Preparation and Application of a Carrier-Free Metal-Organic Small Molecule Supramolecular Hydrogel Derived from Maxing Shigan Decoction with Antipyretic and Anti-Inflammatory Effects

Abstract

The invention discloses a preparation method and antipyretic and anti-inflammatory application of a novel carrier-free metal-organic small molecule supramolecular hydrogel derived from Maxing Shigan Decoction. The hydrogel drug raw materials of the invention include pseudoephedrine hydrochloride in Ephedra sinica in the prescription of Maxing Shigan Decoction, amygdalin in Armeniaca vulgaris, magnesium ions in Gypsum fibrosum, and glycyrrhizic acid in Glycyrrhiza uralensis Fisch. The preparation method of the quaternary magnesium hydrogel drug is simple, green, has good stability and high biocompatibility. In a rat fever model induced by lipopolysaccharide at different doses, the antipyretic effect of the hydrogel drug can last up to 12 hours, and the antipyretic effect is significantly better than that of other metal ions such as quaternary calcium hydrogel, quaternary zinc hydrogel, and quaternary copper hydrogel groups. At the same time, it has a more persistent antipyretic effect than the whole prescription of Maxing Shigan Decoction; the hydrogel drug does not require the addition of excipients, has no toxic and side effects, and has a high drug utilization rate.

Description

Technical Field

[0001] The present invention belongs to the field of novel pharmaceutical preparations, and particularly relates to a supramolecular hydrogel derived from Maxing Shigan Decoction, its preparation and application. Background Art

[0002] Fever is a very common symptom in clinical practice, mostly caused by inflammation and infection. Currently, common clinical antipyretic and anti-inflammatory drugs include non-steroidal antipyretic and anti-inflammatory drugs such as paracetamol, ibuprofen, and aspirin. Their mechanism of action is mainly to inhibit cyclooxygenase and reduce the synthesis of prostaglandins. However, such drugs may increase the risk of cardiovascular diseases, and large or long-term use may cause digestive system ulcers, hypothermia symptoms, etc. There has been no innovative drug research and development and listing in this field in recent decades. Therefore, developing antipyretic and anti-inflammatory drugs with definite curative effects and few side effects is one of the hotspots in the research and development of such drugs. Traditional Chinese medicine has been proven to have definite curative effects after thousands of years of use and has therapeutic effects on various diseases. Many scholars have found that some traditional Chinese medicine compound prescriptions have good antipyretic and anti-inflammatory effects, and various components isolated from these traditional Chinese medicines have also been proven to have good antipyretic and anti-inflammatory effects. As is well known, traditional Chinese medicine has a long history of use and rich clinical experience. Therefore, it is possible to find highly effective, low-toxic and side-effect, and long-lasting antipyretic drugs from traditional Chinese medicine, providing a new direction for the development of new clinical antipyretic and anti-inflammatory drugs. Among them, Maxing Shigan Decoction derived from Treatise on Febrile and Miscellaneous Diseases can not only be widely used for fever symptoms caused by various reasons, but also through long-term clinical application and modern basic research, it has been shown that this drug can significantly reduce the expression of inflammatory factors and regulate body temperature through multiple pathways, with high safety and definite curative effects. Metal elements are one of the important pharmacodynamic components of Maxing Shigan Decoction. In addition to calcium, the content of magnesium element is also relatively high. In addition, there are also trace elements such as zinc and copper, which are the key to the full play of the pharmacodynamic effects of Maxing Shigan Decoction (References: [1] Guo Huaizhong, Wen Qian, Ran Ruixue, Wang Min. Effects of different compatibilities on the dissolution of calcium and magnesium in Maxing Shigan Decoction [J]. China Journal of Chinese Materia Medica, 2010, 35(22): 2985 - 2989. [2] Zhang Wuping, Kou Guang, Rao Yi, Wei Huizhen, Wu Xidong. Determination of the effects of changes in the dosage of almonds in Maxing Shigan Decoction on trace elements by ICP-AES [J]. Journal of Shanxi Medical University, 2018, 49(11): 1319 - 1323. [3] Chen Rui, Chen Zhipeng. New strategies for exploring the antiviral mechanism of Maxing Shigan Decoction using supramolecular assembly theory [J]. Journal of Nanjing University of Traditional Chinese Medicine, 2021, 37(01): 136 - 139.).

[0003] The application of supramolecular materials in drug delivery systems is a current research hotspot, which can be used to improve drug bioavailability, reduce toxic and side effects, and reduce the frequency of drug administration. Commonly used supramolecular drug delivery carriers include liposomes, proteins, polymer micelles, etc. However, these supramolecular carriers also have defects. For example, the surface fusion effect of liposomes will make their drug-loading ability poor, and they are easily phagocytosed by macrophages, thereby affecting the efficacy of the loaded drugs; protein carriers also have disadvantages such as short half-life, poor stability, and sensitization, which limit their application; there are also some drug-loading materials that are prone to cause toxicity in the body during degradation and metabolism, which all limit the application of supramolecular carrier materials. Therefore, the development of carrier-free supramolecular drugs has gradually become a hotspot. Carrier-free supramolecular drugs not only have the same drug delivery advantages as supramolecular materials, but also have many advantages such as small toxic and side effects and high delivery efficiency.

[0004] In addition, a gel is a semi-solid or thick liquid preparation with gel properties made from an active pharmaceutical ingredient or drug extract and a suitable matrix, and it is the mainstream dosage form for mucosal administration in the cavity. Oral gel is a relatively new oral preparation that can adhere to the surface of the stomach to form a protective film, with slow drug release but rapid absorption. In China, paracetamol oral gel has been approved as an antipyretic drug. However, the matrices of existing oral gel preparations are usually polymer hydrogels, aluminum hydroxide and aluminum phosphate inorganic gels. Common hydrogel matrices such as carbomer, cellulose, poloxamer, etc. are inert excipients without pharmacological activity, and often need to swell during the preparation process. Gels based on them are prone to water loss and mildew, and are greatly affected by metal ions in the system. For example, cellulose-based drugs are prone to form insoluble precipitates with cations, significantly affecting drug loading and drug formation. Clinically, there is an urgent need for oral gels that take into account safety, effectiveness, and excellent performance.

[0005] Glycyrrhizic acid is one of the active ingredients of the traditional Chinese medicine licorice. Some scholars have found that glycyrrhizic acid has the effect of self-assembling to form a supramolecular hydrogel, and can form a composite system with cellulose nanocrystals. This system is expected to be used as a stabilizer and thermoresponsive carrier for food and cosmetics because it can improve the stability of emulsions. However, the success of self-assembly is greatly affected by the solvent, temperature, pH value of the system, and the structure and properties of other components / impurities. There are no research reports on co-assembling glycyrrhizic acid with more than two small molecule components into a gel, and there are even fewer reports on applying the obtained gel as an active ingredient without a carrier. Summary of the Invention

[0006] Based on the above background, in view of various problems existing in the aforementioned prior art, the present invention uses representative components of the classic antipyretic formula Maxing Shigan Decoction in Zhang Zhongjing's Treatise on Febrile and Miscellaneous Diseases, namely pseudoephedrine, glycyrrhizic acid, amygdalin, and soluble inorganic metal salts that can dissociate into calcium, magnesium, zinc, or copper ions, as raw materials to prepare a quaternary carrier-free supramolecular hydrogel. This hydrogel has no toxic side effects, has a significant and long-lasting antipyretic and anti-inflammatory effect, and has great potential for development into a new drug.

[0007] The hydrogel is self-assembled from the representative active components of four traditional Chinese medicines in Maxing Shigan Decoction, namely glycyrrhizic acid, pseudoephedrine, amygdalin, and magnesium / calcium / zinc / copper ions, and does not contain excipients, being a quaternary carrier-free hydrogel.

[0008] The inventors also discovered a method for preparing a self-assembled hydrogel, which can be used to prepare the aforementioned quaternary supramolecular hydrogel, and confirmed the assembly mechanism, the structure of the hydrogel formed after assembly, and the material properties by means such as infrared, nuclear magnetic resonance, and rheology.

[0009] In addition, the inventors also evaluated its antipyretic and anti-inflammatory activities through a rat fever model induced by different doses of lipopolysaccharide (LPS), and found that the prepared quaternary magnesium, quaternary calcium, quaternary zinc, and quaternary copper supramolecular hydrogels have various advantages such as high drug loading, no toxic side effects, high bioavailability, and long-lasting antipyretic and anti-inflammatory effects. Surprisingly, it was found that this hydrogel has significantly better antipyretic and anti-inflammatory effects compared with the original formula Maxing Shigan Decoction, and among them, the quaternary magnesium hydrogel has the best antipyretic and anti-inflammatory effects. This invention has great research significance for discovering and developing drugs with significant antipyretic and anti-inflammatory effects from traditional Chinese medicine compounds.

[0010] Therefore, the objectives of the present invention include providing a carrier-free supramolecular hydrogel.

[0011] The objectives of the present invention include providing a method for preparing a supramolecular hydrogel.

[0012] The objectives of the present invention include providing a new pharmaceutical preparation with good antipyretic effects and high safety.

[0013] The objectives of the present invention include providing the application of preparing a drug for antipyretic treatment using the aforementioned supramolecular hydrogel.

[0014] The objectives of the present invention include providing the application of preparing a drug for anti-inflammatory treatment using the aforementioned supramolecular hydrogel.

[0015] To achieve the above invention objectives, the present invention adopts the following technical solutions:

[0016] A supramolecular hydrogel is self-assembled from glycyrrhizic acid, pseudoephedrine or its salt, amygdalin, and inorganic magnesium salt.

[0017] The pseudoephedrine or its salt is preferably pseudoephedrine hydrochloride or pseudoephedrine oxalate, more preferably pseudoephedrine hydrochloride.

[0018] The inorganic magnesium salts described above are common soluble inorganic magnesium salts in the art that can dissociate into magnesium ions, including but not limited to magnesium sulfate, magnesium chloride, magnesium dihydrogen phosphate, etc.; soluble inorganic calcium salts that can dissociate into calcium ions, including but not limited to calcium chloride, calcium dihydrogen phosphate, etc.; soluble inorganic zinc salts that can dissociate into zinc ions, including but not limited to zinc chloride, zinc sulfate, etc.; soluble inorganic copper salts that can dissociate into copper ions, including but not limited to copper chloride, copper sulfate, etc.

[0019] The self-assembly preparation method of the aforementioned supramolecular hydrogel includes the following steps (taking the preparation of a quaternary magnesium hydrogel as an example):

[0020] (1) Heat to dissolve glycyrrhizic acid in water.

[0021] (2) Heat to dissolve pseudoephedrine or its salt in water.

[0022] (3) Heat to dissolve amygdalin in water.

[0023] (4) Heat to dissolve the inorganic magnesium salt in water.

[0024] (5) Mix and heat the aqueous solutions of glycyrrhizic acid, pseudoephedrine hydrochloride, amygdalin, and magnesium ions prepared in steps (1), (2), (3), and (4), and let it stand and cool.

[0025] Preferably, the heating temperature in steps (1) and (5) is 60 - 100 °C, such as 60 °C, 65 °C, 70 °C, 80 °C, 100 °C.

[0026] Preferably, the heating temperature in steps (2), (3), and (4) is 25 - 100 °C, such as 25 °C, 30 °C, 50 °C, 80 °C, 100 °C.

[0027] The preferably weighed molar ratio of glycyrrhizic acid, pseudoephedrine or its salt, amygdalin, and inorganic magnesium salt is 1 - 10∶1 - 10∶1 - 10∶0.1 - 2, preferably 1 - 5∶1 - 5∶1 - 5∶0.1 - 1, such as 1∶1∶1∶0.1, 1∶1∶1∶0.2, 1∶1∶1∶0.3, 1∶2∶1∶0.1, 1∶1∶2∶0.1. Experiments have shown that stable hydrogel drugs can be prepared only within the above specific range. When exceeding this range, the hydrogel will disintegrate into a solution state. For example: when the ratio of glycyrrhizic acid to magnesium ions exceeds 1∶0.5, flocculent precipitates will form and a stable hydrogel drug system cannot be formed.

[0028] The present invention provides the application of the hydrogel in the preparation of antipyretic and anti-inflammatory drugs.

[0029] Preferably, the fever model used is a fever model of SD rats induced by intraperitoneal injection of different doses of lipopolysaccharide (LPS) to evaluate the antipyretic and anti-inflammatory effects of the hydrogel.

[0030] The supramolecular hydrogel of the present invention can be prepared into a pharmaceutically acceptable preparation form and administered in an acceptable form. Further, the supramolecular hydrogel can be prepared into oral drugs, gastrointestinal release drugs, sustained and controlled release drugs, topical transdermal drugs, and can be prepared into preparation forms such as oral gels, tablets, capsules, soft capsules, topical gels, injectable hydrogels, etc.

[0031] Advantages of the present invention:

[0032] The hydrogel of the present invention is self-assembled from the representative active ingredients of the four traditional Chinese medicines in Maxing Shigan Decoction, namely glycyrrhizic acid, pseudoephedrine, amygdalin, and soluble inorganic calcium salts that can dissociate into magnesium / calcium / zinc / copper ions. It does not contain drug excipients such as carriers, is not chemically modified, does not use organic solvents, has a simple, safe and green preparation method, and has no toxic and side effects and a significant and lasting antipyretic effect.

[0033] The supramolecular hydrogel of the present invention can also be used as a drug delivery carrier to carry other drugs, such as antipyretic, analgesic and anti-inflammatory drugs, playing a dual role of both exerting pharmacological activity and carrying active ingredients. Brief Description of the Drawings

[0034] Figure 1 It is a macroscopic view of the quaternary magnesium carrier-free supramolecular hydrogel prepared in Example 3 of the present invention.

[0035] Figure 2 It is a scanning electron micrograph of the quaternary magnesium carrier-free supramolecular hydrogel prepared in Example 3 of the present invention.

[0036] Figure 3 It is a rheological property characterization of the binary magnesium carrier-free supramolecular hydrogel prepared in Example 2 and described in Example 6 of the present invention.

[0037] Figure 4 It is a rheological property characterization of the quaternary magnesium carrier-free supramolecular hydrogel prepared in Example 3 and described in Example 6 of the present invention.

[0038] Figure 5 It is the pathological tissue staining of the heart, liver, spleen, lung tissue, and kidney after intragastric administration of the quaternary magnesium hydrogel prepared in Example 3 of the present invention to SD rats at the maximum administration dose (2 ml / 100 g / day) for 3 consecutive days. Detailed Description of the Invention

[0039] The following examples are intended to further illustrate the present invention. Those skilled in the art should understand that the examples are only to assist in understanding the present invention and should not be regarded as specific limitations on the present invention.

[0040] Example 1 Component Analysis of Maxing Shigan Decoction

[0041] The metal elements and small molecule compounds in Maxing Shigan Decoction were detected by the following methods.

[0042] Weigh the medicinal materials of Ephedra, Bitter Apricot Kernel, Honey-Fried Licorice Root, and Gypsum Fibrosum in a weight ratio of 9:9:6:18, add 10 times the amount of deionized water to decoct and filter, then obtain the water decoction of Maxing Shigan Decoction, and freeze-dry the water decoction to obtain the freeze-dried powder. Take 50.00 mg of the freeze-dried powder of Maxing Shigan Decoction, dissolve it in 5 mL of ultrapure water, add 10 mL of concentrated nitric acid, heat it on an electric heating mantle until there is no white smoke and the liquid is clear and transparent, and make up the volume to 100 mL in a volumetric flask with 2% dilute nitric acid, and detect it with an inductively coupled plasma optical emission spectrometer (ICP-OES).

[0043] Take the freeze-dried powder of Maxing Shigan Decoction and dissolve it in ultrapure water to make the concentration 1 mg / mL, and perform ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry (UPLC-QE / MS) tests under the following conditions. Chromatographic conditions: Chromatographic column: TC-C18 (4.6 mm × 250 mm, 5 μm, Agilent); Mobile phase: 0.1% formic acid water (A) and acetonitrile (B); Injection volume: 5 μL; Gradient elution: 0 - 30 min, 4 - 98% B; Flow rate: 0.3 mL / min. Mass spectrometry conditions: The ion source uses an ESI source; Collect information in positive and negative ion modes; Nebulization pressure: 45 psi; The drying gas is nitrogen, flow rate: 1.0 mL / min, temperature: 350 °C; Capillary voltage: 3500 V.

[0044] Analysis of the test results of ICP-OES and UPLC-QE / MS shows that Maxing Shigan Decoction mainly contains metal ions such as calcium, magnesium, zinc, copper, etc. and small molecule organic compounds such as pseudoephedrine, amygdalin, glycyrrhizic acid, etc.

[0045] Example 2 Preparation of Binary Hydrogel

[0046] Prepare binary magnesium hydrogel by the method of the following steps.

[0047] Weigh glycyrrhizic acid and inorganic magnesium salt in a molar ratio of 1 - 10:0.1 - 2 and dissolve them in water respectively, heat and mix them, and let them stand and cool to obtain the hydrogel. If the ratio of glycyrrhizic acid to magnesium ions exceeds 1:0.7, flocculent precipitation will form and a stable hydrogel drug system cannot be formed.

[0048] According to the aforementioned method, prepare binary calcium / zinc / copper hydrogels in sequence.

[0049] Example 3 Preparation of Quaternary Magnesium Hydrogel

[0050] The quaternary magnesium hydrogel was prepared by the following method.

[0051] Weigh glycyrrhizic acid, pseudoephedrine or its salt, amygdalin, and inorganic magnesium salt in a molar ratio of 1:1:1:0.2, dissolve them in water respectively, heat and mix them, and let them stand and cool to obtain the quaternary magnesium hydrogel. The morphological appearance is as Figure 1 shown.

[0052] Example 4 Structural Characterization of Quaternary Hydrogel

[0053] (1) Observe the structure of the hydrogel prepared in Example 1 by scanning electron microscopy, as Figure 2 shown.

[0054] (2) Perform infrared analysis on the quaternary magnesium hydrogel prepared in Example 3 and its monomer components. The infrared analysis conditions are as follows: the scanning range is 4000 cm -1 -400 cm -1 , the resolution is 4 cm -1 , the scanning speed is 7.5 KHz. The peak positions of the infrared peaks obtained by infrared analysis are as follows:

[0055] Infrared attribution of quaternary magnesium hydrogel: 3292 cm -1 (-OH stretching vibration), 2926 cm -1 (-CH symmetric and asymmetric stretching vibrations), 1723 cm -1 (-C=O stretching vibration), 1653 cm -1 (C=C stretching vibration), 1591 cm -1 、1422 cm -1 (-COO symmetric and asymmetric stretching vibrations), 1030 cm -1 (-C-O vibration peak).

[0056] Infrared attribution of glycyrrhizic acid: 3251 cm -1 (-OH stretching vibration), 2924 cm -1 (-CH symmetric and asymmetric stretching vibrations), 1723 cm -1 (-C=O stretching vibration), 1653 cm -1 (C=C stretching vibration), 1590 cm -1 、1424 cm -1 (-COO- symmetric and asymmetric stretching vibrations), 1039 cm -1 (-C-O vibration peak).

[0057] Infrared attribution of pseudoephedrine: 3265 cm -1(-OH stretching vibration), 2724 cm -1 (-NH stretching vibration), 1587 cm -1 , 1455 cm -1 (C=C stretching vibration on the benzene ring), 1371 cm -1 (-OH bending vibration).

[0058] Infrared attribution of amygdalin: 3241 cm -1 (-OH stretching vibration), 2880 cm -1 (-CH symmetric and asymmetric stretching vibrations), 1618 cm -1 , 1450 cm -1 (C=C stretching vibration on the benzene ring), 1016 cm -1 (-C-O stretching vibration).

[0059] From the above analysis of the infrared spectra of the quaternary magnesium hydrogel and its monomer components, it can be seen that the -NH of pseudoephedrine disappears, and the stretching vibration of -OH shifts from 3265 cm -1 to 3292 cm -1 , and the -OH peak moves 27 cm to a higher wavenumber -1 ; for amygdalin, the stretching vibration of -OH shifts from 3241 cm -1 to 3292 cm -1 , and the stretching vibration of -C-O shifts from 1016 cm -1 to 1030 cm -1 ; for glycyrrhizic acid, the stretching vibration of -OH shifts from 3251 cm -1 to 3292 cm -1 , the peak intensity at -COO- increases significantly, and the -C-O absorption peak on the sugar shifts from 1039 cm -1 to 1030 cm -1 . The above analysis results suggest that the carboxyl group and sugar part of glycyrrhizic acid, the amino group and hydroxyl group of pseudoephedrine, and the sugar part of amygdalin are the action sites during the formation of the quaternary magnesium hydrogel.

[0060] (3) Characterize the nuclear magnetic resonance of the quaternary magnesium hydrogel prepared in Example 3 and its monomer components, and the results are as follows:

[0061] Nuclear magnetic resonance attribution of the quaternary magnesium hydrogel: 11H NMR (400 MHz, DMSO-d6) δ 7.58 (m, 2H, benzene ring, amygdalin), 7.47 (m, 3H, benzene ring, amygdalin), 7.38 (m, 5H, benzene ring, pseudoephedrine), 6.00 (s, 1H, amygdalin), 5.40 (s, 1H, glycyrrhizic acid), 4.51 (d, 1H, pseudoephedrine), 4.48 (d, 1H, glycyrrhizic acid), 4.42 (d, 1H, amygdalin), 4.34 (d, 1H, glycyrrhizic acid), 4.24 (d, 1H, amygdalin), 4.04 (dd, 1H, amygdalin), 3.67 (dd, 1H, amygdalin), 3.62 (m, 1H, amygdalin), 3 - 3.46 (m, sugar moiety of amygdalin and glycyrrhizic acid), 2.55 (s, 3H, pseudoephedrine).

[0062] Nuclear magnetic resonance assignment of glycyrrhizic acid: 1 1H NMR (400 MHz, DMSO-d6) δ 5.39 (s, 1H, 12-H), 4.47 (d, J = 8.0 Hz, 1H, 1”-H), 4.34 (d, J = 8.0 Hz, 1H, 1’-H), 3.50 - 3.00 (m, 14H, 3-H, Glu-H), 2.55 (d, J = 12.0 Hz, 1H, 1-Hb), 2.32 (s, 1H, 9-H), 2.06 - 0.71 (m, 20H, other H of glycyrrhizic acid nucleus).

[0063] Nuclear magnetic resonance assignment of pseudoephedrine: 1 1H NMR (400 MHz, DMSO-d6) δ 9.00 (brs, 2H, -NH2-), 7.38 (m, 5H, -Ar), 6.36 (d, J = 4.0 Hz, 1H, -OH), 4.58 (dd, J1 = 12 Hz, J2 = 4 Hz, 1H, 1-H), 3.30 (m, 1H, 2-H), 2.55 (s, 3H, 4-CH3), 0.96 (d, J = 4.0 Hz, 3H, 3-CH3).

[0064] Nuclear magnetic resonance assignment of amygdalin: 11H NMR (400 MHz, DMSO-d6) δ 7.47 - 7.58 (m, 5H, -Ar), 6.00 (s, 1H, 7-H), 5.30 (d, J = 4.0 Hz, 1H, 4'-OH), 5.09 (m, 2H, 2'-OH, 3'-OH), 5.00 (d, J = 4.0 Hz, 1H, 4''-OH), 4.95 (d, J = 8.0 Hz, 1H, 3-OH), 4.90 (d, J = 4.0 Hz, 1H, 2''-OH), 4.48 (t, 1H, 6''-OH), 4.41 (d, J = 8.0 Hz, 1H, 1''-H), 4.24 (d, J = 8.0 Hz, 1H, 1'-H), 4.02 (d, J = 12.0 Hz, 1H, 6'a-H), 3.70 (dd, J1 = 10.0 Hz, J2 = 4.0 Hz, 1H, 6''a-H), 3.62 (q, 1H, 6'b-H), 3.46 (m, 1H, 6''b-H), 3.37 (m, 1H, 5'-H), 3.23 (m, 1H, 5''-H), 2.99 - 3.13 (m, 6H, 2', 3', 4', 2'', 3'', 4''-H).

[0065] From the results of the assignment of the 1H NMR signals of the quaternary magnesium hydrogel and its monomer components, it can be seen that the peaks of -OH in pseudoephedrine disappear, and the amino peak at 9.00 ppm also disappears; the H-1 peak of pseudoephedrine shifts to a higher field, and the peak shape changes from a dd peak to a d peak; consistent with the infrared results, it indicates that -OH and -NH- in pseudoephedrine are binding sites. The 6''-OH of amygdalin disappears; the -OH peaks on the sugar change from sharp peaks to broad peaks, and there are no 1H NMR signals for glycyrrhizic acid and pseudoephedrine here, so the binding sites of amygdalin are mainly in the sugar part. The peak shape of the sugar part of glycyrrhizic acid has changed, which may be its binding site, and this analysis result is consistent with the infrared results.

[0066] Example 5 Preparation of Quaternary Calcium / Zinc / Copper Hydrogel

[0067] According to the method of Example 3, quaternary calcium / zinc / copper hydrogels were prepared respectively.

[0068] Weigh glycyrrhizic acid, pseudoephedrine or its salt, amygdalin, and inorganic calcium / zinc / copper salts in a molar ratio of 1 - 10∶1 - 10∶1 - 10∶0.1 - 2, dissolve them in water respectively, heat and mix, and let it stand and cool to obtain 3 kinds of quaternary hydrogels.

[0069] Example 6 Rheological Property Characterization of Magnesium-Containing Hydrogel

[0070] The rheological properties of the hydrogels prepared in Examples 2 and 3 were characterized as follows:

[0071] Prepare a binary / quaternary magnesium hydrogel with a final concentration of 10 mmol / L according to Examples 2 and 3, place it on the rheometer measurement plate, set the gap to 5 mm, and the temperature to 25 °C. Frequency sweep: The strain is kept constant at 0.1%, and the measurement frequency range is 0.1 Hz - 10 Hz. Amplitude sweep: Set the frequency constant value to 1 Hz, and the strain change range is from 0.001% to 10%, and obtain the storage modulus (G′) and loss modulus (G″) that change with strain and frequency.

[0072] Further analyze the rheological properties of the prepared hydrogel. G′ reflects the solid-like properties of the gel, and G″ reflects the liquid-like properties of the gel. The G′ of the binary / quaternary magnesium hydrogel is much greater than G″, and both G′ and G″ are independent of the frequency (0.1 - 10 Hz), proving that the sample exists as a stable hydrogel and there is an elastic network inside. These characteristics are common features of hydrogel materials. The oscillatory stress sweep process of the binary / quaternary magnesium hydrogel shows that when the shear stress increases to break the network structure of the hydrogel, the stress value at this time is the yield stress value of the hydrogel. After that, the G′ of the hydrogel will rapidly decrease. G′ and the yield stress value reflect the mechanical strength of the hydrogel. Comparing with the rheological properties of the binary magnesium hydrogel prepared in Example 2, the G′ and G″ of the quaternary magnesium hydrogel are the highest, showing better mechanical strength, indicating that the synergistic effect of magnesium ions, glycyrrhizic acid, pseudoephedrine, and amygdalin can obtain a hydrogel with better mechanical strength.

[0073] Example 7 Verification of the biological safety of the quaternary magnesium hydrogel

[0074] Determine the biological safety of the quaternary magnesium hydrogel prepared in Example 3. The method is as follows:

[0075] Cytotoxicity experiment: Cell seeding: Take logarithmically growing human normal liver cells L02 and inoculate them into a 96-well plate, with a cell density of 3000 cells per well, and place them in a cell culture incubator for 24 h.

[0076] Cell drug administration: Set up a blank cell group, a blank group, and a drug administration group. The blank cell group only adds the culture medium, and the drug administration group adds the drug-containing culture medium. The concentrations per well are 125, 62.5, 31.25, 15.625, 7.8125 μM in sequence, and continue to place them in the cell culture incubator for 24 h and 72 h.

[0077] MTT experiment: Except for the blank group, add 20 μL of MTT solution to each of the other groups, continue to culture in the incubator for 4 h, change the culture medium in the wells to 150 μL of dimethyl sulfoxide, place it on a shaker and shake well in the dark, then measure the absorbance (OD value) at a wavelength of 490 nm, and calculate the inhibition rate according to formula (1):

[0078] Inhibition rate % = [1 - (OD of drug-administered group - OD of blank group) / (OD of blank cell group - OD of blank group)] × 100%

[0079] HE staining experiment: After intragastric administration of the quaternary magnesium hydrogel drug to SD rats, blood was collected from the abdominal aorta of the rats, and the heart, liver, spleen, lung tissue, and kidney organs of the rats were dissected and fixed and preserved with formalin fixative for HE staining. The rat breed used was SD rats provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., and the animal certificate number was (SCXK (Beijing) 2020-0006).

[0080] The specific results are shown in the following table.

[0081] Table 1: Survival rate of L02 cells treated with quaternary magnesium hydrogel

[0082]

[0083] Animal safety experiment research shows that 8 SD rats were intragastrically administered with the quaternary magnesium hydrogel prepared in Example 3 of the present invention at the maximum administration dose (2 ml / 100 g / day). After continuous administration for 3 days, the heart, liver, spleen, lung tissue, and kidney were taken and subjected to pathological tissue staining and observed under a microscope (HE staining, 10*10). All experimental animals showed normal tissue manifestations and no pathological changes were found, indicating that the hydrogel system has a high safety coefficient in vivo.

[0084] Verification of antipyretic activity of the quaternary magnesium hydrogel in Example 8

[0085] The antipyretic and anti-inflammatory activities of the hydrogel prepared in Example 3 were measured as follows:

[0086] A rat fever model induced by intraperitoneal injection of lipopolysaccharide was used to observe the antipyretic effect after intragastric administration of the hydrogel drug. The rat breed used was SD rats provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., and the animal certificate number was (SCXK (Beijing) 2020-0006).

[0087] Adaptation feeding and adaptation operation of rats: Experimental animals were adaptively fed for 7 days in an environment with a temperature of (23 ± 2) °C, a humidity of (60 ± 5)%, and a 12-hour day-night cycle. During this period, they had free access to water and food and were fed with standard feed throughout the process. Three days before the experiment, the rats were subjected to an adaptation operation of measuring rectal temperature at 8:00 am every day (including catching, fixing, and placing the thermometer). The animals emptied their feces before measuring the rectal temperature.

[0088] Model establishment and drug administration: Rats in the model group and the drug administration group were intraperitoneally injected with LPS (50 and 200 μg·kg -1 ), and the blank group was intraperitoneally injected with 1 mL·kg of 0.9% sodium chloride injection -1, the body temperature of each group of animals was measured starting from 0.5 h after modeling and continuously monitored for 24 h. Administration was given by gavage half an hour before modeling, and normal saline was gavaged to the blank and model groups. Among them, the dosages of the quaternary calcium, quaternary zinc, and quaternary copper hydrogels prepared in Example 5 were the same as those of the quaternary magnesium hydrogel prepared in Example 3 (the dosage was calculated as ephedrine: 18 mg / kg). Since the literature reported that the main active ingredients in Maxing Shigan Decoction that play an antipyretic role are the alkaloid components in ephedra (Reference: Liu Yan, Su Xiangying, Liang Ying. Exploration on the effects of different decocting methods on the components in Maxing Shigan Decoction [J]. Chinese General Practice Medicine, 2021, 24(S2): 182-184.), the whole formula was administered by gavage at the high clinical dose. The dosage was 0.63 g / mL of crude drug, equivalent to 63 mg / mL of freeze-dried powder, and the gavage volume was 10 mL / kg.

[0089] The specific results are shown in the following table.

[0090] Table 2: Antipyretic effect of quaternary magnesium hydrogel in different-dose LPS fever models (I represents the modeling dose LPS = 50 μg·kg -1 , II represents the modeling dose LPS = 200 μg·kg -1 )

[0091]

[0092]

[0093] Continued in the above table

[0094]

[0095] Continued in the above table

[0096]

[0097]

[0098] Verification of the antipyretic and anti-inflammatory activities of the quaternary magnesium hydrogel in Example 9

[0099] In this example, the antipyretic and anti-inflammatory activities of the hydrogel prepared in Example 3 were measured as follows:

[0100] In view of the fact that in Example 8, with two modeling doses of high and low, the quaternary magnesium hydrogel group showed good body temperature regulation and antipyretic effects. In this example, the modeling and administration methods of Example 8 were continued, and the modeling dose was adjusted to LPS (100 μg·kg -1 ), and the antipyretic effect was continuously observed, and the effects of the quaternary magnesium, quaternary calcium, quaternary zinc, quaternary copper hydrogels, the ephedrine monomer group (administered by gavage in an equimolar amount of ephedrine as in the quaternary hydrogel group), and the whole formula of Maxing Shigan Decoction on the fever and inflammatory mediators of the model animals were measured in parallel.

[0101] Experiment for determination of the content of pyrogenic inflammatory mediators: 5 h after intragastric administration of drugs to rats, the rats were anesthetized, blood was collected from the abdominal aorta, the serum part of the rats was obtained by centrifugation, the hypothalamus was removed on ice, and after being divided into portions, it was immediately stored in liquid nitrogen. The contents of IL-1β, IL-6 and TNF-α in the serum of rats and PGE2, COX-2, mPGES-1, NF-κBP65, IKB-α, P38MAPK, JNK, ERK and TLR4 in the hypothalamus of rats were detected by the Elisa competitive method.

[0102] The specific results are shown in the following table.

[0103] Table 3: Comparison of antipyretic effects of quaternary magnesium / calcium / zinc / copper hydrogel, the whole formula of Maxing Shigan Decoction and pseudoephedrine monomer (LPS = 100 μg·kg -1 )

[0104]

[0105]

[0106] As shown in the following table

[0107]

[0108]

[0109] As shown in the following table

[0110]

[0111] Table 4: Results of determination of the contents of IL-1β, IL-6 and TNF-α in the serum of rats (n = 6 - 7)

[0112] Group IL-1β (pg / mL) IL-6 (pg / mL) TNF-α (pg / mL) Normal group 4.08±0.89 29.80±9.47 98.79±14.43 Model group 6.59±0.56 47.03±7.42 119.46±14.55 Maxing Shigan Decoction 4.18±0.57 34.36±3.27 101.95±9.00 Quaternary magnesium hydrogel 4.52±0.32 39.65±1.69 96.21±10.55 Quaternary calcium hydrogel 4.64±0.64 40.13±8.54 103.35±14.53 Quaternary zinc hydrogel 4.66±0.29 41.32±4.40 100.47±12.97 Quaternary copper hydrogel 4.59±0.55 40.89±7.23 106.68±13.51 Pseudoephedrine 3.82±0.64 38.92±3.74 111.83±8.67

[0113] Table 5: Results of determination of the contents of PGE2, COX-2, mPGES-1, NF-κB P65, IKB-α, P38MAPK, JNK, ERK and TLR4 in the hypothalamus of rats (n = 7)

[0114]

[0115]

[0116] As shown in the following table

[0117]

[0118] The results showed that the quaternary magnesium carrier-free supramolecular hydrogel exhibited significant antipyretic effects in the fever models induced by different doses of lipopolysaccharide. For the lipopolysaccharide dose of 100 μg·kg-1 In-depth pharmacodynamic evaluation of the induced rat fever model found that: the quaternary magnesium hydrogel had the best antipyretic effect, with significant differences within 4 - 8 hours compared with the model group; its antipyretic effect was significantly better than that of the quaternary calcium, quaternary zinc, quaternary copper hydrogel groups and the pseudoephedrine group, and it had a more persistent antipyretic effect than the whole formula of Maxing Shigan Decoction. At the same time, it had an obvious inhibitory effect on the fever inflammatory mediators in the serum and hypothalamus; of particular concern was that: the quaternary magnesium hydrogel showed an exact inhibitory effect on 9 fever inflammation-related factors in the hypothalamus. As the body's temperature regulation center, the hypothalamus indicated that the persistent and stable antipyretic and anti-inflammatory effects of the quaternary magnesium hydrogel were closely related to the regulation of the body's hypothalamic temperature center. The above results could all prove that the quaternary magnesium hydrogel had a significant and persistent antipyretic and anti-inflammatory effect, with a high safety factor and the value of further development into a clinical drug.

[0119] Intestinal retention performance of the quaternary magnesium hydrogel in Example 10

[0120] To further reveal the mechanism of the long-acting, persistent and stable antipyretic and anti-inflammatory effects of the quaternary magnesium hydrogel, the present invention deeply explored the behavior of the gel after oral administration into the small intestine. The quaternary magnesium hydrogel was loaded with Cy7 dye. After a certain interval of gavage to rats, a small animal in vivo imager was used to observe the retention effect of the hydrogel in the small intestine by comparing with the blank fluorescent dye group. The results showed that at 5h, 12h and 24h after gavage, the fluorescence intensity in the small intestine of the quaternary magnesium hydrogel group was stronger than that of the blank fluorescent dye group, with significant differences. It indicated that the quaternary magnesium hydrogel could significantly prolong the retention time of the drug in the small intestine, which was beneficial to maintaining the blood drug concentration in a stable state for a long time.

[0121] The specific fluorescence values are shown in the following table.

[0122] Table 6: Fluorescence intensity in the small intestine of the blank dye and the dye-loaded quaternary magnesium hydrogel at different time points (n = 5)

[0123]

[0124] Note: Compared with the blank dye group, *P≤0.05, **P≤0.01, ***P≤0.001

[0125] Preparation of other ratios of the quaternary magnesium hydrogel in Example 11

[0126] (1) Weigh glycyrrhizic acid, pseudoephedrine or its salt, amygdalin, and magnesium chloride in a molar ratio of 1∶2∶1∶0.1, dissolve them in water respectively, heat and mix, and let it stand and cool to obtain a colorless and transparent hydrogel.

[0127] (2) Weigh glycyrrhizic acid, pseudoephedrine or its salt, amygdalin, and magnesium chloride in a molar ratio of 1:1:1:0.3, dissolve them separately in water, heat and mix them, and then let it stand and cool to obtain a colorless and transparent hydrogel.

[0128] (3) Weigh glycyrrhizic acid, pseudoephedrine or its salt, amygdalin, and magnesium chloride in a molar ratio of 1:1:2:0.4, dissolve them separately in water, heat and mix them, and then let it stand and cool to obtain a colorless and transparent hydrogel.

[0129] (4) Through comparative studies, it was found that the ratio of glycyrrhizic acid to magnesium in the four raw materials has a greater impact on the formation of the hydrogel. When the ratio of the two exceeds 1:0.7, a uniform and stable quaternary hydrogel cannot be formed.

Claims

1. A method for self-assembling preparation of a supramolecular hydrogel, characterized in that, The supramolecular hydrogel is made of glycyrrhizic acid, pseudoephedrine or its salt, amygdalin, and inorganic magnesium salt with a molar ratio of 1-5:1-5:1-5: 0.1-1, wherein the molar ratio of glycyrrhizic acid to magnesium ions does not exceed 1:0.

7. The self-assembly preparation method includes: Step 1: Heat to 60-100 °C to dissolve glycyrrhizic acid in water; Step 2: Heat to 25-100 °C to dissolve pseudoephedrine or its salt in water; Step 3: Heat to 25-100 °C to dissolve amygdalin in water; Step 4: Heat to 25-100 °C to dissolve inorganic magnesium salt in water; Step 5: Mix the aqueous solutions prepared in Steps 1, 2, 3, and 4. With glycyrrhizic acid as the basis, the concentration is not less than 5 mmol / L. Heat to 60-100 °C and let it stand and cool to obtain the supramolecular hydrogel; The pseudoephedrine or its salt is pseudoephedrine hydrochloride; The inorganic magnesium salt is magnesium sulfate, magnesium chloride, or magnesium dihydrogen phosphate.

2. Use of the supramolecular hydrogel obtained by the self-assembly preparation method of the supramolecular hydrogel according to claim 1 in the preparation of antipyretic drugs.

3. Use of the supramolecular hydrogel obtained by the self-assembly preparation method of the supramolecular hydrogel according to claim 1 in the preparation of anti-inflammatory drugs.

4. The application according to any one of claims 2-3, characterized in that The supramolecular hydrogel is prepared into a sustained-release or controlled-release drug.

5. The application according to any one of claims 2-3, characterized in that The supramolecular hydrogel is prepared into an oral drug or a transdermal drug.

6. The application according to any one of claims 2-3, characterized in that The supramolecular hydrogel is prepared into an oral gel, tablet, capsule, external gel, or injectable hydrogel.

Images