging collaterals, and is often used to treat symptoms such as headache, dizziness, numbness of limbs, and infantile convulsion. It has mild medicinal properties and can be used alone or combined with other medicinal materials to enhance efficacy. Gastrodia elata grows in a special environment, mostly in humid and cool environments, so its yield 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 should be harvested from the appropriate time period, carefully washed to remove dirt and impurities, and then graded according to size. Gastrodia elata needs to be softened by soaking or steaming, which can make its internal structure soft and facilitate subsequent slicing. After slicing, Gastrodia elata needs to be dried by methods such as airing or drying to achieve the appropriate degree of dryness, which helps to prolong its shelf life. To further enhance its medicinal efficacy, special processing methods such as frying, baking, or frying with ginger juice may be used. The dried and processed Gastrodia elata should be stored in a cool and dry place to prevent moisture and deterioration, ensuring its quality and effectiveness in clinical use. The Gastrodia elata slices used in this study were prepared according to the Chongqing Local Standard "Gastrodia elata Primary Processing Technical Regulations" (No. DB50 / T1498-2023) Figure 2

[0071] 1.2 Extraction of gelatinous polysaccharides from Gastrodia elata slices

[0072] Take 2 kg of Gastrodia elata slices and chop them into small pieces less than 1 cm in size using a hammer or a cutting knife. Place them in a 20 L bucket and add water until the water level is above the Gastrodia elata pieces (more than 15 times the volume). Soak for 4-5 hours. Extract at 80°C for 3 hours in a water bath. While hot, filter the extract using a 300-mesh filter cloth to separate the filtrate and reserve the residue for further extraction. Repeat the extraction process twice more, and then combine the three 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). This will yield a concentrated solution. Cool the concentrated solution to room temperature, and then add ethanol until the ethanol concentration reaches 70%. At this point, observe the precipitation of two types of polysaccharides with different properties: dark block-shaped solids and white gelatinous solids. The white gelatinous solids are the gelatinous Gastrodia elata polysaccharides (Gastrodia elata gel, GGEPs). The two types of polysaccharides have different densities and volumes, so they can be separated by floating and then allowing them to settle. At this point, remove the Gastrodia elata gel from the suspension and filter to remove the ethanol. Dissolve the Gastrodia elata gel in water and repeat the alcohol precipitation process to remove small molecule impurities such as gastrodin and bartzin. After filtering again, freeze-dry the Gastrodia elata gel to obtain a solid product Figure 3

[0073] 1.3 Characterization of Gastrodia elata gel polysaccharide fragments

[0074] ​​The polysaccharide fragments in Gastrodia elata Blume have amyloid paste properties, so the starch identification and determination methods are used to perform physical and chemical research on the prepared colloidal Gastrodia elata Blume polysaccharide GGEPs. The key indicators such as amylose content, starch polymerization degree, branching degree, molecular weight, viscosity and paste properties are used to provide accurate, stable data and reliable technical support for the starch-based material development of Gastrodia elata Blume polysaccharide and other related research.

[0075] 1.3.1 Determination of resistant starch content

[0076] Resistant starch, also known as anti-enzymatic starch and indigestible starch, cannot be enzymatically degraded in the small intestine, but can react with volatile fatty acids in the human intestinal and colon. The content of resistant starch in the sample is mainly determined by using α-amylase and starch glucosidase to remove non-resistant starch, dissolving the residue with KOH, adjusting to neutral with acetic acid buffer, hydrolyzing starch to glucose with starch glucosidase, reacting GOPOD reagent buffer with D-glucose to generate kunya imine dye, and determining the content by colorimetry. No resistant starch is detected in the Gastrodia elata Blume polysaccharide.

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

[0078]

[0079] 1.3.2 Determination of total starch content

[0080] The content of starch in the sample is mainly determined by using heat-resistant α-amylase to hydrolyze starch into branched and unbranched maltodextrin, and then quantitatively hydrolyzing maltodextrin into D-glucose with starch glucosidase. GOPOD reagent buffer reacts with D-glucose to generate kunya imine dye, and the content is determined by colorimetry. The total starch content in the Gastrodia elata Blume polysaccharide is 49.95% after testing.

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

[0082]

[0083] 1.3.3 Determination of amylose content

[0084] The content of amylose in the sample is mainly determined by using the different affinity of amylose and amylopectin to iodine, preparing standard solutions with different proportions of amylose, and preparing a standard curve to quantitatively determine the content of amylose in the sample. Figure 4 ) After testing, no amylose is detected in the Gastrodia elata Blume polysaccharide sample.

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

[0086] No. 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] The molecular weight of starch was detected by gel chromatography, laser light scattering and differential detector. Gel chromatography can elute molecules in solvent according to weight or size. Molecules with larger size can only enter the gel pores with larger aperture, while molecules with smaller size can enter more gel particles. Thus, molecules with larger size move shorter distance in the gel bed, and molecules with smaller size move longer distance. Molecules with larger size are eluted first, and molecules with smaller size are eluted later, so as to achieve the separation effect and the purpose of separation according to the size of molecules. Then, the concentration information of the sample is detected by the differential detector according to the refractive intensity, the light scattering information of macromolecules is detected by the multi-angle laser light scattering instrument, and the molecular weight corresponding to each component is calculated according to the Mark-Houwink equation. The molecular weight of Gastrodia elata gum polysaccharide is Mw=35581.273 kDa.

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

[0090]

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

[0092] In the absolute molecular weight analysis chart ( Figure 5 ), the red line is the multi-angle laser light scattering signal, which reflects the molecular size of the sample; the blue line is the differential signal, which reflects the concentration of the sample; and the black line is the molecular weight fitted from the two signals. The blue line is the differential signal, which represents the concentration of the sample; and the red line is the light scattering signal, which represents the molecular size of the sample. In the molecular configuration chart ( Figure 6 ), the abscissa is the molecular weight (Molar Mass), and the ordinate is the radius of gyration (R.M.S.Radius). The slope can be used as a reference for the molecular configuration. Generally, the slope = 1 indicates a rod-shaped molecule, the slope is 0.5-0.6 indicates a random coil, and the slope = 1 / 3 indicates a spherical shape. The slope of Gastrodia elata gum polysaccharide is 0.16, so it is speculated to be a dense spherical shape.

[0093] 1.3.5 Gelatinization temperature detection

[0094] Starch is a tandem complex of glucose formed by α-1,4 and α-1,6 glycosidic bonds, and has a crystalline or semi-crystalline structure. With increasing temperature, the double helix structure, crystalline structure, and other higher-order structures in starch are disrupted; for native starch, this process is called gelatinization. Different starches have different structures, and their phase transition processes, required temperatures, and energy levels vary. In the study of starch and related polymer derivatives, differential calorimetry (DSC) is used to measure the phase transition process of starch and its derivatives, allowing for the analysis of the structure and physicochemical properties of starch. The gelatinization temperature of Gastrodia elata gum was tested to be: initial temperature 52.6℃, peak temperature 69.5℃, and termination temperature 94.3℃, with a gelatinization enthalpy of 5.533 J / g.

[0095] Table 5. Results of Gelatinization Temperature Detection for Gastrodia elata Gelatinous Polysaccharide

[0096]

[0097] 1.3.6 Branching Degree and Chain Length Distribution

[0098] The position of the proton on the carbon chain can be inferred from the chemical shift and other information provided by the nuclear magnetic resonance (NMR) of hydrogen. The data was analyzed using MestReNova software, and the peak range was selected based on the sample elution time to obtain the final result. The calculation was performed using the following formula:

[0099]

[0100] DB: Branching degree

[0101] A: Peak area of ​​α-1,6 bonds.

[0102] B: Peak area of ​​α-1,4 bonds

[0103] MRI results ( Figure 7 It can be seen that the branching degree of Gastrodia elata gelatinous polysaccharide is 6.65%.

[0104] Chain length distribution ( Figure 8 Standards with different chain lengths have different retention times on chromatography. Qualitative analysis is based on the retention time of the sample on the chromatographic column, and the concentration is calculated from the standard curve based on the peak area of ​​the sample. The test results show an average degree of polymerization of 19.7.

[0105] 1.3.7 Summary of Characterization of Gastrodia elata Gastrodia elata Polysaccharide

[0106] After characterization, it is known that the gum Gastrodia elata polysaccharides GGEPs obtained by extraction and separation of Gastrodia elata decoction pieces are natural polysaccharides with certain branching degree, starch-like properties and high molecular weight. The high molecular weight and branching interaction of the gum Gastrodia elata polysaccharides GGEPs can provide more excellent stability when used as a carrier, and the gelatinization properties of the Gastrodia elata gum also indicate that its solubility in water is limited or the solubility rate is slow, and it needs more time to be metabolized in the digestive juice, so it can be used as a natural intestinal targeted delivery material.

[0107] 1.4 Acetylation modification of Gastrodia elata gum

[0108] Acetylation modification of polysaccharides is an important chemical operation that changes the structure and properties of polysaccharides by introducing acetyl groups into the polysaccharide molecules. After acetylation modification, the hydroxyl groups of the polysaccharides are exposed to the 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 hydrophobicity, hardness and other physicochemical properties of polysaccharides, so that they exhibit better performance in emulsification, foam stabilization, etc. 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 performance of polysaccharides, such as improving stability, enhancing biological activity, etc. Acetylation modification also provides new ideas and methods for the development of new products of polysaccharides.

[0109] Weigh 4g of Gastrodia elata gum polysaccharides and 10ml of water into a 200ml conical flask, ultrasonic dissolution, heated to 50℃, add magnetic stirring to 1000r / min stirring speed. Prepare 20ml of 6M sodium hydroxide solution. Add a small amount of acetic anhydride to the Gastrodia elata gum solution, and at the same time add sodium hydroxide solution for neutralization, detect pH value with pH test paper, make pH value equal to 9, continue to add acetic anhydride and sodium hydroxide until acetic anhydride is added to 4ml. After 4 hours of reaction, cool to room temperature, and then place the reaction solution in a 2.5kD dialysis bag under water flow or water bath (frequently change water) for 3-5 days. Freeze-dry the reaction solution after dialysis to obtain acetylated Gastrodia elata gum (Ac-GGEPs). The preparation process is shown in Figure 9 .

[0110] 1.5 Repeated freezing and thawing of acetylated Gastrodia elata gum

[0111] Take 0.5 g of acetylated Gastrodia elata gum (dry sample), add 5 mL of ultrapure water to make a 10% aqueous solution, and place it in a suitable size surface dish. Equilibrate at room temperature for 0.5 h, then place the surface dish in a -20°C environment for 4 h. Thaw (or melt) at 25°C until the ice crystals inside the Gastrodia elata gum completely melt, completing one freeze-thaw cycle. Repeat the freeze-thaw process 6 times. After the freeze-thaw process is complete, freeze-dry the sample to obtain a porous structure. Store at 4°C for later use. See the preparation process diagram Figure 10 .

[0112] 1.6 Preparation of OLSA-loaded acetylated Gastrodia elata gum complex (double solvent method)

[0113] Take 100 mg of acetylated Gastrodia elata gum after repeated freeze-thawing, and grind it into small particles. Add 30 mL of n-hexane to a 50 mL centrifuge tube, and add a suitable size stir bar to stir at 800 r / min. Dissolve 20 mg of OLSA in 0.5 mL of water using ultrasonic waves. Use a plastic dropper to add the OLSA solution dropwise to the n-hexane while stirring. Place the mixture in a magnetic stirrer or ultrasonic crusher and stir overnight. Remove the stir bar after stirring, and slowly pour out most of the n-hexane. Remove the small amount of n-hexane remaining on the product by drying in a vacuum drying oven at 40°C for 2 hours. This yields OLSA-loaded acetylated Gastrodia elata gum (OLSA@Ac-GGEPs) prepared by the double solvent method. No OLSA is detected in the n-hexane solution by UV detection, so the drug loading is approximately 20%. See the preparation process diagram Figure 11 .

[0114] 1.7 Characterization of OLSA@Ac-GGEPs

[0115] 1.7.1 Nuclear magnetic resonance characterization

[0116] Take 0.009 g of OLSA@Ac-GGEPs and 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: AVANCE 500) to detect the 1 1H NMR spectrum of the test sample. Test the 1H NMR spectrum of pure OLSA according to the above procedure. As shown in Figure 12 , OLSA can be loaded into acetylated Gastrodia elata polysaccharides using the double solvent method, successfully preparing the OLSA@Ac-GGEPs delivery system.

[0117] 1.7.2 Scanning electron microscopy

[0118] A small amount of OLSA@Ac-GGEPs sample was weighed and dried in an oven at 40°C, fixed on the sample stage, and sprayed with gold for 30 s at a voltage of 15 kV. The microstructure of the sample was observed under a scanning electron microscope (model: Quattro S) and an image was collected. Figure 13 The OLSA@Ac-GGEPs after repeated freezing and thawing can form relatively uniform pores, which is conducive to subsequent drug loading.

[0119] 1.8 Drug content determination in OLSA@Ac-GGEPs

[0120] Currently, the first choice for the treatment of mild to moderate UC patients in clinical practice is aminosalicylic acid drugs, such as mesalazine and olsalazine. Mesalazine, also known as 5-aminosalicylic acid (5-ASA), mainly inhibits leukotriene synthase, reduces the production of leukotrienes, and inhibits the synthesis of prostaglandins and the formation of inflammatory mediators leukotrienes, thereby significantly inhibiting the inflammation of intestinal mucosa; olsalazine is an isomer of 5-aminosalicylic acid, which is coupled by two molecules of 5-ASA to release two molecules of 5-aminosalicylic acid in the colon to exert its effect, and has very low toxicity, and is suitable for long-term treatment of ulcerative colitis. Olsalazine can also be partially converted to mesalazine in water, so the drug content determination in the above prepared complex OLSA@Ac-GGEPs needs to include both olsalazine and mesalazine.

[0121] 1.8.1 Olsalazine content determination

[0122] (1) Preparation of standard solution

[0123] 10 mg of olsalazine was accurately weighed into a 50 ml volumetric flask and diluted with pH 6.8 phosphate buffer to prepare a 0.2 mg / ml stock solution.

[0124] (2) Determination of wavelength

[0125] The aqueous solution of olsalazine sodium salt or phosphate buffer has a maximum absorption in the ultraviolet region. 1.0 ml of olsalazine stock solution was accurately measured into a 50 ml volumetric flask and diluted to the mark with pH 6.8 phosphate buffer. The pH 6.8 phosphate buffer was used as a reference. Scanning was performed in the wavelength range of 200-700 nm, Figure 14 Results: There is a maximum absorption at 360 nm. Therefore, 360 nm is determined as the detection wavelength.

[0126] (3) Establishment of standard curve determination

[0127] Accurately pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0 ml stock solution into 10 ml volumetric flask, dilute to the mark with pH = 6.8 phosphate buffer. With pH = 6.8 phosphate buffer as reference, determine the absorbance at 360 nm wavelength, make linear regression of concentration C against absorbance A, the regression equation is C = 72.925 x A - 0.0448, R2= 0.999( Figure 15 ). It shows that oxazepam is in good linear relationship in the range of 4-20 μg·ml. Obtain standard solution of different concentration gradient, determine the ultraviolet absorption at 360 nm wavelength.

[0128] (4) Sample detection

[0129] Take 1.27 mg OLSA@Ac-GGEPs sample in 10 ml volumetric flask, dilute to the mark with pH 6.8 phosphate buffer, ultrasonic for 10 min, configure into 0.127 mg / ml solution, determine the ultraviolet absorption at 360 nm as 1.544, calculate the oxazepam concentration as 0.0216 mg / ml according to the standard curve, the drug loading is 17.01%. The drug loading measured by spectrophotometry is less than the theoretical value, because oxazepam is encapsulated in the polysaccharide phase with large molecular weight, which is limited by diffusion and cannot be completely dissolved in phosphate buffer in a short time, and even if waiting for a long time for sufficient diffusion, oxazepam will be partially converted to mesalamine in water, so the actual measured drug loading of oxazepam is less than 20% of the true value.

[0130] 1.8.2 Content determination of mesalamine

[0131] (1) Preparation of standard solution

[0132] Accurately weigh 10 mg mesalamine into 50 ml volumetric flask, dilute to the mark with pH 6.8 phosphate buffer, prepare 0.2 mg / ml stock solution.

[0133] (2) Determination of wavelength

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

[0135] (3) Establishment of standard curve

[0136] Same as 1.8.1 (3)

[0137] (4) Sample detection

[0138] No mesalamine was detected in the initial sample of 1.8.1 (4) OLSA@Ac-GGEPs.

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

[0140] 1.9.1 Establishment of standard curve of olsalazine

[0141] Accurately weigh 10 mg of olsalazine into a 50 ml volumetric flask, and dilute to the mark with pH 6.8 phosphate buffer to prepare a 0.2 mg / ml stock solution. Accurately 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. With the corresponding simulated liquid as reference, the absorbance was measured at 360 nm wavelength, and the concentration C was linearly regressed against the absorbance A. The fitting curve R in the four different media 2 were all around 0.999, Figures 17-20 indicating that olsalazine showed good linear relationship in the range of 4-20 μg·ml.

[0142] 1.9.2 Establishment of standard curve of mesalamine

[0143] Accurately weigh 10 mg of mesalamine into a 10 ml volumetric flask, and dilute to the mark with pH 6.8 phosphate buffer to prepare a 1.6 mg / ml stock solution. Accurately 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. With the corresponding simulated liquid as reference, the absorbance was measured at 303 nm wavelength, and the concentration C was linearly regressed against the absorbance A. The fitting curve R in the four different media 2 were all around 0.99, Figures 21-24 indicating that mesalamine showed good linear relationship in the range of 20-100 μg·ml.

[0144] 3.9.3 Obtaining of in vitro release experiment samples

[0145] (1) Pretreatment of dialysis bag

[0146] Take an appropriate amount of dialysis bag, cut it to the appropriate length, and 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 weigh 10 mg of OLSA@GGEPs into 10 ml of distilled water to make a drug solution with a concentration of 1 mg / ml. Inject the solution into the dialysis bag through the end without knot, and then knot the end to seal the drug in the dialysis bag, and try to avoid air bubbles.

[0149] (3) Preparation of digestive juice

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

[0151] Artificial gastric juice: take 16.4 ml of dilute hydrochloric acid, add 800 ml of water, and add 10 g of pepsin, shake well, and then dilute with water to 1000 ml to obtain; wherein the dilute hydrochloric acid is 234 ml of concentrated hydrochloric acid diluted with water to 1000 ml to obtain 9.5%-10.5% dilute hydrochloric acid.

[0152] Duodenal juice: weigh 6.8 g of sodium chloride, 0.4 g of potassium chloride, and 0.06 g of potassium dihydrogen phosphate, dissolve in water, and then dilute to 1000 ml to obtain solution A; weigh 10.0 g of sodium bicarbonate, dissolve and dilute to 100 ml to obtain solution B; weigh 0.04 g of calcium chloride, dissolve and dilute 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 salt, stir to dissolve, and transfer to a 1000 ml volumetric flask to dilute to volume.

[0153] Artificial small intestinal juice: take 6.8 g of potassium dihydrogen phosphate, add 500 ml of water to dissolve, and adjust the pH to 6.8 with 0.1 mol / L

[0154] hydrogen chloride solution to obtain; take 10 g of trypsin, add an appropriate amount of water to dissolve, mix the two solutions, and then dilute with water to 1000 ml to obtain.

[0155] Artificial colon juice: take 5.59 g of potassium phosphate dibasic and 0.41 g of potassium dihydrogen phosphate, add water to dissolve to 1000 ml to obtain.

[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 in the conical flask, ensuring that the dialysis bag is completely immersed in the gastric juice. Place the conical flask in a constant temperature water bath shaker, set the temperature to 37°C, and the shaking speed to 100 rpm, and avoid light. Within 2 hours, take 2 ml of gastric juice at time points of 0.5 hour, 1 hour, 1.5 hours, and 2 hours for drug concentration detection, and supplement 2 ml of fresh simulated gastric juice after each sampling.

[0158] (5) Transfer to simulated duodenal phase (2 hours)

[0159] After 2 hours, transfer the dialysis bag and all the gastric fluid in 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 shaker, maintain the temperature at 37°C, the shaking speed at 100 rpm, and avoid light. During these 2 hours, take 2 ml of the mixture at 0.5 hour, 1 hour, 1.5 hour, respectively, for drug concentration detection. After each sampling, supplement 2 ml of the solution mixed by simulated duodenal fluid and simulated gastric fluid at a volume ratio of 30:30 (to keep the total volume and the proportion of ingredients relatively stable).

[0160] (6) Transfer to simulated small intestinal phase (4 hours)

[0161] After the 2-hour simulated duodenal fluid phase is completed, transfer the mixture containing the dialysis bag to a conical flask containing 30 ml of simulated small intestinal fluid. Continue the release experiment in a constant temperature water bath shaker at 37°C, 100 rpm. Take 2 ml of the sample at 0.5 hour, 1 hour, 2 hours, 3 hours, respectively, for drug concentration detection. After each sampling, supplement 2 ml of the solution mixed by simulated small intestinal fluid and the previous mixture at a volume ratio of 30:(30+30).

[0162] (7) Transfer to simulated colon phase (8 hours)

[0163] After the small intestinal fluid phase is completed, transfer the dialysis bag and the mixture to a conical flask containing 30 ml of simulated colon fluid. Continue the release at 37°C, 100 rpm. Take 2 ml of the sample at 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, respectively, for drug concentration detection. After each sampling, supplement 2 ml of the solution mixed by simulated colon fluid and the previous mixture at 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] According to the determined data, draw the concentration-time curve. Determine the ultraviolet absorption of the sample taken at each time point at a wavelength of 360 nm on the ultraviolet spectrophotometer (Shimadzu UV2600i), calculate the concentration according to the corresponding simulated release medium calibration curve, and calculate the cumulative release rate (%) according to the formula: cumulative release rate (%) = (V0xC t +V∑C i )×100% / m, where: V0is the total volume of the release medium. C t is the concentration of the drug in the release medium at the last sampling time point (ug·mL^-1); V is the volume of each sampling; ΣCi is the sum of the concentrations of the drug released from the medium at all sampling time points 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 periodically sampling ( Figure 26 ) and measuring the concentration of the drug in the samples during the experiment, the cumulative release ( Figure 27 ) can be calculated, thereby evaluating the release performance of the drug.

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

[0169] From the in vitro release distribution of olsalazine from OLSA@Ac-GGEPs in the simulated digestive tract environment, it can be seen that the release amount of olsalazine@Gastrodine polysaccharide complex in gastric juice is about 0.5% in 2h. It can be seen that gastrodine polysaccharide wraps the drug molecules, greatly reducing the release of the drug in gastric juice. When the complex enters the small intestine juice, the release is accelerated, and the release amount reaches about 40%. When the complex reaches the colon site, the drug is continuously released, and gradually tends to be balanced in about 20h.

[0170] In addition, olsalazine can be broken down by specific azo reductase in the colon site, and reduced to two molecules of mesalazine, which plays a therapeutic role in the form of mesalazine in the colon site. Therefore, the drug concentration of mesalazine is synchronously collected during the drug release process. From the in vitro release distribution of mesalazine from OLSA@Ac-GGEPs in the simulated digestive tract environment, it can be seen that 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 is detected to be about 70% in the colon environment.

[0171] In summary, it is confirmed that gastrodine polysaccharide as a drug carrier can effectively protect the drug from being released too early in the upper and middle sections of the digestive tract, and can continuously release in the colon lesion site, having the effects of colon targeting and sustained release.

[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 polysaccharide sulfate, which has a toxic effect on intestinal epithelial cells due to its negative charge, can destroy its barrier integrity and increase the permeability of the colon epithelium, and induce intestinal injury. When the antioxidant capacity of cells decreases and is insufficient to remove excessive accumulation of lipid ROS, oxidative stress occurs in cells. In order to evaluate this, the effect of OLSA@Ac-GGEPs on the ROS level of DSS-induced Caco-2 cells was tested, with Ac-GGEPs, OLSA, and MSZ (mesalamine) as controls. The specific steps are as follows:

[0175] (1) Stimulation

[0176] 6-well plate, 2.5 x 10 5 After 12 h of adherent cell growth in the cell plate, 2.5% DSS was added to the cell well, and cell culture medium containing 10 μg / mL OLSA@Ac-GGEPs, 10 μg / mL Ac-GGEPs, 10 μg / mL OLSA, and 10 μg / mL MSZ, respectively, was added to the cell well, and a blank treatment group was set up to add normal culture medium. Continue to culture in the incubator for 24 h, and then perform probe staining to detect the change in intracellular ROS.

[0177] (2) Staining

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

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

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

[0181] D. Directly observe and photograph (200x) the living cells with a fluorescence microscope. Green light: excitation light set to 490 nm, emission light set to 530 nm.

[0182] The results are shown in Figures 28-29From the results: the fluorescence intensity of DSS group is significantly higher than that of the blank group, indicating that 2.5% DSS induces 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 is significantly reduced. And the intracellular ROS level of OLSA@Ac-GGEPs group is more significantly reduced than that of Ac-GGEPs, OLSA and MSZ groups, which indicates that OLSA@Ac-GGEPs treatment can 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 shows 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] 6-well plates, 2.5 x 10 5 After 12 h of cell growth, 2.5% DSS was added to the cell wells, and cell culture medium containing 10 μg / mL OLSA@Ac-GGEPs, 10 μg / mL Ac-GGEPs, 10 μg / mL OLSA and 10 μg / mL MSZ was added, respectively. The blank treatment group was added with normal culture medium, and continued to be cultured in the incubator for 24 h, and then the probe staining was performed to detect the changes in mitochondrial membrane potential of cells.

[0187] (2) Staining

[0188] A, take appropriate amount of JC-1 (200x), dilute JC-1 according to the ratio of 8 mL ultrapure water per 50 μl JC-1 (200x). Vortex to dissolve and mix well. Then add 2 mL JC-1 staining buffer (5x), mix well to get 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, mix well. Incubate at 37°C in the cell incubator for 20 minutes.

[0191] D, During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1x) according to the ratio of 4 mL distilled water per 1 mL of JC-1 staining buffer (5x) and place it in an ice bath.

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

[0193] F, Live cells are observed and photographed directly under a fluorescence microscope (200x). Green light: excitation light set to 490 nm, emission light set to 530 nm; red light: excitation light set to 525 nm, emission light set to 590 nm.

[0194] Results are shown in Figures 30-31 . From the results, it can be seen that compared with the blank control group, the cell membrane potential of the DSS group decreased significantly, indicating that DSS induced mitochondrial polarization in cells; compared with the DSS group, the red / green fluorescence ratio in cells treated with 10 μg / mL OLSA@Ac-GGEPs, 10 μg / mL Ac-GGEPs, 10 μg / mL OLSA and 10 μg / mL MSZ decreased significantly, indicating that the above drug treatments can significantly inhibit the decrease in mitochondrial membrane potential of cells induced by DSS, and OLSA@Ac-GGEPs has a more significant effect.

[0195] 2. Conclusion

[0196] In this study, the preparation method of OLSA@Ac-GGEPs complex is as follows: crude polysaccharides are extracted from Gastrodia elata by hot water alcohol precipitation method; the gelatinous part is obtained by floating and washing, and freeze-dried into powder; through chemical modification, repeated freeze-thawing and double solvent method, OLSA@Ac-GGEPs is prepared; through in vitro release experiment, it is confirmed that OLSA@Ac-GGEPs has colon targeting and sustained release effect; through DSS-induced Caco-2 cell oxidative stress experiment, it is confirmed that OLSA@Ac-GGEPs has a synergistic effect on the protection of colon cells.

[0197] In summary, the drug-loaded Gastrodia elata gum complex is a targeted delivery of olsalazine to the colon and a combination of traditional Chinese and Western medicine to treat Gastrodia elata polysaccharide itself, aiming at treating clinically common ulcerative colitis. The innovative therapy of the application improves the bioavailability of olsalazine and reduces adverse reactions. At the same time, olsalazine and Gastrodia elata gum complex produce 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 pharmaceutical complex for the treatment of inflammatory bowel disease, characterized in that: It is composed of acetylated gastrodia elata gum and olsalazine; The mass ratio of acetylated gastrodia elata gelatin to olsalazine is 100~150:20; The acetylated gastrodia elata gum is a gelatinous white solid obtained by water extraction and alcohol precipitation of gastrodia elata, which is reacted with acetic anhydride. The method for preparing the gelatinous white solid is as follows: take Gastrodia elata, soak it in water to extract it, concentrate the extract and add ethanol, and collect the gelatinous white solid; The ethanol is added to the solution to achieve an ethanol concentration of 70%.

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

20.

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

4. A method for preparing the drug complex according to any one of claims 1 to 3, characterized in that: Includes the following steps: 1) Take Gastrodia elata, soak it in water to extract it, concentrate the extract and add ethanol, collect the gelatinous white solid to obtain Gastrodia elata gelatinous polysaccharide; the ethanol is added to the solution to reach an ethanol concentration of 70%; 2) Take the gastrodia elata gelatin polysaccharide obtained in step 1), dissolve it in water, add acetic anhydride dropwise to the solution to react, dialyze the liquid after the reaction is complete, and dry it to obtain acetylated gastrodia elata gelatin; 3) Acetylated gastrodia gum is dissolved in water, and freeze-thawed to form a porous acetylated gastrodia gum; 4) Take the porous acetylated gastrodin obtained in step 3), grind it into powder, add n-hexane and stir, and simultaneously add olsalazine aqueous solution dropwise. After the addition is completed, continue stirring for 6-8 hours, and remove n-hexane to obtain the gastrodin-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 piece of Gastrodia elata with a diameter of less than 1 cm; the soaking time is 4-5 hours, the extraction is water bath extraction, the number of water bath extractions is 3 times, the temperature is 70-90℃ each time, and the time is 2-4 hours; the concentration is vacuum concentration, the temperature is 40-60℃, and the concentration is made up to a relative density of 1.05; the ethanol is added to the solution to reach an ethanol concentration of 70%.

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

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

8. The preparation method according to claim 4, characterized in that: Step 4) The mass-to-volume ratio of the porous acetylated gastrodia elata gelatin, 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 pharmaceutical complex according to any one of claims 1 to 3 in the preparation of a medicament for treating inflammatory diseases of the colon.

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

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