A kind of Euphorbia-Glycyrrhiza supramolecular hydrogel and its preparation method and use

Abstract

The present invention belongs to the field of medicine, and specifically relates to a Euphorbia licorice supramolecular hydrogel and a preparation method and use thereof. The supramolecular hydrogel comprises Euphorbia ethyl acetate extract and glycyrrhizic acid, and the Euphorbia ethyl acetate extract and the glycyrrhizic acid interact with each other to form nanoparticles by self-assembly. The preparation method of the Euphorbia licorice supramolecular hydrogel comprises the following steps: weighing the Euphorbia ethyl acetate extract and glycyrrhizic acid and placing them in a round-bottom flask, adding an appropriate amount of deionized water, and decocting for 0.5 to 2 hours. Experimental results show that the Euphorbia licorice supramolecular hydrogel of the present invention has a good anti-hepatoma ascites effect and has excellent safety compared with conventional chemotherapy drugs.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a euphorbia-licorice supramolecular hydrogel and a preparation method and application thereof. Background Art

[0002] Euphorbia is a poisonous Chinese medicine of the Euphorbia genus of the Euphorbia family included in the 2020 edition of the Chinese Pharmacopoeia. It has the effects of purging water, reducing swelling and dispersing nodules. It can be used for edema, hydrothorax and ascites, phlegm and fluid accumulation, etc. It can be used clinically for the treatment of cancerous ascites and ascites due to cirrhosis. However, studies have found that most oral anti-tumor Chinese medicines have gastrointestinal irritation side effects, which limits their clinical application. Therefore, in recent years, some researchers have found that intraperitoneal chemotherapy has a better effect on malignant ascites of liver cancer by changing the route of administration, and helps to improve the survival and quality of life of patients.

[0003] Intraperitoneal chemotherapy represents a specific local chemotherapy strategy, which has the advantage of expanding the contact range between the drug and the peritoneum, thereby increasing the drug concentration in the local area, and at the same time, compared with systemic treatment, its systemic toxic side effects are relatively low. Studies have found that intraperitoneal chemotherapy shows significant advantages in pharmacokinetics, among which the efficiency of intraperitoneal administration far exceeds that of intravenous administration, reaching 10 to 1000 times that of the latter. This mode of administration allows the drug to enter the cancer cell mass directly through surface diffusion. With the help of the barrier effect between the peritoneum and the plasma, the drug can maintain a high concentration in the peritoneal cavity and exert its effect for a long time. The drug is slowly absorbed from the peritoneal surface into the systemic circulation, first entering the portal vein through the lymphatic system and small blood vessels, and then reaching the liver. In clinical practice, how to effectively maintain the high concentration and long-term effect of the drug in the peritoneal cavity, while reducing or avoiding possible local and systemic side effects, has become an urgent problem to be solved.

[0004] Recent studies have shown that by combining specific drug carriers to form a drug-carrier system, the absorption rate of chemotherapeutic drugs can be reduced and their duration of action in tumor tissue can be prolonged, further improving the therapeutic effect. In particular, hydrogels have become an important material in the field of intratumoral injection due to their ability to carry drugs, inject, implant, biodegrade, and respond to stimuli, showing excellent application potential. Using injection methods, a variety of hydrogel preparations can be directly placed into the affected area, thus avoiding the need for the drug to be absorbed by the blood circulation first. In addition, by adjusting the structure of the hydrogel, the drug release rate can be controlled, thereby realizing a target-oriented and sustained release treatment strategy.

[0005] The chemical composition of traditional Chinese medicine is remarkable for its unique structural properties and diverse modification sites. These molecules tend to self-recognize and self-assemble through interactions. Studies have revealed that in traditional Chinese medicine compound, specific ingredients can form so-called "potential preparations" or co-assemblies with specific structures through self-assembly or co-assembly with other ingredients. This process can improve key biopharmaceutical properties of drugs, such as solubility and stability, thereby improving the bioavailability of drugs and enhancing their efficacy.

[0006] Based on the above analysis, the inventors developed a novel Euphorbia-Glycyrrhiza supramolecular hydrogel drug sustained-release system and successfully used it to treat ascites of liver cancer. Summary of the invention

[0007] The purpose of the present invention is to solve the deficiencies of the prior art and to provide a novel Euphorbia-Glycyrrhiza supramolecular hydrogel and a preparation method and use thereof.

[0008] Specifically, the present invention is realized through the following technical solutions:

[0009] In a first aspect, the present invention provides a Euphorbia-Glycyrrhiza supramolecular hydrogel, wherein the supramolecular hydrogel comprises Euphorbia ethyl acetate extract and glycyrrhizic acid, wherein the Euphorbia ethyl acetate extract and the glycyrrhizic acid interact with each other to form nanoparticles through self-assembly.

[0010] As an optional manner, in the supramolecular hydrogel, the weight ratio of the Euphorbia ethyl acetate extract to glycyrrhizic acid is 1:10 to 10:1.

[0011] As an optional manner, in the supramolecular hydrogel, the weight ratio of the Euphorbia ethyl acetate extract to glycyrrhizic acid is 1:5~5:1.

[0012] As an optional embodiment, in the supramolecular hydrogel, the weight ratio of the Euphorbia ethyl acetate extract to glycyrrhizic acid is 1:1.

[0013] As an optional manner, in the supramolecular hydrogel, the average diameter of the nanoparticles is 100-250 nm.

[0014] As an optional mode, in the supramolecular hydrogel, the glycyrrhizic acid promotes the dissolution of the effective ingredient euphorbia dienol in the ethyl acetate extract of Euphorbia rapa.

[0015] As an optional method, in the above-mentioned supramolecular hydrogel, the preparation method of the ethyl acetate extract of Euphorbia rapa comprises the following steps: weighing an appropriate amount of Euphorbia rapa slices and placing them in a round-bottom flask, adding 8 to 10 times the amount of 75% to 95% ethanol, refluxing and decocting for 0.5 to 2 h to obtain decoction I; then adding 8 to 10 times the amount of 75% to 95% ethanol, refluxing and decocting for 0.5 to 2 h to obtain decoction II; filtering while hot and combining the two decoctions, concentrating under reduced pressure, placing in a separatory funnel, adding ethyl acetate for extraction, and vacuum freeze-drying to obtain freeze-dried powder of ethyl acetate extract of Euphorbia rapa for later use.

[0016] Preferably, the preparation method of the ethyl acetate extract of Euphorbia rapa comprises the following steps: weighing 50 g of Euphorbia rapa slices and putting them into a round-bottom flask, adding 10 times the amount of 95% ethanol, refluxing and decocting for 1 h to obtain decoction I; then adding 8 times the amount of 95% ethanol, refluxing and decocting for 45 min to obtain decoction II; filtering while hot and combining the two decoctions, concentrating under reduced pressure to 20 mL, placing in a separatory funnel, adding ethyl acetate to extract 3 times, and vacuum freeze-drying to obtain freeze-dried powder of ethyl acetate extract of Euphorbia rapa for later use.

[0017] In a second aspect, the present invention provides a method for preparing the supramolecular hydrogel described in the first aspect, characterized in that the preparation method comprises the following steps:

[0018] Weigh the ethyl acetate extract of Euphorbia and glycyrrhizic acid and place them in a round-bottom flask, add appropriate amount of deionized water, and boil for 0.5-2 h.

[0019] As an optional manner, in the above preparation method, the ratio of the total weight of the Euphorbia ethyl acetate extract and the glycyrrhizic acid to the volume of the deionized water is (1~10) mg: (1~10) mL.

[0020] As an optional manner, in the above preparation method, the ratio of the total weight of the Euphorbia ethyl acetate extract and the glycyrrhizic acid to the volume of the deionized water is 50 mg:5 mL.

[0021] As an optional manner, in the above preparation method, the decoction time is 30 min.

[0022] In a third aspect, the present invention provides use of the supramolecular hydrogel described in the first aspect or the supramolecular hydrogel prepared by the preparation method described in the second aspect in preparing a drug for treating malignant ascites caused by cancer.

[0023] As an alternative, in the above use, the malignant ascites caused by cancer is liver cancer ascites.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The inventors have developed a novel Euphorbia-Glycyrrhiza supramolecular hydrogel drug sustained-release system and successfully used it to treat liver cancer ascites. Experimental results show that the Euphorbia-Glycyrrhiza supramolecular hydrogel of the present invention has a good anti-liver cancer ascites effect and has excellent safety compared with conventional chemotherapy drugs. Therefore, the Euphorbia-Glycyrrhiza supramolecular hydrogel of the present invention has high clinical significance and commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Preparation of Euphorbia-Glycyrrhiza supramolecular. A: Euphorbia ethyl acetate decoction; B: Euphorbia-Glycyrrhiza supramolecular hydrogel.

[0027] Figure 2 : Morphological characterization of Euphorbia-Glycyrrhiza supramolecular and Euphorbia decoction. A: SEM image of Euphorbia ethyl acetate decoction; B: SEM image of Euphorbia-Glycyrrhiza supramolecular; C: Particle size distribution of Euphorbia-Glycyrrhiza supramolecular.

[0028] Figure 3 : Infrared absorption spectra of Euphorbia rapa decoction and Euphorbia rapa-Glycyrrhiza supramolecular.

[0029] Figure 4 : Liquid chromatograms of Euphorbia ethyl acetate decoction and Euphorbia-Glycyrrhiza supramolecular. Wherein: AB: Euphorbia ethyl acetate decoction; CD: Euphorbia-Glycyrrhiza supramolecular; E: solubilization rate of different ratios.

[0030] Figure 5 :In vitro experimental results of hepatocellular carcinoma ascites cells H22.

[0031] Figure 6 :Establishment of subcutaneous ascites tumor model of H22 hepatocarcinoma in Balb / c mice and the results of drug efficacy.

[0032] Figure 7 :Tumor volume growth curve and tumor mass results of each group. Compared with the model group, the positive drug group, glycyrrhizic acid group, Euphorbia decoction group, Euphorbia-glycyrrhizic acid group P <0.01, P <0.001.

[0033] Figure 8 :Effects of Euphorbia-Glycyrrhiza supramolecular hydrogel on organ indexes of mice with subcutaneous tumors. ### P <0.001; compared with the positive drug group, the model group, glycyrrhizic acid group, Euphorbia decoction group, Euphorbia-glycyrrhizic acid group P <0.05, P <0.01, P <0.001.

[0034] Fig. 9 :H&E staining sections of the organs of mice in each group. Among them, blank group-Control, model group-Model, positive drug-CTX, Euphorbia ethyl acetate decoction group-DA, glycyrrhizic acid-GA, Euphorbia ethyl acetate part-glycyrrhizic acid-DA-GA.

[0035] Fig.10 : Comparison of ascites changes in mice in the normal control group, model group and each drug-treated group.

[0036] Fig.11 :Effects of Euphorbia-Glycyrrhiza supramolecular hydrogel on body weight and abdominal circumference of mice. Compared with the normal group, the model group # P <0.05, ## P <0.01; compared with the model group, the positive drug group and the Euphorbia-Glycyrrhizic acid group P <0.05.

[0037] Fig.12 :H&E stained sections of liver, spleen and kidney of mice in each group. Among them, blank group-Control, model group-Model, positive drug-CTX, ethyl acetate part of Euphorbia cerevisiae-DA-GA. DETAILED DESCRIPTION

[0038] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0039] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0040] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0041] Unless otherwise specified, all percentages and parts in the present invention are percentages and parts by weight.

[0042] In this article, "Euphorbia-Glycyrrhizic Acid Supramolecular Hydrogel", "Euphorbia Ethyl Acetate Part-Glycyrrhizic Acid", "Euphorbia ... and "Euphorbia-Glycyrrhizic Acid Supramolecular" all have the same or similar meanings, and these terms can be used interchangeably in this article, all referring to nanoparticles formed by self-assembly of Euphorbia Ethyl Acetate Extract and Glycyrrhizic Acid.

[0043] Example 1: Preparation and characterization of Euphorbia-Glycyrrhiza supramolecular hydrogel

[0044] 1. Experimental Methods

[0045] 1.1 Preparation of test samples

[0046] Euphorbia ethyl acetate part: Weigh 50 g of Euphorbia decoction pieces (purchased from Beijing Dongzhou Traditional Chinese Medicine Clinic Co., Ltd.) and put them into a round-bottom flask, add 10 times the amount of 95% ethanol, reflux and boil for 1 h to obtain decoction I; then add 8 times the amount of 95% ethanol, reflux and boil for 45 min to obtain decoction II; filter while hot and combine the two decoctions, concentrate under reduced pressure to 20 mL, place in a separatory funnel, add ethyl acetate to extract 3 times, and vacuum freeze-dry to obtain the ethyl acetate part of Euphorbia lyophilized powder for use.

[0047] Decoction of ethyl acetate part of Euphorbia (single decoction of Euphorbia): Weigh different proportions of ethyl acetate part of Euphorbia, 5 mg, 10 mg, 15 mg, 20 mg, and 25 mg, put them into round-bottom flasks respectively, add 5 mL of deionized water and reflux and decoct for 30 min to obtain the decoction for later use.

[0048] Euphorbia ethyl acetate-glycyrrhizic acid (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) decoction (Euphorbia-Glycyrrhizic acid): fix glycyrrhizic acid 25 mg, weigh different proportions of Euphorbia ethyl acetate (5 mg, 10 mg, 15 mg, 20 mg, 25 mg), put them in round-bottom flasks, add 5 mL of deionized water, and boil for 30 min to obtain the decoction. Set aside.

[0049] Glycyrrhizic acid solution: Weigh 20 mg of glycyrrhizic acid and add 10 mL of deionized water to completely dissolve it to obtain a glycyrrhizic acid solution.

[0050] 1.2 Determination of particle size

[0051] Take samples of Euphorbia monodecocted and Euphorbia-Glycyrrhiza uralensis, turn on the Malvern particle size analyzer and preheat it for 15 min, set the measurement parameters, measure each sample three times in parallel, and determine the particle size of the sample.

[0052] 1.3 Field emission scanning electron microscope (SEM)

[0053] The decoctions of Euphorbia-Glycyrrhiza and Euphorbia monodecocted were heated in a water bath at 60°C for 0.5 h to promote dispersion. A pipette was used to accurately take 2 μL of the suspension and drop it on a silicon wafer cleaned with a wipe paper. A water droplet sample was formed during the drop addition. After the drop addition was completed, the sample was naturally air-dried at room temperature for 4 h. The sample was gold-sprayed and placed under a field emission scanning electron microscope with an operating voltage of 15.0 kV to observe the surface morphology and size of the sample.

[0054] 1.4 Ultraviolet-visible spectrometry (UV-vis)

[0055] Take Euphorbia decoction alone and Euphorbia-Glycyrrhiza decoction in different proportions, dissolve each sample fully in deionized water, put them into the cleaned cuvette, set the detection wavelength of the UV-Vis spectrophotometer to 190~600 nm, use deionized water as the blank control, and perform full wavelength scanning.

[0056] 1.5 Fourier Transform Infrared Spectroscopy (IR)

[0057] Glycyrrhizic acid powder, Euphorbia decoction powder, and Euphorbia-licorice powder were accurately weighed and placed in centrifuge tubes, mixed with spectral grade potassium bromide (KBr) powder in an agate mortar at an appropriate mass ratio and ground into fine powder. The powder was pressed into tablets at a pressure of 80 MPa for 5 min. The scanning range of the Fourier transform infrared spectrometer (Shimadzu IR Prestige-21) was 400-4000 cm -1 Each sample was scanned 200 times on average, with a spectral resolution of 4 cm -1 , air as background.

[0058] 1.6 Establishment of high performance liquid chromatography (HPLC) detection method for the index components of Euphorbia

[0059] The Chinese Pharmacopoeia uses euphorbia dienol as an indicator component for the determination of the content of euphorbia medicinal materials. After heating different proportions of euphorbia-licorice hydrogel, mix them evenly and filter them through a 0.45 μm microporous filter membrane to obtain the product.

[0060] HPLC conditions: Agilent 300SB-C8 column (150 mm×4.6 mm, 5 μm), mobile phase acetonitrile (A) ~ water (B), elution gradient: 0-20 min, 40% A; 20-30 min, 40-80% A; 30-35 min, 80-85% A; 35-50 min, 85-40% A; 50-50 min, 40% A. Flow rate: 0.1 mL• min -1 ; Column temperature: 25℃; Injection volume: 10 μL.

[0061] 2. Experimental Results

[0062] 2.1 Preparation of Euphorbia-Glycyrrhiza supramolecular

[0063] As shown in the figure, the extract of the ethyl acetate part of Euphorbia rapa appears as a light yellow suspension, and the color gradually deepens with the increase of the dosage ( Figure 1 A); The extract of Euphorbia-Glycyrrhiza uralensis was in the form of brown gel, and the co-decoction promoted the formation of glycyrrhizic acid hydrogel. The color of the Euphorbia-Glycyrrhiza uralensis group was darker than that of Euphorbia alone, which was probably due to the increased dissolution of chemical components in Euphorbia. At the same time, as the ratio of the dose of Euphorbia ethyl acetate increased, the solubilization capacity of glycyrrhizic acid was limited ( Figure 1 B).

[0064] The surface morphology of Euphorbia ethyl acetate decoction alone and co-decoction with glycyrrhizic acid was observed by Malvern particle size analyzer and SEM. Figure 2 A- Figure 2 C). From the SEM results, it can be seen that the decoction of Euphorbia dahurica alone presents particles with uneven morphology and irregular shapes ( Figure 2 A); while the morphology of Euphorbia-Glycyrrhiza uralensis is uniformly distributed and regularly shaped spherical nanoparticles, with an average particle diameter of about 200 nm and a particle size of 133 nm ( Figure 2 B- Figure 2 C). The experimental results show that the addition of glycyrrhizic acid can form nanoparticles with uniform morphology and distribution in the ethyl acetate part of Euphorbia rapa, thus exerting its medicinal properties.

[0065] 2.2 Study on the supramolecular assembly mechanism of Euphorbia pulex-Glycyrrhiza uralensis

[0066] The Euphorbia monodecocted group had obvious characteristic absorption peaks at 247 nm and 359 nm, and the Euphorbia-Glycyrrhiza group had obvious characteristic absorption peaks at 256 nm and 360 nm. It can be seen that after adding glycyrrhizic acid, the characteristic absorption peaks of Euphorbia-Glycyrrhiza all moved to higher wavelengths. It was preliminarily judged that an interaction occurred between the ethyl acetate part of Euphorbia and glycyrrhizic acid.

[0067] like Figure 3 As shown, the changes in the peak shape and peak position of the infrared spectrum indicate that after the ethyl acetate part of Euphorbia cerevisiae and glycyrrhizic acid are decocted together, the supramolecular part has characteristic absorption peaks of the ethyl acetate part of Euphorbia cerevisiae and glycyrrhizic acid, the chemical components collide with each other, and weak bonds are formed between the functional groups of the molecules, indicating that the supramolecular part of the ethyl acetate part of Euphorbia cerevisiae and glycyrrhizic acid co-decocted is a new material state, not a simple addition of the two.

[0068] 2.3 Chemical analysis of the Euphorbia-Glycyrrhiza supramolecular complex

[0069] HPLC was used to determine the content of euphordienolide in the single decoction of ethyl acetate part of Euphorbia rapa (Euphorbia rapa single decoction) and Euphorbia rapa-Glycyrrhiza uralensis test samples. Figure 4As shown, in order to determine the optimal supramolecular solubilization rate, 25 mg-30 mg and 25 mg-35 mg were added. Figure 4 A and 4B show that the peak area of ​​euphordienolide in Euphorbia monodecocted increases with the increase of dosage, and the peak area increases from 31.2 to 359.3 after automatic integration; Figure 4 C-4E shows that after the ethyl acetate part of Euphorbia ulmoides was decocted with glycyrrhizic acid, glycyrrhizic acid promoted the dissolution of the effective ingredient euphordienolide, which also confirmed the above 2.1 experimental speculation: the deepening of the color of Euphorbia ulmoides-licorice was due to the increased dissolution of chemical components including euphordienolide in Euphorbia ulmoides. It can also be seen that the optimal ratio was reached at 25 mg-25 mg, and then a downward trend appeared. The above results show that the ethyl acetate part of Euphorbia ulmoides and glycyrrhizic acid co-decocted to form supramolecular molecules are conducive to the increased dissolution of euphordienolide, and the ratio of 25 mg-25 mg is the optimal supramolecular ratio for subsequent in vitro and in vivo experiments.

[0070] Example 2: In vitro anti-ascites effect of Euphorbia-Glycyrrhiza supramolecular hydrogel

[0071] In this example, the in vitro anti-tumor effects of four test products, namely, the optimal supramolecule of Euphorbia-Glycyrrhiza (25 mg-25 mg ratio), Euphorbia + Glycyrrhizic acid (mechanical mixing, 25 mg+25 mg ratio), Euphorbia decoction (ethyl acetate fraction, 25 mg), and Glycyrrhizic acid (25 mg), were verified by in vitro experiments to observe the effect of the supramolecule on the survival rate of liver cancer ascites cells.

[0072] 1. Experimental Methods

[0073] 1.1 Cell plating

[0074] Mouse H22 liver cancer ascites cells in the logarithmic growth phase (purchased from Shanghai Fuheng Biotechnology Co., Ltd.) were collected and the cell suspension was adjusted to a concentration of 5×10 4 The cells were plated in 96-well plates with 100 μL per well, and 3 replicates were plated in a 5% CO2 sterile incubator at 37°C for 12 h to allow the cells to fully adhere to the plate.

[0075] 1.2 Effect of Euphorbia-Glycyrrhiza uralensis optimal supramolecular on the survival rate of H22 hepatocarcinoma ascites cells

[0076] The samples were divided into Euphorbia decoction group (Euphorbia ethyl acetate decoction, 25 mg), Euphorbia-Glycyrrhizic acid group (Euphorbia-Glycyrrhizic acid supramolecular group, 25 mg-25 mg ratio), Euphorbia decoction + Glycyrrhizic acid group (Euphorbia ethyl acetate + Glycyrrhizic acid mechanical mixing group, 25 mg+25 mg ratio), Glycyrrhizic acid group (25 mg), and three replicate experimental wells were set in each group. The concentration of Euphorbia-Glycyrrhizic acid group was used as the standard for each group, and the corresponding multiples were diluted to compare the efficacy differences in parallel. 100 μL of culture medium containing the corresponding active ingredient was added to each well, and the 96-well culture plate was placed in a 37°C, 5% CO2 cell culture incubator for 48 h. 20 μL of CCK-8 solution was added to each well, and then the culture was continued for 2 h in a 37°C constant temperature incubator containing 5% CO2, and the absorbance (A) value of each well was measured at a wavelength of 450 nm. The cell viability was calculated according to the following formula:

[0077] Cell survival rate % = (A 450 Drug group-A 450 Blank control group) / (A 450 Cell control group-A 450 Blank control group) × 100%.

[0078] 1.3 Data processing and analysis

[0079] IBM SPSS 20.0 was used for statistical analysis, and the t-test was used to compare the significance of the difference between the two groups of data. P < 0.05 indicated a significant difference.

[0080] 2. Experimental Results

[0081] 2.1 Effect of Euphorbia-Glycyrrhiza supramolecular on the survival rate of H22 cells

[0082] Depend on Figure 5 It can be seen that the Euphorbia decoction group (Euphorbia ethyl acetate decoction, 25 mg), Euphorbia-Glycyrrhizic acid group (Euphorbia-Glycyrrhizic acid supramolecular group, 25 mg-25 mg ratio), Euphorbia decoction + Glycyrrhizic acid group (Euphorbia ethyl acetate + Glycyrrhizic acid mechanical mixing group, 25 mg + 25 mg ratio) were all lower than the Glycyrrhizic acid group at 48h, showing a certain inhibitory effect on the activity of liver cancer ascites cells, and was positively correlated with the dosage. In addition, the survival rate of liver cancer ascites cells in the Euphorbia-Glycyrrhizic acid group was lower than that of Euphorbia decoction + Glycyrrhizic acid (mechanical mixing group) and Euphorbia decoction group, indicating that the two did not simply play a physical role by mechanical mixing. Instead, through decoction, the addition of glycyrrhizic acid promoted the dissolution of the active ingredients of the Euphorbia ethyl acetate part, formed a supramolecular hydrogel, and then enhanced the inhibitory effect on H22 cells. At the same time, it can also be seen that glycyrrhizic acid has a high cell survival rate at different concentrations, indicating that it is highly safe and will not affect the growth of H22 cells.

[0083] Example 3: In vivo anti-ascites effect of Euphorbia-Glycyrrhiza supramolecular hydrogel

[0084] 1. Experimental Materials

[0085] 1.1 Experimental animals

[0086] SPF healthy Balb / c mice, male, weighing 19-21 g, were purchased from Beijing Weitonglihua Experimental Animal Co., Ltd. (Beijing, China, SCXK (Beijing) 2021-0006). They were kept in the Institute of Basic Theory of Traditional Chinese Medicine, China Academy of Chinese Medical Sciences (Experimental Animal Laboratory License No. SYXK (Beijing) 2021-0017). The breeding environment: humidity 40-70%, room temperature 23±2℃, natural light day and night. Check water and feed daily to keep the environment dry. Adaptive breeding for 7 days.

[0087] 1.2 Main reagents and materials

[0088] Cyclophosphamide for injection was purchased from Beijing Jikangduo Pharmacy, batch number: 22120325; formaldehyde was purchased from Tianjin Damao Chemical Reagent Factory.

[0089] 2. Experimental Methods

[0090] 2.1 Preparation of main solutions

[0091] 2.1.1 Cyclophosphamide Solution Dissolve 46 mg of cyclophosphamide powder for injection in 10 mL of deionized water to prepare a cyclophosphamide solution with a concentration of 4.6 mg / mL. Wrap in tin foil to protect from light and store at 4°C.

[0092] 2.2 Preparation of H22 Hepatocellular Carcinoma Ascites Cells

[0093] Logarithmic phase H22 cells were collected under sterile conditions and placed in sterile centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and an appropriate amount of RPMI 1640 complete medium was added and gently pipetted to mix well. The cell suspension concentration was adjusted to 5 × 10 4 pcs / hole, store in ice box for later use.

[0094] 2.3 Establishment of the mouse model of liver cancer ascites

[0095] 2.3.1 Establishment of subcutaneous tumor model and grouping and drug administration

[0096] The dorsal side of the right hind lower limb of the mouse was depilated and disinfected with alcohol, and the H22 cell suspension was shaken and subcutaneously inoculated with 0.2 mL of cell suspension per mouse. The injection site was pressed for a moment to prevent leakage of cell fluid; to ensure the activity of the cells, the operation was controlled within 30 min. After 1 week, the mice that failed to establish the model were eliminated and randomly divided into the model group, the positive drug group, the glycyrrhizic acid group (25 mg), the Euphorbia decoction group (Euphorbia ethyl acetate decoction, 25 mg), and the Euphorbia-Glycyrrhizic acid supramolecular group (25 mg-25 mg ratio), with 5 mice in each group. The normal group and the model group were intraperitoneally injected with 0.1 mL / mouse normal saline; the positive drug group was intraperitoneally injected with cyclophosphamide at a dose of 20 mg / kg·d (0.1 mL / mouse); the Euphorbia-Glycyrrhizic acid supramolecular group was intraperitoneally injected with 75 μL / mouse, once a day for 10 consecutive days.

[0097] 2.3.2 Establishment of ascites tumor model and grouping and drug administration

[0098] The abdomen of the mice was depilated and disinfected with alcohol, and 0.2 mL / mouse of H22 cell suspension was injected into the left lower abdomen. The mice with unsatisfactory modeling were eliminated, and the mice with successful modeling were randomly divided into normal group, model group, positive drug group, and Euphorbia-Glycyrrhizic acid supramolecular group (Euphorbia ethyl acetate 25 mg-Glycyrrhizic acid 25 mg group), 5 mice in each group, and the dosage and feeding time were the same as those of subcutaneous tumor.

[0099] 2.4 Indicator Observation

[0100] (1) During the drug administration period, the diet, activity and mental state of mice in each group were observed.

[0101] (2) Subcutaneous tumor inhibition experiment: The changes in mouse tumor volume were recorded using a vernier caliper. The tumor diameter was measured at the same time every day after drug administration, and the tumor volume was calculated using the following formula: V = (L × W2) / 2, (L is the length of the tumor, W is the width of the tumor). At the end of the experiment, a tumor volume growth curve was drawn. After the treatment plan was completed, the mice were killed by cervical dislocation, the tumor was completely excised on ice, excess tissue was removed, and the tumor was washed with saline. The tumor was dried with filter paper, and the excised tumor weight was weighed and recorded. The tumor inhibition rate was calculated as (1-tumor in drug administration group / tumor in model group) × 100%.

[0102] (3) Ascites tumor model ascites inhibition experiment: Before drug administration, the abdominal circumference and weight of mice were measured daily, and a growth trend chart of abdominal circumference and weight was drawn based on this. In order to accurately measure the abdominal circumference, the mouse was laid flat on a horizontal surface, ensuring that it was in a supine position, and then the circumference of the most protruding part of the abdomen above the hip joint was measured.

[0103] 2.5 Impact of organ index

[0104] 24 hours after the last administration of the drug, the eyeballs of the mice were removed and blood was collected. The organs and tumors were dissected on ice, and the excess tissues were removed. The organs were washed with 0.9% NS, and the water and blood stains were absorbed with filter paper. The organs were weighed and recorded, and the organ index was calculated (organ index = organ weight / mouse body weight × 100%). The organs were preserved in 10% formaldehyde for preparation of paraffin-embedded pathological sections, and the pathological sections were stained with HE.

[0105] 2.6 Statistical analysis

[0106] IBM SPSS 20.0 was used for data statistical analysis, using one-way analysis of variance and non-parametric tests: p <0.05 was considered statistically significant. p <0.01 was considered a significant difference. p <0.001 was considered a highly significant difference.

[0107] 3. Experimental Results

[0108] 3.1 Subcutaneous tumor results

[0109] 3.1.1 Establishment of subcutaneous tumor model in Balb / c mice

[0110] The subcutaneous inoculation of H22 cells into Balb / c mice successfully established a subcutaneous hepatocellular carcinoma model. Figure 6 As shown, tumor cells were injected into the dorsal side of the right hind limb of mice. After 7 days, a soybean-sized mass could be felt, indicating that the H22 subcutaneous tumor mouse model was successfully established.

[0111] 3.1.2 Effect on tumor development speed

[0112] Tumor growth curve was drawn using tumor volume ( Figure 7 A) To explore the effects of different drug administration regimens on the tumor development rate in tumor-bearing mice, and to plot the tumor inhibition rate using tumor mass ( Figure 7 B), the results are shown in Table 1 and Figure 7 As shown in Table 1, compared with the model group, the positive drug, glycyrrhizic acid, Euphorbia decoction alone and Euphorbia-glycyrrhizic acid group all have the effect of inhibiting tumor growth. Among them, the tumor development speed of tumor-bearing mice in the cyclophosphamide group and Euphorbia-glycyrrhizic acid group is relatively slow, and the Euphorbia-glycyrrhizic acid group has the most significant effect. Figure 6 The tumor volume of the drug-treated group was significantly smaller than that of the model group. The tumor tissue mass of each mouse in the model group and the drug-treated group was weighed with an analytical balance and found that the average mass of the tumor tissue of the drug-treated group was significantly lower than that of the model group ( p<0.01). From the perspective of tumor inhibition rate, the average tumor inhibition rate of cyclophosphamide was 74.98%, and the calculated tumor inhibition rate of the Euphorbia-Glycyrrhizic acid group was 68.63%, which was equivalent to the effect of the cyclophosphamide group. The experimental results showed that the Euphorbia-Glycyrrhizic acid supramolecule had a good anti-tumor effect at a certain proportion and dose, and the effect could reach the anti-tumor effect of the common anti-tumor drug cyclophosphamide.

[0113] Table 1: Tumor mass and tumor inhibition rate of H22 liver cancer model

[0114]

[0115] Note: Compared with the model group, p <0.01, p <0.001.

[0116] 3.1.3 Effects on mouse organ indexes

[0117] like Figure 8 As shown in Figure 2, there was no significant difference in the liver, kidney, spleen, and thymus indexes of the mice in the Euphorbia-Glycyrrhiza supramolecular treatment group compared with the blank group, indicating that the Euphorbia-Glycyrrhiza supramolecular did not damage the organs of the mice (see Figure 8 A- Figure 8 D). It was also found that the spleen and thymus indexes of mice injected with cyclophosphamide were significantly lower, even lower than those of the blank group, indicating that cyclophosphamide not only inhibits tumor growth, but also affects the function of immune organs, causing atrophy of the spleen and thymus immune organs. The Euphorbia-Glycyrrhiza supramolecular group significantly improved the toxic side effects of cyclophosphamide ( p <0.01), which can play a certain regulatory role in the atrophy of immune organs.

[0118] 3.1.4 H&E staining

[0119] like Fig. 9 As shown, from the organ section staining results between the groups, there was no significant difference between the Euphorbia-Glycyrrhiza supramolecular group and the blank group, which was consistent with the organ index results, indicating that the administration of the Euphorbia-Glycyrrhiza supramolecular group did not cause damage to the organs of mice.

[0120] 3.2 Ascites tumor results

[0121] 3.2.1 Effects of Euphorbia-Glycyrrhiza supramolecular hydrogel on body weight and abdominal circumference of mice

[0122] During the whole experimental period, the weight changes and abdominal circumference of mice were as follows: Fig.11 As shown (see Fig.11 A- Fig.11B). Compared with the normal group, the body weight and abdominal circumference of the model group mice increased significantly after being inoculated with H22 tumor cells ( p <0.01), indicating that the modeling was successful. Compared with the model group, the mice in the positive drug group and the Euphorbia-Glycyrrhizic acid group had lighter weight and smaller abdominal circumference ( p <0.05). It is speculated that the reason is that after the model group mice were inoculated with H22 cells, although the body's ability to absorb nutrients became poor, the cancerous ascites increased rapidly, resulting in excessive weight and abdominal circumference, while after the Euphorbia-Glycyrrhizic Acid group was treated, the proliferation of tumor cells was inhibited, resulting in a lighter weight and smaller abdominal circumference of the mice, which was not significantly different from the normal group.

[0123] 3.2.2 H&E staining

[0124] like Fig.12 As shown, from the results of section staining of the liver, kidney and spleen, there was no significant difference between the Euphorbia-Glycyrrhiza supramolecular group and the normal group, which indicates that the administration of the Euphorbia-Glycyrrhiza supramolecular group did not cause damage to the liver, spleen and kidney of mice.

[0125] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A Euphorbia-Glycyrrhiza supramolecular hydrogel, characterized in that: The supramolecular hydrogel comprises ethyl acetate extract of Euphorbia and glycyrrhizic acid, wherein the ethyl acetate extract of Euphorbia and the glycyrrhizic acid interact with each other and form nanoparticles through self-assembly. The preparation method of the ethyl acetate extract of Euphorbia comprises the following steps: weighing an appropriate amount of Euphorbia decoction pieces and putting them into a round-bottom flask, adding 8 to 10 times the amount of 75% to 95% ethanol, reflux and decoct for 0.5 to 2 h, and obtaining decoction liquid I; then adding 8 to 10 times the amount of 75% to 95% ethanol, reflux and decoct for 0.5 to 2 h, and obtaining decoction liquid II; filtering while hot and combining the two decoction liquids, concentrating under reduced pressure, placing in a separating funnel, adding ethyl acetate for extraction, and vacuum freeze-drying to obtain freeze-dried powder of ethyl acetate extract of Euphorbia for standby use.

2. The supramolecular hydrogel according to claim 1, characterized in that: The weight ratio of the ethyl acetate extract of Euphorbia pulegosa to glycyrrhizic acid is 1:10~10:

1.

3. The supramolecular hydrogel according to claim 1, characterized in that: The weight ratio of the ethyl acetate extract of Euphorbia pulegosa to glycyrrhizic acid is 1:5~5:

1.

4. The supramolecular hydrogel according to claim 1, characterized in that: The average diameter of the nanoparticles is 100-250 nm.

5. The supramolecular hydrogel according to claim 1, characterized in that: The glycyrrhizic acid promotes the dissolution of the effective ingredient euphorbia dienol in the ethyl acetate extract of the euphorbia.

6. The method for preparing the supramolecular hydrogel according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: Weigh the ethyl acetate extract of Euphorbia and glycyrrhizic acid and place them in a round-bottom flask, add appropriate amount of deionized water, and boil for 0.5~2h.

7. The preparation method according to claim 6, characterized in that: The usage ratio of the total weight of the Euphorbia ethyl acetate extract and the glycyrrhizic acid to the volume of the deionized water is (1-10) mg: (1-10) mL.

8. Use of the supramolecular hydrogel according to any one of claims 1 to 5 or the supramolecular hydrogel prepared by the preparation method according to claim 6 or claim 7 in preparing a drug for treating malignant ascites caused by cancer.

9. The use according to claim 8, characterized in that: The malignant ascites caused by the cancer is liver cancer ascites.

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