Glycyrrhizic acid metal complex, preparation method thereof and application of glycyrrhizic acid metal complex in preparation of anti-tumor and anti-inflammatory drugs

Abstract

The invention provides a glycyrrhizic acid metal complex, a preparation method thereof and application of the glycyrrhizic acid metal complex in preparation of anti-tumor and anti-inflammatory drugs, and belongs to the technical field of anti-tumor drugs. The hexokinase inhibitor is constructed by performing metal complexing on glycyrrhizic acid, cobalt, nickel and rubidium. Results of embodiments show that compared with an NS group, the glycyrrhizic acid metal complex provided by the invention shows a significant hexokinase inhibition effect, and has a good anti-proliferation effect on A549 (human non-small cell lung cancer cells), MCF-7 (human breast cancer cells), SK-Mel-28 (human malignant melanoma cells) and SW480 (human pancreatic cancer cells). In addition, the glycyrrhizic acid metal complex provided by the invention improves the inhibitory activity of glycyrrhizic acid on OH free radicals, and has stronger anti-inflammatory activity compared with glycyrrhizic acid.

Description

Technical Field

[0001] The invention relates to the technical field of anti-tumor drugs, and in particular to a glycyrrhizic acid metal complex and a preparation method thereof and application thereof in the preparation of anti-tumor and anti-inflammatory drugs. Background Art

[0002] Hexokinases (HKs) are rate-limiting enzymes in the glycolysis process. They convert glucose into 6-glucose phosphate, allowing it to participate in subsequent metabolic processes to provide energy for the body. Hexokinase 2 is mainly found in insulin-sensitive tissues such as adipose, skeletal muscle and myocardium. One of its functions is to directly regulate cell apoptosis. It is considered a potential target for preventing organelle damage and maintaining cell vitality after apoptosis. Hexokinase is one of the key enzymes that affects glycolysis and produces cell apoptosis. Since hexokinase is highly expressed in cancer cells, hexokinase has also become a marker for cancer cell development and prognosis, as well as a target for anticancer treatment. Therefore, hexokinase inhibitors are expected to be a new type of anticancer drug that interferes with tumor metabolism and achieves clinical treatment of cancer.

[0003] 2-Deoxy-D-glucose (2-DG) is a hexokinase inhibitor that has been used in tumor treatment research (see: Shao Xia, Wang Ting. Research progress on the anti-tumor mechanism of 2-deoxy-D-glucose and its combined application in the treatment of tumors [J]. Medical Review, 2016, 22(17): 5. DOI: 10.3969 / j.issn.1006-2084.2016.17.013.). However, 2-DG must be used at relatively high concentrations to compete with glucose, and 2-DG has a short half-life, which limits its monotherapy in clinical practice.

[0004] Therefore, the development of new hexokinase inhibitors is of great significance for enriching tumor treatment methods and improving tumor treatment effects. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a glycyrrhizic acid metal complex and its preparation method and use in the preparation of anti-tumor and anti-inflammatory drugs. The glycyrrhizic acid metal complex provided by the present invention has a good inhibitory effect on hexokinase, good anti-tumor cell proliferation activity and anti-inflammatory effect.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a glycyrrhizic acid metal complex, comprising glycyrrhizic acid and a metal salt complexed with the glycyrrhizic acid; the metal salt comprises cobalt chloride, nickel chloride or rubidium nitrate;

[0008] The glycyrrhizic acid metal complex has a structure shown in any one of Formulas 1 to 3:

[0009]

[0010] Preferably, the particle size of the glycyrrhizic acid metal complex is 177 to 250 nm.

[0011] The present invention provides a method for preparing the glycyrrhizic acid metal complex, comprising the following steps:

[0012] Glycyrrhizic acid, metal salt and organic solvent are mixed, the pH value of the obtained mixed solution is adjusted to alkaline, and complex reaction is carried out to obtain glycyrrhizic acid metal complex.

[0013] Preferably, the molar ratio of glycyrrhizic acid to metal salt is 1:(1.0-1.5).

[0014] Preferably, the alkaline pH value is 8.

[0015] Preferably, the complexation reaction is carried out under ultrasonic conditions;

[0016] The complexing reaction time is 10 to 450 minutes.

[0017] The present invention provides application of the glycyrrhizic acid metal complex in preparing a hexokinase inhibitor.

[0018] The present invention provides application of the glycyrrhizic acid metal complex in preparing anti-tumor drugs.

[0019] Preferably, the anti-tumor drug includes one or more of an anti-lung cancer drug, an anti-pancreatic cancer drug, an anti-breast cancer drug and an anti-melanoma drug.

[0020] The present invention provides application of the glycyrrhizic acid metal complex in preparing anti-inflammatory drugs.

[0021] The present invention provides a glycyrrhizic acid metal complex, including glycyrrhizic acid and a metal salt complexed with glycyrrhizic acid; the metal salt includes cobalt chloride, nickel chloride or rubidium nitrate; the glycyrrhizic acid metal complex has a structure shown in any one of formulas 1 to 3. Glycyrrhizic acid, as one of the main components of licorice, has multiple pharmacological effects such as anti-tumor and anti-inflammatory; in anti-tumor research, glycyrrhizic acid requires a higher concentration to achieve anti-tumor effects, and the solubility of glycyrrhizic acid in water is poor. The present invention metal complexes glycyrrhizic acid with cobalt, nickel or rubidium to construct a hexokinase inhibitor. The results of the embodiment show that compared with the NS group, the glycyrrhizic acid metal complex provided by the present invention exhibits a significant inhibitory effect on hexokinase, and has a good anti-proliferation effect on A549 (human non-small cell lung cancer cells), MCF-7 (human breast cancer cells), SK-Mel-28 (human malignant melanoma cells), and SW480 (human pancreatic cancer cells). In addition, the glycyrrhizic acid metal complex provided by the present invention improves the inhibitory activity of glycyrrhizic acid on OH free radicals and has stronger anti-inflammatory activity than glycyrrhizic acid.

[0022] The invention provides a method for preparing the glycyrrhizic acid metal complex, which has the advantages of simple operation, low cost and easy industrial batch production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the mass spectrum of CoCl2(Gly);

[0024] Figure 2 is the mass spectrum of NiCl2(Gly);

[0025] Figure 3 is the mass spectrum of RbNO3(Gly);

[0026] Figure 4 is the infrared spectrum of compound Gly;

[0027] Figure 5 is the infrared spectrum of the compound CoCl2(Gly);

[0028] Figure 6 is the infrared spectrum of the compound NiCl2(Gly);

[0029] Figure 7 is the infrared spectrum of the compound RbNO3(Gly);

[0030] Figure 8 The test results of Tyndall effect before and after laser irradiation;

[0031] Fig. 9 The results of scavenging free radicals by different compounds;

[0032] Fig.10The results of the in vivo mouse ear swelling experiment. DETAILED DESCRIPTION

[0033] The present invention provides a glycyrrhizic acid metal complex, comprising glycyrrhizic acid and a metal salt complexed with the glycyrrhizic acid; the metal salt comprises cobalt chloride, nickel chloride or rubidium nitrate;

[0034] The glycyrrhizic acid metal complex has a structure shown in any one of Formulas 1 to 3:

[0035]

[0036] In the present invention, the particle size of the glycyrrhizic acid metal complex is preferably 177 to 250 nm, more preferably 200 to 220 nm.

[0037] The present invention provides a method for preparing the glycyrrhizic acid metal complex, comprising the following steps:

[0038] Glycyrrhizic acid, metal salt and organic solvent are mixed, the pH value of the obtained mixed solution is adjusted to alkaline, and complex reaction is carried out to obtain glycyrrhizic acid metal complex.

[0039] Unless otherwise specified, the raw materials used in the present invention are all commercially available.

[0040] In the present invention, the metal salt includes cobalt chloride, nickel chloride or rubidium nitrate. In the present invention, the molar ratio of glycyrrhizic acid to the metal salt is preferably 1:1.

[0041] In the present invention, the organic solvent is preferably one or more of methanol, ethanol and dioxane. The present invention has no special requirements for the specific amount of the organic solvent, as long as it can completely dissolve glycyrrhizic acid and the metal salt. The present invention has no special requirements for the mixing method, and a mixing method well known to those skilled in the art can be used.

[0042] In the present invention, the alkaline agent used to adjust the pH value of the obtained mixed solution to alkaline is preferably a sodium hydroxide aqueous solution, and the concentration of the sodium hydroxide aqueous solution is preferably 1 mmol / L. In the present invention, the alkaline pH value is preferably 8.

[0043] In the present invention, the complexation reaction is carried out under ultrasonic conditions, and the power of the ultrasound is preferably 1000-2500 W. In the present invention, the temperature of the complexation reaction is preferably 20-25 degrees, and the time is preferably 20 minutes.

[0044] After the complexation reaction, the present invention preferably concentrates the obtained complexation reaction solution under reduced pressure. The present invention has no special limitation on the process of the reduced pressure concentration, and the process can be carried out by a process well known to those skilled in the art.

[0045] The present invention provides application of the glycyrrhizic acid metal complex in preparing a hexokinase inhibitor.

[0046] The present invention provides the use of the glycyrrhizic acid metal complex in the preparation of anti-tumor drugs. In the present invention, the anti-tumor drug preferably includes one or more of anti-lung cancer drugs, anti-pancreatic cancer drugs, anti-breast cancer drugs and anti-melanoma drugs.

[0047] The present invention provides the use of the glycyrrhizic acid metal complex in the preparation of an anti-inflammatory drug. In the present invention, the dosage of the glycyrrhizic acid metal complex in the preparation of the anti-inflammatory drug is preferably 0.1 mg / kg.

[0048] The glycyrrhizic acid metal complex provided by the present invention, its preparation method and its application in the preparation of anti-tumor and anti-inflammatory drugs are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0049] In the following examples, the hexokinase (HK) activity detection kit was purchased from Beijing Solebow Technology Co., Ltd., model number BC0745;

[0050] A549 (KG007, human non-small cell lung cancer cells): cultured in DMEM medium containing 10% FBS, purchased from Jiangsu KeyGen Biotech Co., Ltd.;

[0051] MCF-7 (KG031, human breast cancer cells): cultured in DMEM medium containing 10% FBS, purchased from Jiangsu KeyGen Biotech Co., Ltd.;

[0052] SK-Mel-28 (CL-0717, human malignant melanoma cells): cultured in DMEM medium containing 10% FBS, purchased from Wuhan Pusino Biotechnology Co., Ltd.;

[0053] SW480 (KGG3319-1, human pancreatic cancer cells): cultured in DMEM medium containing 10% FBS, purchased from Jiangsu KeyGen Biotech Co., Ltd.

[0054] Example 1

[0055] Preparation of compound CoCl2(Gly):

[0056]

[0057] Weigh 16.01 mg (0.12 mmol / L) of CoCl2 and 104.8 mg (0.12 mmol / L) of glycyrrhizic acid (Gly), dissolve them in methanol, adjust the pH to 8 with 1 mmol / L sodium hydroxide aqueous solution, and react with ultrasound for 20 minutes to obtain a glycyrrhizic acid-CoCl2 complex, recorded as CoCl2 (Gly). After adjusting the pH to 8 and reacting with glycyrrhizic acid, the color of anhydrous CoCl2 changes significantly from light brown to purple, and it is dried by spin drying after the reaction is completed. The mass spectrum of the obtained CoCl2 (Gly) is shown in the figure. Figure 1 As shown. ESI + -MS(m / e):889.72[M+H] + ;Mp270.4~271.6℃;[α] 25 D =+22.3(c 0.5,H2O).

[0058] Depend on Figure 1 It can be inferred that two molecules of glycyrrhizic acid are complexed with one molecule of cobalt chloride.

[0059] Example 2

[0060] Preparation of compound NiCl2(Gly):

[0061]

[0062] Weigh 8.08 mg (0.06 mmol) of NiCl2 and 51.56 mg (0.06 mmol) of Gly, dissolve them in methanol, adjust the pH to 8 with 1 mmol / L sodium hydroxide aqueous solution, and react with ultrasound for 20 minutes to obtain glycyrrhizic acid-NiCl2 complex, which is recorded as NiCl2 (Gly). After adjusting the pH to 8 and reacting with glycyrrhizic acid, the color of anhydrous NiCl2 changes significantly from light brown to green. After the reaction is completed, it is dried by rotation. The mass spectrum of the obtained NiCl2 (Gly) is shown in the figure. Figure 2 As shown. ESI + +MS(m / e):889.37[M+H] + , Mp>280℃; [α] 25 D =+22.4 (c 0.5, H2O).

[0063] Depend on Figure 2 It can be inferred that two molecules of glycyrrhizic acid were complexed with one molecule of nickel chloride.

[0064] Example 3

[0065] Preparation of compound RbNO3(Gly):

[0066]

[0067] Weigh 28.2 mg (0.19 mmol) of RbNO3 and 162.2 mg (0.20 mmol) of Gly, dissolve them in methanol, adjust the pH to 8 with 1 mmol / L sodium hydroxide aqueous solution, and react with ultrasound for 20 minutes to obtain a glycyrrhizic acid-RbNO3 complex, which is recorded as RbNO3 (Gly). The color of anhydrous RbNO3 changes from colorless to yellow after adjusting the pH to 8 and reacting with glycyrrhizic acid. After the reaction is completed, it is dried by rotation. The mass spectrum of the obtained RbNO3 (Gly) is shown in the figure. Figure 3 As shown. ESI - -MS(m / e):931.16[MH] - ,Mp267.9~268.4℃;[α] 25 D =+28.0(c 0.5,H2O).

[0068] Depend on Figure 3 It can be inferred that two molecules of glycyrrhizic acid are complexed with one molecule of rubidium nitrate.

[0069] Structural characterization

[0070] (1) Infrared spectroscopy

[0071] The infrared spectrum of compound Gly is as follows Figure 4 As shown, the infrared spectrum of the compound CoCl2(Gly) is as follows Figure 5 As shown. It can be seen that CoCl2(Gly) and compound Gly have a higher -1 Therefore, it was determined that the metal was complexed with the carbonyl part of glycyrrhizic acid.

[0072] The infrared spectrum of the compound NiCl2(Gly) is as follows Figure 6 As shown in the figure, it can be seen that NiCl2(Gly) has a higher peak at 1710 cm-1 than compound Gly. -1 The absorption of is shifted, which is the absorption peak of carbonyl group, so it is determined that the metal is complexed with the carbonyl group of glycyrrhizic acid.

[0073] The infrared spectrum of the compound RbNO3(Gly) is as follows Figure 7 As shown in the figure, it can be seen that RbNO3(Gly) has a higher peak at 1700 cm-1 than compound Gly. -1 The shift in absorption of , proves the formation of complex bond.

[0074] (2) Nanostructure characterization

[0075] A laser pen (λ=650nm) was used to irradiate a glass bottle containing a solution, and water was used as a control group to observe the Tyndall effect produced in each group. The Tyndall effect test results before and after laser irradiation are shown in the figure below. Figure 8 As shown, from left to right are pure water, CoCl2(Gly)(10 -5 M), NiCl2(Gly)(10 -6 M), RbNO3(Gly)(10 -6 M) in aqueous solution, all showed obvious Tyndall effect, indicating that the complex can self-assemble to form a nanosystem.

[0076] (3) Particle size and potential measurement

[0077] CoCl2(Gly), NiCl2(Gly) and RbNO3(Gly) were prepared with pure water to form 10 -5 The particle size and Zeta potential of the tested samples were measured at 25° C. using a laser nanoparticle size analyzer and DTS (Nano) software. The particle size and Zeta potential of different compounds are shown in Table 1.

[0078] Table 1 Particle size and Zeta potential of the compounds

[0079] Test samples PDI Particle size (nm) Zeta(mV) <![CDATA[CoCl2(Gly)]]> 0.593 284.6±89.48 -36.4±4.12 <![CDATA[NiCl2(Gly)]]> 0.431 177.1±66.50 -52.2±8.03 <![CDATA[RbNO3(Gly)]]> 0.492 190.6±54.63 -16.8±4.62

[0080] As can be seen from Table 1, the particle size of the glycyrrhizic acid metal complex provided by the present invention is in the range of 177-250 nm, and the PDI value is small and stable. It is found that the maximum Zeta potential of NiCl2(Gly) is -52.2±8.03, which has good nano properties.

[0081] Test Example 1

[0082] In vitro hexokinase inhibition assay:

[0083] Glycyrrhizic acid, CoCl2(Gly), NiCl2(Gly) and RbNO3(Gly) were used as test samples. 5 μL of the compound was incubated with 10 μL of hexokinase (0.25 U) at 37°C for 15 min, and 85 μL of a mixed solution was added. The mixed solution included 100 mM TrisHCl pH 8.0, 5 mM MgCl2, 200 mM glucose, 0.8 mM ATP, 1 mM oxidized coenzyme I (NAD + , purchased from Beijing Solebow Technology Co., Ltd., IN0010), 0.25Units of glucose-6-phosphate dehydrogenase (G6P-DH), and the microplate reader was preheated to 37°C in advance. The absorption value was measured at 340nm every 1min for a total of 5min, and the IC was calculated based on the rate of change of the absorption value. 50 .

[0084] The in vitro inhibitory effects of different compounds on hexokinase are shown in Table 2.

[0085] Table 2 In vitro inhibition of hexokinase by different compounds (n=3, IC 50 ±SD, μM)

[0086] Compound <![CDATA[IC 50 ]]> <![CDATA[CoCl2(Gly)]]> 16.67±1.76* <![CDATA[NiCl2(Gly)]]> 9.31±2.57 Gly 11.28±0.97

[0087] As shown in Table 2, compared with glycyrrhizic acid, the inhibitory activity of NiCl2(Gly) was significantly different, p<0.01, and the activity of NiCl2(Gly) was improved compared with Gly.

[0088] Test Example 2

[0089] In vitro tumor cell antiproliferation assay:

[0090] Weigh 0.1mmol of Gly, CoCl2(Gly), NiCl2(Gly), and RbNO3(Gly) respectively, add 100μL of DMSO to dissolve, add 9.9mL of PBS solution to prepare a 10mM solution, add 25μL to 100μL of culture medium during cell culture, the final concentration is 2.5mM, dilute the concentration according to the preliminary experimental results, and set the concentration gradient. Use PBS as a blank control.

[0091] A549 (KG007, human non-small cell lung cancer cells), MCF-7 (KG031, human breast cancer cells), SK-Mel-28 (CL-0717, human malignant melanoma cells) and SW480 (KGG3319-1, human pancreatic cancer cells) were used as test cells, and all of the above four cell lines are adherent cells.

[0092] The cell line culture method is as follows:

[0093] Cell recovery: quickly thaw the cells frozen in liquid nitrogen in a 37°C water bath, aspirate the cell suspension in a safety cabinet and transfer it to a centrifuge tube, add 5 mL of complete culture medium, set the centrifuge to 1000 rpm, centrifuge for 3 min, discard the supernatant, add new complete culture medium and transfer to a T25 cell culture flask containing 5 mL of new culture medium, culture in an incubator at 37°C, 5% CO2 for 24 h, observe the cell growth, and subculture according to the cell number.

[0094] Cell passaging: Cells can be passaged when the healing degree is above 90%. Discard the old culture, add 2 mL of PBS buffer to rinse, add 3 mL of trypsin (KGL2101-100, purchased from Jiangsu KeyGen Biotech Co., Ltd.), and place in a 37°C, 5% CO2 incubator for digestion for 3 to 5 minutes until most of the cells are floating in a spherical state. Add 3 mL of new complete culture medium to terminate digestion, gently blow up with a pipette, transfer the cell suspension to a centrifuge tube, set the centrifuge to 1000 rpm, centrifuge for 3 minutes, discard the supernatant, add new complete culture medium, transfer to a T25 cell culture flask, culture in a 37°C, 5% CO2 incubator for 24 hours, and observe the cell growth.

[0095] Seed plate: Take adherent cells in logarithmic phase growth, pour out the old culture medium in the culture flask, add 2mL of PBS buffer to rinse, add 3-5mL of trypsin to the culture flask, put it in an incubator at 37℃, 5% CO2 to digest for 3-5min, until most of the cells are floating in a spherical state, blow it up gently with a pipette, transfer the cell suspension to a centrifuge tube, set the centrifuge to 1000 rpm, centrifuge for 3min, remove the supernatant, then add 2mL of fresh complete culture medium to the tube, blow it evenly with a sterilized pipette tip, and then count the cells.

[0096] The density of the seed plate is 50,000 cells / mL. 100 μL is drawn from each well and placed in a cell culture incubator at 37°C and 5% CO2 for culture. After 12 hours of culture, the cell adhesion is observed under a microscope. After all cells are attached, 25 μL of the prepared test sample is added. Each test sample has 6 wells and a negative control well and a blank control well are set. No drug is given to the negative control well, only an equal volume of PBS is added. The blank control well does not contain cells, only culture medium and an equal volume of PBS are added, and the well is placed in a cell culture incubator at 37°C and 5% CO2 for culture for 24 hours. After 24 hours, 25 μL of the prepared MTT solution (concentration of 5 mg / mL) is added to each well plate, and then placed in a cell culture incubator for culture. After 4 hours of culture, the culture is terminated. Take out the 96-well plate, remove the culture solution in the well, and then add 150 μL of DMSO to each well, place it on a flat plate oscillator and shake it for 15 minutes to fully dissolve the crystal formazan in DMSO. Finally, the wavelength was set to 490nm and 570nm on a multifunctional microplate reader, the absorbance value of each well was measured, and the inhibition rate of cells was calculated. Repeat three times and then take the average value.

[0097] Inhibition rate = [(OD value of the test sample - average OD value of the blank control) / (average OD value of the negative control group - average OD value of the blank control)] × 100%.

[0098] The present invention evaluated the in vitro anti-cell proliferation activity of the test samples against four cell lines, A549, MCF-7, SK-Mel-28, and SW480, and calculated the IC by linear regression curve fitting of logarithmic concentration and cell survival percentage using GraphPadPrism8. 50 , the results are shown in Table 3.

[0099] Table 3 Effects of compounds on antiproliferative ability of cancer cells (IC 50 ,Mean±SD, mM,n=18)

[0100] Test samples A549 SW480 MCF-7 SK-Mel-28 <![CDATA[CoCl2(Gly)]]> 0.69±0.065 0.55±0.11 0.75±0.16 0.42±0.015 <![CDATA[NiCl2(Gly)]]> 0.82±0.055 0.43±0.010 0.69±0.045 0.75±0.0075 <![CDATA[RbNO3(Gly)]]> >2.5 >2.5 >2.5 >2.5 Glycyrrhizic acid >2.5 >2.5 >2.5 >2.5

[0101] The experimental results showed that both CoCl2(Gly) and NiCl2(Gly) could inhibit the proliferation of A549, SW480, MCF-7 and SK-Mel-28 cells. 50 The value is between 0.42 and 0.82 mM. Compared with glycyrrhizic acid, the antiproliferative activity is improved.

[0102] Test Example 3

[0103] Hexokinase activity assay:

[0104] Glycyrrhizic acid, CoCl2(Gly), NiCl2(Gly) and RbNO3(Gly) were used as the test samples. CoCl2(Gly) and NiCl2(Gly) were tested according to the IC 50 The NS group was given an equal volume of PBS, and the control group was given deionized water. The experimental method is as follows:

[0105] The plate was seeded with 100,000 cells per well in 1 mL, and cultured in a cell culture incubator at 37°C and 5% CO2. After 12 hours of culture, the cells adhered to the wall and 250 μL of the prepared test sample was added. Three wells were set for each test sample and a control well. No drug was given to the control well, but only PBS was added. After culturing in a cell culture incubator at 37°C and 5% CO2 for 48 hours, the cells were collected and the number of cells was calculated. The assay was performed according to the procedure in the hexokinase activity assay kit (Solabo, China) (i.e., the collected cells were added to the extract solution 1 at a ratio of 5 million cells: 1 mL, and the cells were broken using a non-contact DNA fragmentor (power: 22.5 kW, time: working 3 s, interval 7 s, total time 10 min), centrifuged at 8000g and 4°C for 10 min, the supernatant was collected, and placed on ice for testing. According to the sample volume, a mixed solution was prepared in the ratio of Reagent 2A: Reagent 2B: Reagent 2C: Reagent 2D = 100 μL: 500 μL: 150 μL: 150 μL (5T), and it was prepared before use). During the test, the amount used was 1 / 2 of the prescribed amount; the microplate reader was preheated to 37°C, and the absorbance value A1 at 340 nm at 20 s was measured, and the absorbance value A2 at 5 min 20 s was measured. Compared with the control group, the inhibition ratio of the test sample on the hexokinase in the cells was calculated (inhibition rate = ΔA 样品 / ΔA 水 ).

[0106] ΔA=A2-A1

[0107] HK activity (U / 10 4 cell)=[ΔA×V total÷(ε×d)×10 9 ]÷(N×V total)÷T=1071.7×ΔA÷N

[0108] N is the total number of bacteria or cells, in tens of thousands.

[0109] Unit definition: One unit of enzyme activity is defined as 1 nmol of NADPH produced per 10,000 bacteria or cells per minute.

[0110] The inhibitory effects of different compounds on hexokinase in MCF-7, SW380, A549, and SK-Mel-28 cells are shown in Table 4.

[0111] Table 4 Inhibitory effects of different compounds on hexokinase in MCF-7, SW380 and A549 cells (n=9, U / 10 4 cell)

[0112] Compound MCF-7 SW480 A549 NS 1.34±0.02 1.03±0.24 0.93±0.21 Gly 0.93±0.16 0.68±0.06 0.46±0.15 <![CDATA[CoCl2(Gly)]]> 0.60±0.02* 0.29±0.07* 0.50±0.05 <![CDATA[NiCl2(Gly)]]> 0.68±0.09* 0.42±0.04* 0.30±0.03* <![CDATA[RbNO3(Gly)]]> 0.76±0.12* 0.25±0.09* 0.38±0.04*

[0113] Note: *, compared with Gly group, p<0.05.

[0114] It can be seen that in MCF-7 cells, RbNO3 (Gly) and CoCl2 (Gly) showed an inhibitory effect on hexokinase and had a significant difference compared with the NS group. In SW480 and A549 cells, compared with the NS group, the three metal complexes showed a significant inhibitory effect on hexokinase.

[0115] Test Example 4

[0116] Free radical scavenging experiment:

[0117] Accurately weigh Gly, RbNO3(Gly), CoCl2(Gly), and NiCl2(Gly) to prepare a mother solution with a concentration of 1mM. The experimental method is as follows:

[0118] OH free radical determination method: Use a pipette to transfer 5 μL of ferrous sulfate solution into a 0.5 mL EP tube, add 5 μL of DMPO aqueous solution, add 5 μL of blank solvent / sample, and add 5 μL of 1% hydrogen peroxide aqueous solution. Mix well with a vortex mixer, siphon the sample with a capillary tube, seal it with sealing glue, put it into a paramagnetic tube, keep the sample in the reaction for 2 minutes, and then test it on the machine. Repeat six times to get the average peak height.

[0119] The results of free radical scavenging by different compounds are as follows Fig. 9 As shown. It can be seen that compared with the blank group, glycyrrhizic acid did not show a significant inhibitory effect on OH free radicals, but the metal complexes (RbNO3 (Gly), CoCl2 (Gly), NiCl2 (Gly) showed a significant inhibitory effect compared with the blank control group, among which, compared with glycyrrhizic acid, CoCl2 (Gly), NiCl2 (Gly) groups had significant differences; therefore, after modification with CoCl2 and NiCl2, the inhibitory activity of glycyrrhizic acid on OH free radicals was improved.

[0120] Test Example 5

[0121] Mouse ear swelling experiment:

[0122] The xylene-induced acute inflammatory model of mouse ear swelling refers to applying xylene to the mouse ears. Under its stimulation, it causes local capillary congestion, increases the permeability of the mouse ears, and increases the infiltrated tissue fluid, resulting in visible redness and swelling. The anti-inflammatory ability of the drug is evaluated by comparing the degree of ear swelling.

[0123] Male ICR mice (SPF grade) weighing 20±5 g were selected as experimental animals. They were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and raised in the Experimental Animal Department of Capital Medical University.

[0124] Target compound group: 2.20 mg of glycyrrhizic acid, 2.37 mg of RbNO3(Gly), 2.39 mg of CoCl2(Gly), and 8 mL of 0.1% sodium carboxymethyl cellulose aqueous solution were added to prepare suspensions.

[0125] Blank control group: 0.1% sodium carboxymethylcellulose aqueous solution (NS), the administration volume was calculated as 0.5 mL / 20 g body weight, n=6.

[0126] The experimental model was established as follows:

[0127] 0.5 h after intragastric administration at a volume of 20 g / 0.5 mL, 20 μL of xylene solution was evenly applied to both sides of the right ear of the mouse. The mouse was deprived of water for 2 h, blood was collected and the mouse was killed. The left and right ears of the mouse were removed, holes were pierced in alignment, and the ears were weighed. The swelling rate was calculated according to the following formula.

[0128]

[0129] In vivo mouse ear swelling experiment results Fig.10 As shown (n=6). Fig.10 Compared with Control at the same time point ( * P<0.05; compared with Gly at the same time point ( # P<0.05); compared with CoCl2(Gly) at the same time point ( & P<0.05).

[0130] It can be seen that compared with the NS group, the swelling rates of the Gly, RbNO3(Gly) and CoCl2(Gly) groups after administration were statistically different (P<0.05). Compared with the glycyrrhizic acid Gly group, the swelling rate of the CoCl2(Gly) group was smaller, with statistical differences (P<0.05), showing stronger anti-inflammatory activity.

[0131] In summary, the present invention uses glycyrrhizic acid as a modified compound to synthesize and identify three target compounds, RbNO3 (Gly), CoCl2 (Gly) and NiCl2 (Gly). The target compounds are characterized by infrared and ultraviolet spectra to determine that their structures are correct and exhibit good nano properties. The MTT method is used to evaluate the antiproliferative ability of the target compounds on A549, SW480, MCF-7, and SK-Mel-28 cells and the hexokinase inhibition ability. Among them, CoCl2 (Gly) and NiCl2 (Gly) have improved antiproliferative ability on the four cells, but in A549 (0.38 ± 0.04 U / 10 4 cell)、SW480(0.25±0.09U / 10 4cell), MCF-7 (0.762±0.22U / 10 4 cell) showed an inhibitory effect on hexokinase and was superior to glycyrrhizic acid (1.16±0.17 U / 10 4 CoCl2(Gly) showed an inhibitory effect on hexokinase in SW480 and MCF-7, and NiCl2(Gly) showed an inhibitory effect on hexokinase in MCF-7 (1.18±0.09U / 10 4 cell) exhibited an inhibitory effect on hexokinase.

[0132] Inflammation is closely related to the occurrence, development and therapeutic effect of tumors. In the mouse ear swelling model, compared with the blank group (CMCNa, 81.93%±20.66%), the compounds glycyrrhizic acid (48%.40±9.08%), CoCl2(Gly) (19.40%±15.42%), and NiCl2(Gly) (51.41%±14.34%) all showed lower swelling rates and exhibited anti-inflammatory activity, among which CoCl2(Gly) and glycyrrhizic acid showed significant differences.

[0133] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A glycyrrhizic acid metal complex, comprising glycyrrhizic acid and a metal salt complexed with glycyrrhizic acid; the metal salt comprises cobalt chloride, nickel chloride or rubidium nitrate; The glycyrrhizic acid metal complex has a structure shown in any one of Formulas 1 to 3:

2. The glycyrrhizic acid metal complex according to claim 1, characterized in that The particle size of the glycyrrhizic acid metal complex is 177-250 nm.

3. The method for preparing the glycyrrhizic acid metal complex according to claim 1 or 2, comprising the following steps: Glycyrrhizic acid, metal salt and organic solvent are mixed, the pH value of the obtained mixed solution is adjusted to alkaline, and complex reaction is carried out to obtain glycyrrhizic acid metal complex.

4. The preparation method according to claim 3, characterized in that: The molar ratio of the glycyrrhizic acid to the metal salt is 1:(1.0-1.5).

5. The preparation method according to claim 3, characterized in that: The alkaline pH is 8.

6. The preparation method according to claim 3 or 5, characterized in that: The complexation reaction is carried out under ultrasonic conditions; The complexing reaction time is 10 to 45 minutes.

7. Use of the glycyrrhizic acid metal complex according to claim 1 or 2 or the glycyrrhizic acid metal complex prepared by the preparation method according to any one of claims 3 to 6 in the preparation of a hexokinase inhibitor.

8. Use of the glycyrrhizic acid metal complex according to claim 1 or 2 or the glycyrrhizic acid metal complex prepared by the preparation method according to any one of claims 3 to 6 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The anti-tumor drug includes one or more of an anti-lung cancer drug, an anti-pancreatic cancer drug, an anti-breast cancer drug and an anti-melanoma drug.

10. Use of the glycyrrhizic acid metal complex according to claim 1 or 2 or the glycyrrhizic acid metal complex prepared by the preparation method according to any one of claims 3 to 6 in the preparation of anti-inflammatory drugs.

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