In-situ phase change gel as well as preparation method and application thereof

Abstract

The invention provides an in-situ phase change gel and a preparation method and application thereof, the in-situ phase change gel comprises rhizoma bletillae polysaccharide, codonopsis pilosula polysaccharide, a bleaching assisting auxiliary material and a solvent, the concentration of the rhizoma bletillae polysaccharide is 10-20 mg / mL, and the concentration ratio of the rhizoma bletillae polysaccharide to the codonopsis pilosula polysaccharide is 1: (1-5); the bleaching assisting auxiliary material is liquid, and the volume fraction is 10-30%; or the bleaching assisting auxiliary material is a solid, and the concentration is 10-25 mg / mL. The in-situ phase change gel provided by the invention can be retained for several hours without being damaged in the stomach, plays roles in protecting gastric mucosa and slowly disintegrating and releasing, can enter the intestinal tract along with the promotion of physiological rhythm to protect the intestinal mucosa, can also reduce the alcohol level in blood, and can be used for preventing and treating the gastric mucosa. Therefore, the in-situ phase change gel can be used for preventing and / or treating gastrointestinal injury caused by acute alcoholism. The in-situ phase change gel contains traditional Chinese medicine composite polysaccharide components, is safe and high in compliance, and has good clinical advantages.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to an in-situ phase change gel, a preparation method and use thereof. Background Art

[0002] According to the classification of the International Agency for Research on Cancer (IARC), alcohol is a Class 1 carcinogen and is causally related to seven types of cancer, including esophageal cancer, liver cancer, colorectal cancer, prostate cancer and breast cancer, and can cause toxicity, psychoactivity and dependence in the human body. More and more studies have shown that alcohol abuse is inextricably linked to gastrointestinal diseases.

[0003] Acute alcohol poisoning refers to a state of central nervous system dysfunction after consuming a large amount of alcohol or alcoholic beverages in a short period of time. The clinical manifestations are mainly characterized by behavioral and neurological symptoms. Severe alcohol poisoning can damage the functions of important organs such as the stomach, intestines, liver and kidneys, leading to respiratory and circulatory failure, and then endangering life. It is one of the most common toxic diseases in the emergency department. Alcohol can affect a variety of neurotransmitter systems and interact with the γ-aminobutyric acid (GABA) neurotransmitter system, noradrenergic neurotransmitter system, 5-hydroxytryptamine neurotransmitter system and neuroendocrine system. After alcohol enters the human body, it can enter the liver through the portal vein, diffuse through the blood in the stomach and small intestine to different organs of the body, such as the brain, kidneys and lungs, which are rich in blood. In these organs, alcohol is metabolized into acetaldehyde and acetate through the ethanol oxidation system catalyzed by alcohol dehydrogenase (ADH), the microsomal ethanol oxidation system (MEOS), the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-catalase system and the xanthine oxidase-catalase system, and is finally excreted through the urinary system, respiratory system and metabolism. Acetaldehyde has been classified as a Class 1 human carcinogen by the International Agency for Research on Cancer. It is highly reactive to deoxyribonucleic acid (DNA), can bind to DNA, block DNA synthesis and repair, and cause DNA-protein cross-linking, DNA chain breaks, DNA adducts and other phenomena in eukaryotic cells. It is also genotoxic, mutagenic and carcinogenic. Ethanol and its metabolic derivative acetaldehyde can activate reduced nicotinamide adenine dinucleotide (NADH) and NADPH oxidase, increase the production of reactive oxygen species (ROS) and lipid peroxidation in the body, and intestinal oxidative stress will further damage the tight junctions of the intestine, increase intestinal permeability, and eventually cause intestinal leakage. Although the stomach is not the main organ for ethanol metabolism, about 70% of ethanol is absorbed in the stomach. Large amounts of alcohol intake will change gastric acid secretion, gastric motility, and damage the esophagus and gastric mucosa, leading to epithelial layer destruction, mucosal edema, leukocyte infiltration, congestion and bleeding, and even gastric ulcers, which ultimately destroy the barrier function of the gastric mucosa, the so-called "alcoholic gastritis", and further cause diseases such as gastroesophageal reflux disease and erosive esophagitis. In the intestine, alcohol can also damage the intestinal barrier, change the composition of the intestinal flora, reduce the proportion of key bacteria such as Lactobacillus and Bifidobacterium, increase the abundance of Proteus related to human pathogens such as Escherichia coli, and activate systemic inflammatory cascade pathways, affecting human immune function.

[0004] According to the "Expert Consensus on the Diagnosis and Treatment of Acute Alcohol Poisoning" issued by the Emergency Medicine Branch of the Chinese Medical Association, clinically used drugs for the treatment of acute alcohol poisoning can be roughly divided into three categories: alcohol metabolism-promoting drugs, wakefulness-promoting drugs, and gastrointestinal mucosal protective drugs. Metadoxine, as a commonly used prescription drug that promotes alcohol metabolism, can selectively antagonize 5-hydroxytryptamine (5-HT) 2B receptors. It is commonly used in the clinic to treat alcoholic liver disease and has the effect of promoting alcohol metabolism. Metadoxine can accelerate the metabolism of ethanol, acetaldehyde and ketone bodies in the body and inhibit oxidative stress, reduce the concentration of ethanol in the blood, and improve the symptoms of alcohol poisoning. It is a representative drug that promotes alcohol metabolism. However, metadoxine is only registered for use in a few countries and cannot be widely recognized. Opioid receptor antagonists are a class of compounds that are similar in structure to opioids, but they themselves have no agonist effect on opioid receptors. Instead, they can antagonize opioid analgesics, remove opioid analgesics bound to receptors or competitively bind to analgesics, and eliminate side effects such as gastrointestinal dysfunction and respiratory depression caused by the use of some opioid analgesics. They can be used clinically to treat various poisoning symptoms, including alcohol poisoning and other drug poisoning. Representative drugs include naloxone, naltrexone and nalmefene. Naloxone is a semi-synthetic, lipophilic, competitive and non-selective opioid receptor antagonist that can reverse and block the effects induced by opioids. It is often used to treat alcoholism and drug addiction. By antagonizing the effects of β-endorphin, it can quickly relieve these symptoms and promote patient awakening. However, patients are prone to adverse reactions such as opioid withdrawal, hypertension, irritability, nausea, vomiting, and anorexia. Studies have shown that high doses of naloxone or rapid injection of naloxone can lead to the release of catecholamines, which can lead to life-threatening pulmonary edema and arrhythmias, and their clinical application has certain limitations. Naltrexone and nalmefene have similar chemical structures to naloxone, and their oral half-life is longer than naloxone. They have fewer adverse reactions during treatment, and most patients have high tolerance. They are more widely used in clinical practice, but they still have adverse effects including liver damage, allergic reactions, and withdrawal symptoms. In addition, some patients may be at risk of addiction during use and need to be closely monitored during treatment. Gastrointestinal mucosal protective drugs mainly include acid suppressants (H2 receptor blockers and proton pump inhibitors (PPIs)), antacids, gastric mucosal protective drugs, gastrointestinal prokinetic drugs, anti-Helicobacter pylori drugs, etc. Proton pump inhibitors such as omeprazole are one of the most commonly prescribed drugs. However, a large number of studies have shown that long-term use of proton pump inhibitors can cause some inevitable side effects, including trace element deficiency, increased risk of infection, gastrointestinal diseases, kidney diseases, and cardiovascular diseases. Long-term use of PPIs by pregnant women may also cause congenital malformations of the fetus. Long-term use of PPIs can cause histopathological changes, such as protrusion of parietal cells into the glandular cavity and cystic dilatation of fundic glands. Endoscopy can reveal the formation of fundic gland polyps, hyperplastic polyps, multiple white and flat raised lesions, cobblestone mucosa, and black spots.In addition, hypochlorhydria may eventually induce hypergastrinemia, which may eventually lead to gastric polyps, gastric carcinoid, and gastric cancer.

[0005] Compared with chemical drugs, traditional Chinese medicine has the characteristics of multi-target, multi-pathway and multi-level effects, outstanding efficacy, high cure rate, small toxic and side effects, and strong anti-recurrence. It is of great significance to find effective and safe drugs for the treatment of alcohol poisoning. Summary of the invention

[0006] The purpose of this application is to provide an in-situ phase change gel, its preparation method and use, for preventing and / or treating gastrointestinal damage caused by acute alcohol poisoning. The specific technical solution is as follows:

[0007] The first aspect of the present application provides an in-situ phase change gel, which comprises Bletilla striata polysaccharide, Codonopsis pilosula polysaccharide, a bleaching auxiliary material and a solvent, wherein the bleaching auxiliary material is selected from at least one of monoolein, sodium carbonate, sodium bicarbonate and sodium lauryl sulfate, and the solvent is selected from at least one of deionized water, distilled water and ultrapure water;

[0008] The concentration of the Bletilla striata polysaccharide is 10-20 mg / mL, and the concentration ratio of the Bletilla striata polysaccharide to the Codonopsis pilosula polysaccharide is 1:(1-5); the bleaching auxiliary material is liquid with a volume fraction of 10-30%; or, the bleaching auxiliary material is solid with a concentration of 10-25 mg / mL.

[0009] In some embodiments of the first aspect of the present application, the concentration ratio of the Bletilla striata polysaccharide to the Codonopsis pilosula polysaccharide is 1:(1-3).

[0010] In some embodiments of the first aspect of the present application, the volume fraction of the bleaching-aiding auxiliary material is 15-25%, or the concentration of the bleaching-aiding auxiliary material is 15-25 mg / mL.

[0011] In some embodiments of the first aspect of the present application, the purity of the Bletilla striata polysaccharide is 95-99%.

[0012] In some embodiments of the first aspect of the present application, the purity of the Codonopsis pilosula polysaccharide is 95-99%.

[0013] In some embodiments of the first aspect of the present application, the Codonopsis pilosula polysaccharide is prepared by the following method:

[0014] Reflux extraction of Codonopsis pilosula and water at a solid-liquid ratio of 1: (4-7) for 1-3 times to obtain a total Codonopsis pilosula extract, with each reflux time being 1-3 hours; add macroporous resin to the total Codonopsis pilosula extract for shaking adsorption, then separate the mixture of Codonopsis pilosula and the macroporous resin through macroporous resin column chromatography, and collect the effluent; add 4-6 times the volume of anhydrous ethanol to the effluent, let it stand, filter, and dry to obtain the Codonopsis pilosula polysaccharide.

[0015] In some embodiments of the first aspect of the present application, the tensile strength of the in-situ phase change gel is 4-7 gf.

[0016] The second aspect of the present application provides a method for preparing the in-situ phase change gel provided in the first aspect of the present application, comprising: mixing the Bletilla striata polysaccharide and the Codonopsis pilosula polysaccharide, adding the solvent to dissolve and swell for 50-70 minutes, and then adding the bleaching auxiliary material to obtain the in-situ phase change gel.

[0017] The third aspect of the present application provides a use of the in-situ phase change gel provided in the first aspect of the present application in the preparation of a medicament for preventing and / or treating gastrointestinal damage caused by acute alcohol poisoning.

[0018] The present application provides an in-situ phase change gel, a preparation method and use thereof, wherein the in-situ phase change gel comprises Bletilla striata polysaccharide, Codonopsis pilosula polysaccharide, a bleaching aid and a solvent, wherein the concentration of Bletilla striata polysaccharide is 10-20 mg / mL, and the concentration ratio of Bletilla striata polysaccharide to Codonopsis pilosula polysaccharide is 1:(1-5); the bleaching aid is a liquid with a volume fraction of 10-30%; or, the bleaching aid is a solid with a concentration of 10-25 mg / mL. The in-situ phase change gel provided by the present application is formed into a phase change gel in an appropriate proportion by regulating the codonopsis polysaccharide and the bletilla polysaccharide to form a fluid that is easy to take, and the in-situ phase change gel in the stomach is formed by the properties of the active ingredients of traditional Chinese medicine, and a continuous gel layer is constructed by using bleaching auxiliary materials, so that the in-situ phase change gel can be retained in the stomach for several hours without being destroyed, and plays a role in protecting the gastric mucosa and slowly disintegrating and releasing. At the same time, the in-situ phase change gel can be pushed into the intestine with the physiological rhythm to protect the intestinal mucosa. The in-situ phase change gel can also reduce the alcohol level in the blood. In summary, the in-situ phase change gel of the present application can be used to prevent and / or treat gastrointestinal damage caused by acute alcohol poisoning. The in-situ phase change gel of the present application contains a Chinese medicine composite polysaccharide component, is safe, and has high compliance. It fills the treatment gap of the problem of gastrointestinal damage caused by alcohol poisoning caused by the floatable and in-situ phase change hydrogel preparations of traditional Chinese medicine, and has good clinical advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0020] Figure 1 It is a top view of the floating state of the in-situ phase change gel of Example 3 in different physiological media at different times;

[0021] Figure 2 It is a front view of the floating state of the in-situ phase change gel of Example 3 in different physiological media at different times;

[0022] Figure 3 The floating results of the in-situ phase change gel of Example 3 in artificial gastric fluid at different times;

[0023] Figure 4 The floating results of the in-situ phase change gel in Example 3 in artificial intestinal fluid at different times;

[0024] Figure 5 This is a surface SEM photo of the in-situ phase change gel of Example 3, with a scale of 500 μm;

[0025] Figure 6 This is a surface SEM photo of the in-situ phase change gel of Example 3, with a scale of 200 μm;

[0026] Figure 7 This is a cross-sectional SEM photograph of the in-situ phase change gel of Example 3, with a scale of 500 μm;

[0027] Figure 8 This is a cross-sectional SEM photograph of the in-situ phase change gel of Example 3, with a scale of 200 μm;

[0028] Fig. 9 The infrared spectra of sample A, sample B, sample C, sample D, and sample E in test example 4;

[0029] Fig.10 The mechanical strength change result of the in-situ phase change gel in Example 3 within 24 hours;

[0030] Fig.11 The viscosity of the in-situ phase change gelling agent of Example 3 varies with shear rate;

[0031] Fig.12 The shear stress of the in-situ phase change gelling agent of Example 3 varies with the shear rate;

[0032] Fig.13 The stomach tissue photos of rats in each group after drug administration and ethanol administration in Test Example 7;

[0033] Fig.14 The results of the contents of IL-1β, IL-6, TNF-α and PGE2 in the gastric tissues of rats in each group after administration and ethanol in Test Example 7;

[0034] Fig.15 These are the intestinal permeability results of rats in each group after administration of drug and ethanol in Test Example 8. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art based on the present application belong to the scope of protection of the present application.

[0036] The first aspect of the present application provides an in-situ phase change gel, which comprises Bletilla striata polysaccharide, Codonopsis pilosula polysaccharide, a bleaching auxiliary material and a solvent, wherein the bleaching auxiliary material is selected from at least one of monoolein, sodium carbonate, sodium bicarbonate and sodium lauryl sulfate, and the solvent is selected from at least one of deionized water, distilled water and ultrapure water;

[0037] The concentration of the Bletilla striata polysaccharide is 10-20 mg / mL, and the concentration ratio of the Bletilla striata polysaccharide to the Codonopsis pilosula polysaccharide is 1:(1-5); the bleaching auxiliary material is liquid with a volume fraction of 10-30%; or, the bleaching auxiliary material is solid with a concentration of 10-25 mg / mL.

[0038] The present application provides a floatable composite polysaccharide in-situ phase change gel having the function of preventing and protecting gastrointestinal damage. The phase change gel is formed by regulating Codonopsis pilosula polysaccharide and Bletilla striata polysaccharide in an appropriate proportion to make a fluid that is easy to take. The properties of the active ingredients of traditional Chinese medicine are used to form an in-situ phase change gel in the stomach. The above-mentioned bleaching auxiliary materials are used to construct a continuous gel layer, so that the in-situ phase change gel can be retained in the stomach for several hours without being destroyed, thereby protecting the gastric mucosa and slowly disintegrating and releasing. At the same time, the in-situ phase change gel can be pushed into the intestine with the physiological rhythm to protect the intestinal mucosa. The in-situ phase change gel of the present application can neutralize and reduce the alcohol content in the body, reduce the risk of gastrointestinal damage caused by acute alcohol poisoning, reduce the release of inflammatory factors in gastrointestinal tissues, improve the immunity of alcoholics and alcohol poisoning patients, and increase the safety of preventive or long-term therapeutic use by patients, so that it can be used to prevent and / or treat gastrointestinal damage caused by acute alcohol poisoning. The in situ phase change gel provided by the present application is composed of a composite polysaccharide component of traditional Chinese medicine, is safe and has high compliance, fills the therapeutic gap of traditional Chinese medicine floatable, in situ phase change hydrogel preparations for preventing and / or treating gastrointestinal damage problems caused by alcohol poisoning, and has good clinical advantages.

[0039] In some embodiments of the first aspect of the present application, the concentration ratio of the Bletilla striata polysaccharide to the Codonopsis pilosula polysaccharide is 1:(1-3). Regulating the concentration ratio of Bletilla striata polysaccharide to Codonopsis pilosula polysaccharide within this range can make the in-situ phase change gel more uniform and have better floating performance.

[0040] In some embodiments of the first aspect of the present application, the volume fraction of the bleaching auxiliary material is 15-25%, or the concentration of the bleaching auxiliary material is 15-25 mg / mL. The present application regulates the volume fraction or concentration of the bleaching auxiliary material within the above range to enable the in-situ phase change gel to have better floating performance.

[0041] In some embodiments of the first aspect of the present application, the purity of the Bletilla striata polysaccharide is 95-99%.

[0042] The present application has no particular limitation on the composition of the Bletilla striata polysaccharide, as long as the purpose of the present application can be achieved. For example, the Bletilla striata polysaccharide is composed of mannose, glucose and galactose, with a molar ratio of 9.4:2.6:1.0 and a molecular weight of about 2.35×10 5 Da.

[0043] In some embodiments of the first aspect of the present application, the purity of the Codonopsis pilosula polysaccharide is 95-99%.

[0044] In some embodiments of the first aspect of the present application, the Codonopsis pilosula polysaccharide is prepared by the following method:

[0045] Reflux extraction of Codonopsis pilosula and water at a solid-liquid ratio of 1: (4-7) for 1-3 times to obtain a total Codonopsis pilosula extract, with each reflux time being 1-3 hours; add macroporous resin to the total Codonopsis pilosula extract for shaking adsorption, then separate the mixture of Codonopsis pilosula and the macroporous resin through macroporous resin column chromatography, and collect the effluent; add 4-6 times the volume of anhydrous ethanol to the effluent, let it stand, filter, and dry to obtain the Codonopsis pilosula polysaccharide. The present application has no particular limitation on the amount of the macroporous resin added, as long as the purpose of the present application can be achieved. It is a conventional technology in the field, and the amount added is not overloaded, so that it can achieve the purpose of enriching the traditional Chinese medicine components. Exemplarily, the mass ratio of the total extract of Codonopsis pilosula to the added macroporous resin is 5:1-1:5; the volume ratio of the effluent to the anhydrous ethanol is 1:(4-6); there is no particular limitation on the filtering method, as long as the purpose of the present application can be achieved, for example, the filtration is suction filtration; there is no particular limitation on the drying method, as long as the purpose of the present application can be achieved, for example, the drying is freeze-drying.

[0046] In the present application, the composition of the Codonopsis pilosula polysaccharide includes mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose, with a molar ratio of 1.00:1.76:18.48:69.81:6.06:5.14, and a molecular weight of about 2.65×10 5 Da.

[0047] In some embodiments of the first aspect of the present application, the tensile strength of the in-situ phase change gel is 4-7 gf.

[0048] The second aspect of the present application provides a method for preparing the in-situ phase change gel provided in the first aspect of the present application, comprising: mixing the Bletilla striata polysaccharide and the Codonopsis pilosula polysaccharide, adding the solvent to dissolve and swell for 50-70 minutes, and then adding the bleaching auxiliary material to obtain the in-situ phase change gel.

[0049] The third aspect of the present application provides a use of the in-situ phase change gel provided in the first aspect of the present application in the preparation of a medicament for preventing and / or treating gastrointestinal damage caused by acute alcohol poisoning.

[0050] The in-situ phase-change gel provided by the present application overcomes the disadvantage that the general tablet-type gastric stasis drug delivery system preparation is not friendly to patients with dysphagia. It uses natural and safe traditional Chinese medicine ingredients to prepare a drug that is easy to swallow and can phase-change to form a floating gel after in-situ administration. It can isolate gastric contents, protect the irritation of the gastric mucosa and / or intestinal mucosa, and reduce the alcohol level in the blood. Therefore, it can be used to prevent and / or treat gastrointestinal damage caused by acute alcohol poisoning, and further used to prepare drugs for preventing and / or treating gastrointestinal damage caused by acute alcohol poisoning.

[0051] Terms used herein, unless otherwise specifically described or defined, have their general meanings well known to those skilled in the art.

[0052] Example

[0053] Hereinafter, embodiments and comparative examples are given to more specifically describe the embodiments of the present application. Various tests and evaluations were performed according to the following methods, and the experimental materials and methods used were conventional materials and methods unless otherwise specified. In addition, "parts" and "%" are weight references unless otherwise specified. Experimental instruments are shown in Table 1, and materials or reagents are shown in Table 2.

[0054] Table 1

[0055] Instrument Name Manufacturer Milli-QⅡ Ultrapure Water Analyzer MILLIPORE, USA RW20 Overhead Mixer Shanghai Tuohe Electromechanical Technology Co., Ltd. AX205 1 / 100,000 Balance MettlerToledo, Switzerland AX224ZH 1 / 10,000 Balance Ohaus Instruments (Changzhou) Co., Ltd. Freeze Dryer Shanghai Bilang Instrument Manufacturing Co., Ltd. Tensonor27 Infrared Spectrometer Bruker NTV-P3 Rheometer Shanghai Nirun Intelligent Technology Co., Ltd. SZCL-4B Intelligent Magnetic Heating Stirrer Gongyi Yuhua Instrument Co., Ltd. TM3000 Scanning Electron Microscope Hitachi Corporation PT-501B Sept single arm tensile testing machine Dongguan Saipute Testing Equipment Co., Ltd. TecanSpark Multi-function Microplate Reader Swiss TECAN Three-purpose constant temperature water tank Changzhou Noki Instrument Co., Ltd.

[0056] Table 2

[0057]

[0058]

[0059] Example 1

[0060] <Preparation of Codonopsis pilosula polysaccharide>

[0061] The medicinal material of Codonopsis pilosula was subjected to two distilled water reflux extractions (the material-liquid ratio was 1kg:5.5kg, and the reflux time was 2h each time) to obtain a total extract of Codonopsis pilosula. Subsequently, 1000g of macroporous resin was added to the extract, mixed and shaken for adsorption. The mixture of Codonopsis pilosula and macroporous resin was separated by macroporous resin column chromatography, and the effluent was collected. Five volumes of anhydrous ethanol were added to the effluent, and the effluent was filtered after standing overnight. The precipitate obtained by filtration was evaporated, and a freeze-drying method was used to obtain Codonopsis pilosula polysaccharide with a purity of 95-98%, and the composition included mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose, with a molecular weight of about 2.65×10 5 Da.

[0062] <Preparation of in-situ phase change gelling agent>

[0063] 1.5 g of Bletilla striata polysaccharide with a purity of 98% and 1.5 g of Codonopsis pilosula polysaccharide prepared above were mixed, 80 mL of deionized water was added, stirred to dissolve and fully swelled for 60 min, and then 20 mL of bleaching auxiliary material GMO was added to prepare an in-situ phase change gel.

[0064] Embodiment 2-7

[0065] Except for adjusting the corresponding preparation parameters as shown in Table 3, the rest is the same as Example 1.

[0066] Example 8

[0067] 1.5 g of Bletilla striata polysaccharide with a purity of 98% and 4.5 g of Codonopsis pilosula polysaccharide prepared in Example 1 were mixed, 100 mL of deionized water was added, stirred to dissolve and fully swelled for 60 min, and then 2.0 g of sodium dodecyl sulfate as a bleaching auxiliary was added to prepare an in-situ phase change gel.

[0068] Comparative Examples 1-6

[0069] Except for adjusting the corresponding preparation parameters as shown in Table 3, the rest is the same as Example 1.

[0070] Comparative Example 7

[0071] 1.5 g of Bletilla striata polysaccharide with a purity of 98% and 4.5 g of Codonopsis pilosula polysaccharide prepared in Example 1 were mixed, 100 mL of deionized water was added, stirred to dissolve and fully swelled for 60 min, and then 3.0 g of sodium bicarbonate as a bleaching auxiliary was added to prepare an in-situ phase change gel.

[0072] Table 3

[0073]

[0074] Note: “ / ” indicates no corresponding preparation parameters.

[0075] In-situ phase change gel floating performance test

[0076] Test Example 1 Floating performance of different in-situ phase change gelling agents

[0077] The floating time is a direct reflection of the floating performance of the floatable in-situ phase change gel. The present application prepares a composite polysaccharide floatable in-situ phase change gel by adding an appropriate amount of bleaching auxiliary material to the composite gel of Codonopsis pilosula and Bletilla striata, and investigates the floating performance of different in-situ phase change gels. The uniformity of the in-situ phase change gel was observed. Under the physiological temperature of 37±0.1°C, 10 mL of the in-situ phase change gel was drawn with a syringe and placed in a beaker containing 90 mL of artificial gastric juice at a rotation speed of 100 rpm. The time required for the in-situ phase change gel to float on the surface of the gastric juice was set as the floating lag time, and the continuous floating time of the in-situ phase change gel on the surface of the gastric juice was set as the total floating time. The floating performance test results of the in-situ phase change gels of Examples 1-8 and Comparative Examples 1-7 are shown in Table 4.

[0078] Table 4

[0079]

[0080]

[0081] It can be seen from the observation that the in-situ phase change gel of the present application is more uniform, delicate and easy to take. According to Examples 1-5 and Comparative Examples 1-4, it can be seen that the in-situ phase change gel using the concentration ratio of Bletilla striata polysaccharide and Codonopsis pilosula polysaccharide within the scope of the present application has a shorter floating lag time, a longer total floating time, and better floating performance. According to Examples 3, 6-8 and Comparative Examples 5-7, it can be seen that the in-situ phase change gel using the type and content of the bleaching auxiliary material within the scope of the present application can maintain the floating of the in-situ phase change gel in simulated gastric juice for a long time, and has better floating performance. At the same time, the type of bleaching auxiliary material used in the present application is safe and has no effect on the safety and compliance of the patient's medication. The above results show that the in-situ phase change gel including the composition and content of the present application has better floating performance.

[0082] Test Example 2: Floating performance in different physiological media

[0083] 10 mL of the in-situ phase change gel of Example 3 was drawn with a syringe and placed in beakers containing artificial intestinal fluid (pH=6.8) and artificial gastric fluid (pH=1.2), respectively. The diameter of the beaker was R=4.7 cm. The thickness and the flat area were used as indicators. The floating performance of the in-situ phase change gel in different physiological media was observed and recorded every 1 h. The top view of the floating state of the in-situ phase change gel in different physiological media at different times is shown in FIG. Figure 1 As shown, the front view is as Figure 2 As shown, the floating results in artificial gastric fluid at different times are as follows Figure 3As shown in Figure 2, the floating results in artificial intestinal fluid at different times are as follows: Figure 4 As shown. Figure 1-4 The results show that the in-situ phase change gel of the present application can form a gel structure with a certain physical volume in situ in different physiological media, and the floating performance is good.

[0084] Structural analysis of in-situ phase change gel

[0085] Test Example 3 Scanning Electron Microscope (SEM) Analysis

[0086] The structural morphology of the floatable in-situ phase change gel is closely related to its in vivo floating performance, drug release rate and surface mechanical strength. The surface and cross-section of the in-situ phase change gel were subjected to SEM analysis. The clear internal microstructure can prove the formation of the in-situ hydrogel and preliminarily characterize its possible physical strength and its supporting capacity on the surface of gastric contents. The in-situ phase change gel of Example 3 was adhered to a conductive tape, the excess powder was blown off and gold was sprayed. The operating acceleration voltage of the SEM was 10.0 kV. The surface and cross-section morphology of the in-situ phase change gel sample were observed under a scanning electron microscope, and the surface SEM photo of the in-situ phase change gel of Example 3 was obtained as shown in the figure. Figure 5 and Figure 6 The cross-section SEM photo is shown in Figure 7 and Figure 8 As shown. Figure 5-Figure 8 It can be seen that the surface morphology of the in-situ phase change gel shows a certain pore structure, and the internal pore structure of the gel is multi-layered and relatively regular, which is manifested in the increase of surface pores, more uniform pore size, and more orderly arrangement. The pore diameter is about 80μm. This neatly arranged and uniform pore structure is conducive to the long-term floating of the in-situ phase change gel in the body, and the porous structure will provide a larger surface area, accelerate the dissolution and swelling process, and thus promote the disintegration and release of the drug in the body. Figure 7-Figure 8 It can be seen that the cross-section of the in situ phase change gel forms a uniform layered structure, which may be caused by the ordered polymerization between multiple types of polysaccharide macromolecules. This polymerization increases the mechanical strength of the in situ phase change gel, thereby helping the in situ phase change gel to remain floating in the body for a long time and enhance the adsorption effect on the biological mucosal surface.

[0087] Test Example 4 Infrared (IR) Analysis

[0088] The oil-free composite gel was prepared by the following steps: 100 mL of deionized water was added and GMO was not added, and the rest was the same as in Example 3 to obtain the oil-free composite gel. 2 mg of each of sample A: powder of polysaccharide of Bletilla striata, sample B: powder of polysaccharide of Codonopsis pilosula, sample C: freeze-dried powder of the in-situ phase change gel of Example 3, sample D: freeze-dried powder of the oil-free composite gel, and sample E: a mixed powder of composite polysaccharides with a mass ratio of 1:3 of polysaccharide of Bletilla striata and polysaccharide of Codonopsis pilosula were mixed evenly with 200 mg of potassium bromide, pressed into transparent sheets, and heated at 4000-400 cm -1 Infrared spectrum scanning is performed within the range, and infrared spectra are recorded to obtain infrared spectra of sample A-sample E as shown in Fig. 9 shown. Fig. 9 Sample A: The infrared spectrum of Bletilla striata polysaccharide powder shows 1628 cm -1 The peak at 2920cm indicates that the sample contains water, which is related to the detection of water; -1 and 2852cm -1 The stretching vibration absorption peak of methylene -CH2 is at 3442cm -1 There is a broad hydroxyl-OH stretching vibration absorption peak at 930 cm. Sample B: Codonopsis pilosula polysaccharide powder -1 The peak at 1030 cm represents the asymmetric stretching vibration of the D-glucose pyranose ring. -1 The peak at 869cm represents the stretching vibration of the pyranose ring CO; -1 The absorption band at 595 cm -1 The absorption bands at 3442 cm-1 represent the formation vibration of α-CH in the pyranose ring and the pyranose ring skeleton; the -OH stretching vibration absorption peak at 3442 cm-1 represents ... -1 at 1628cm -1 The location may be related to the detection of water; 2920cm -1 and 2852cm -1 The peak at 1741cm is the stretching vibration of methylene -CH2. Compared with sample D of the freeze-dried powder of the composite gel without oil, sample C of the freeze-dried powder of the in-situ phase change gel with monooleyl glycerol added has a peak at 1741cm -1 A strong absorption peak of the carbonyl C=O stretching vibration of the ester appears at 1628 cm -1 The peaks at the bottom are the absorption peaks of bound water or the CO vibration of carboxyl groups. The changes in these peaks may be due to the addition of monoolein, so it can be considered that the Bletilla striata polysaccharide-Codonopsis pilosula polysaccharide and the bleaching auxiliary materials in the in-situ phase change gel are combined through physical effects such as intermolecular hydrogen bonds.

[0089] Mechanical strength test of in-situ phase change gel

[0090] Test Example 5 Mechanical Strength Test of In-Situ Phase Change Gel

[0091] The mechanical strength of the in-situ phase change gel is the key property for it to play a physical barrier role and resist the upwelling of gastric contents. 30 mL of the in-situ phase change gel of Example 3 was taken and placed in a 100 mL beaker filled with 70 mL of hydrochloric acid (pH = 1.2, temperature 37°C) and allowed to stand to form. The in-situ phase change gel is surrounded by an L-shaped stainless steel wire probe (long arm 90 mm, short arm 30 mm) standing upright in the beaker. Place the beaker on the sample table of a single-arm tensile testing machine, and adjust the position of the tensile arm so that the stainless steel probe is immersed in the in-situ phase change gel. Clear all the values ​​sensed by the tensile machine, use a preload speed of 300 mm / min and stretch upward at a uniform speed. After the in-situ phase change gel is completely separated from the stainless steel probe, stop the experiment, record the maximum tension, and draw a curve of the change in tension (gf) and time (h). Each experiment is measured three times in parallel and the average value is recorded. The change in the mechanical strength of the in-situ phase change gel within 24 hours is obtained as follows Fig.10 As shown, it can be seen that the in-situ phase change gel of the present application has strong tensile strength, strong ability to resist the upwelling of gastric contents in the stomach, and good floating performance.

[0092] Analysis of rheological properties of in-situ phase change gel

[0093] Test Example 6 Rheological properties analysis of in-situ phase change gel

[0094] Use an overhead stirrer to stir thoroughly for 10 minutes at 1000 rpm until the in-situ phase change gel is uniform. Take 40 mL of the in-situ phase change gel of Example 3 and place it in a 50 mL centrifuge tube (height 11.40 cm, diameter 2.80 cm), and use a rheometer (rotor No. 6) to measure the viscosity and shear stress of the in-situ phase change gel at different speeds. The results are shown in Table 5. The viscosity of the in-situ phase change gel varies with the shear rate. Fig.11 The shear stress changes with the shear rate as shown in Fig.12 As shown in Table 5, Fig.11 and Fig.12 It can be seen that the viscosity of the in-situ phase change gel of the present application decreases with increasing shear rate and increases with decreasing shear rate, the shear stress increases with increasing shear rate and decreases with decreasing shear rate, and returns to the initial state after stopping stirring. It can be seen that the in-situ phase change gel of the present application has positive thixotropy and stable quality.

[0095] Table 5

[0096] Speed ​​(rpm) <![CDATA[Shear rate (s -1 )]]> Viscosity (mPa·s) <![CDATA[Shearing stress (dyne / cm 2 )]]> 110 23 7536.66 157.5 120 25.1 6869.69 158.0 130 27.2 6402.77 160.7 140 29.3 6046.15 164.4 150 31.4 5873.8 172.1 150 31.4 5819.04 170.4 140 29.3 5966.66 162.2 130 27.2 6405.55 160.7 120 25.1 6851.51 157.5 110 23 7506.66 156.8

[0097] Pharmacological efficacy experiment of in-situ phase change gel

[0098] Test Example 7 Effect of in-situ phase change gel on ethanol-induced gastrointestinal damage

[0099] 36 SD rats (weight 300-350g, source: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal license number SCXK (Beijing) 20210006) were randomly divided into model group, sucralfate group, medium-dose group, high-dose group, Bletilla striata group, Codonopsis pilosula group, 6 rats in each group. The rats were kept in cages at room temperature of 20-25℃, with a light-dark cycle ratio of 12h:12h. They were adaptively fed for 3 days before the experiment, the litter was changed every 2 days, and food and water were supplied normally. Fasting but not water was allowed 24 hours before the start of the experiment to avoid rats from swallowing feces and eliminating the interference of feces on the induction of gastrointestinal damage in rats. The specific dosing regimen is shown in Table 6. The model group rats were given 5 mL / kg of normal saline, the medium-dose group rats were given 5 mL / kg of the in-situ phase change gel of Example 3, the high-dose group rats were given 10 mL / kg of the in-situ phase change gel of Example 3, the positive drug sucralfate group was given 200 mg / kg.bw of sucralfate oral suspension (200 mg per kilogram of body weight), the Bletilla striata group was given 5 mL / kg of the in-situ phase change gel of Comparative Example 1, and the Codonopsis pilosula group was given 5 mL / kg of the in-situ phase change gel of Comparative Example 2, and the administration was continued for two days. One hour after the end of the administration on the second day, the rats in each group were given 5 mL / kg of anhydrous ethanol. One hour after the administration of anhydrous ethanol, all animals were injected with 10% chloral hydrate 4 mL·kg -1 After anesthesia, the entire stomach and intestines were immediately removed, and the stomach was cut open along the greater curvature of the stomach for analysis of gastric and intestinal lesions. Finally, the rats were euthanized by cervical decapitation. The gastric contents and colon contents were collected and stored in a -80℃ refrigerator.

[0100] Table 6

[0101] Grouping Intragastric administration Oral dose Modeling dose Model Group Normal saline 5mL / kg Anhydrous ethanol 5mL / kg Sucralfate group Sucralfate oral suspension 200mg / kg.bw Anhydrous ethanol 5mL / kg Medium dose group Example 3 In-situ phase change gel 5mL / kg Anhydrous ethanol 5mL / kg High dose group Example 3 In-situ phase change gel 10mL / kg Anhydrous ethanol 5mL / kg Bletilla striata group Comparative Example 1 In-situ phase change gel 5mL / kg Anhydrous ethanol 5mL / kg Codonopsis pilosula group Comparative Example 2 In-situ phase change gel 5mL / kg Anhydrous ethanol 5mL / kg

[0102] In the above steps, the stomach was cut open along the greater curvature of the stomach, and then rinsed with saline to remove the stomach contents and dried with filter paper. After spreading, the length and width of the bleeding point or bleeding band in the stomach were measured with a vernier caliper. The length was from the esophagus to the pylorus and the width was the direction of the cut and spread along the greater curvature of the stomach. The area of ​​gastric mucosal injury induced by ethanol was expressed in mm 2 High-definition photos of gastric tissue were analyzed by Image J 1.46r software (Wayne Rasband, NIH, USA). ELISA kits were used to determine the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and prostaglandin E2 (PGE2) in gastric tissue, and SPSS software was used for statistical analysis of the quantitative data. Fig.13The results of the gastric injury area of ​​each group of rats are shown in Table 7, wherein the left area refers to the left area of ​​the stomach divided along the central axis, and the right area refers to the right area of ​​the stomach divided along the central axis. Fig.13 The results of IL-1β, IL-6, TNF-α and PGE2 in each group of rats are shown in Fig.14 shown.

[0103] Table 7

[0104]

[0105]

[0106] Note: *P<0.05, #P<0.05, &P<0.05, ^P<0.05, * indicates significant difference between each group and model group, # indicates significant difference between each group and positive drug sucralfate group, & indicates significant difference between each group and Bletilla striata group, ^ indicates significant difference between each group and Codonopsis pilosula group.

[0107] Fig.13 The results in Table 7 show that compared with the model group, the gastric mucosal injury area of ​​rats in the medium-dose group and the high-dose group was significantly reduced, especially the high-dose group, which almost completely inhibited the ethanol-induced gastric mucosal injury, and the effect was better than that of the sucralfate group, the Bletilla striata group and the Codonopsis pilosula group. Fig.14 Compared with the model group, **P<0.01, ***P<0.001; compared with the sucralfate group, #P<0.05, ##P<0.01, ###P<0.001; It can be seen that compared with the model group, the IL-1β, IL-6, and TNF-α levels in the gastric tissue of rats in the medium-dose group and the high-dose group were significantly reduced, and the PGE2 content was significantly increased. The above results show that the in situ phase change gel provided by the present application can reduce the level of inflammatory factors in gastric tissue, promote the secretion of gastric mucosal PGE2, and thus reduce gastric mucosal damage.

[0108] The intestinal segment from the duodenum to the small intestine of 10 cm was observed for bleeding and swelling, and the intestinal damage was analyzed. The results showed that the intestinal bleeding and swelling of the rats administered with the in situ phase change gel of the present application were significantly alleviated.

[0109] Test Example 8 Intestinal Permeability Test

[0110] In test example 7, after removing the entire stomach and intestines, 7 cm of the rat small intestine was taken at about 6 cm away from the stomach, the intestinal cavity was flushed with phosphate buffered solution (PBS), and after ligating one end, 0.2 mL of 1.5% Evans blue was injected, and the other end was tied. Place it in a beaker with 20 mL of Gram-Heinz solution, bathe at 37°C, take out the intestinal sac tissue after 30 minutes, rinse the intestinal cavity with normal saline, dry at 37°C, and weigh the intestinal segment. Then add 2 mL of formamide solution, incubate at 37°C for 24 hours, take the supernatant, dilute it 3 times with PBS, and measure the absorbance (OD) value at 650 nm on an enzyme reader. The results of intestinal permeability of each group of rats are shown below. Fig.15 As shown, where the ordinate is the absorbance value of the intestinal segment per unit weight (g), compared with the model group, **P<0.01, ***P<0.001; compared with the sucralfate group, #P<0.05, ###P<0.001, it can be seen that the intestinal permeability of rats in the medium-dose group and the high-dose group of the present application was significantly reduced. The results show that the in situ phase change gel of the present application helps to maintain the intestinal barrier function.

[0111] The above results show that the in-situ phase change gel of the present application has a significant preventive and protective effect on ethanol-induced acute gastric mucosal and intestinal damage. The in-situ phase change gel provided by the present application can float rapidly in the stomach, isolate the gastric contents from further damaging the gastrointestinal mucosa, effectively reduce gastric mucosal bleeding and inflammatory secretions, and has good clinical application prospects.

[0112] It should be noted that, in this article, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method or article that includes a series of elements includes not only those elements, but also includes other elements that are not explicitly listed, or also includes elements that are inherent to such process, method or article.

[0113] Each embodiment in this specification is described in a related manner, and the same or similar parts between the embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0114] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. An in-situ phase change gel, comprising Bletilla striata polysaccharide, Codonopsis pilosula polysaccharide, a bleaching auxiliary material and a solvent, wherein the bleaching auxiliary material is selected from at least one of monoolein, sodium carbonate, sodium bicarbonate and sodium lauryl sulfate, and the solvent is selected from at least one of deionized water, distilled water and ultrapure water; The concentration of the Bletilla striata polysaccharide is 10-20 mg / mL, and the concentration ratio of the Bletilla striata polysaccharide to the Codonopsis pilosula polysaccharide is 1:(1-5); the bleaching auxiliary material is liquid with a volume fraction of 10-30%; or, the bleaching auxiliary material is solid with a concentration of 10-25 mg / mL.

2. The in-situ phase change gel according to claim 1, wherein the concentration ratio of the Bletilla striata polysaccharide to the Codonopsis pilosula polysaccharide is 1:(1-3).

3. The in-situ phase change gelling agent according to claim 1, wherein: The volume fraction of the bleaching auxiliary material is 15-25%, or the concentration of the bleaching auxiliary material is 15-25 mg / mL.

4. The in-situ phase change gel according to any one of claims 1 to 3, wherein The purity of the Bletilla striata polysaccharide is 95-99%.

5. The in-situ phase change gel according to any one of claims 1 to 3, wherein: The purity of the codonopsis pilosula polysaccharide is 95-99%.

6. The in-situ phase change gelling agent according to claim 5, wherein: The codonopsis polysaccharide is prepared by the following method: Reflux extraction of Codonopsis pilosula and water at a solid-liquid ratio of 1: (4-7) for 1-3 times to obtain a total Codonopsis pilosula extract, with each reflux time being 1-3 hours; add macroporous resin to the total Codonopsis pilosula extract for shaking adsorption, then separate the mixture of Codonopsis pilosula and the macroporous resin through macroporous resin column chromatography, and collect the effluent; add 4-6 times the volume of anhydrous ethanol to the effluent, let it stand, filter, and dry to obtain the Codonopsis pilosula polysaccharide.

7. The in-situ phase change gel according to any one of claims 1 to 6, wherein: The tensile strength of the in-situ phase change gel is 4-7gf.

8. A method for preparing an in-situ phase change gel according to any one of claims 1 to 7, comprising: The Bletilla striata polysaccharide and the Codonopsis pilosula polysaccharide are mixed, and the solvent is added to dissolve and swell for 50-70 minutes, and then the bleaching auxiliary material is added to obtain the in-situ phase change gel.

9. Use of the in-situ phase change gel according to any one of claims 1 to 7 in the preparation of a medicament for preventing and / or treating gastrointestinal damage caused by acute alcohol poisoning.

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