Glycyrrhizin metal complex, its preparation method and application in preparation of anti-tumor and anti-inflammatory drugs

Abstract

The application provides a glycyrrhizin metal complex, a preparation method thereof and application thereof in preparation of anti-tumor and anti-inflammatory drugs, and belongs to the technical field of anti-tumor drugs. The glycyrrhizin is metal-complexed with cobalt, nickel and rubidium to construct a hexokinase inhibitor. The results of examples show that, compared with the NS group, the glycyrrhizin metal complex provided by the application 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 glycyrrhizin metal complex provided by the application improves the inhibitory activity of glycyrrhizin on OH free radicals, and has stronger anti-inflammatory activity than glycyrrhizin.

Description

Technical Field

[0001] This invention relates to the field of antitumor drug technology, specifically to a glycyrrhizic acid metal complex, its preparation method, and its application in the preparation of antitumor and anti-inflammatory drugs. Background Technology

[0002] Hexokinases (HKs) are rate-limiting enzymes in glycolysis, converting glucose into phosphate-6-glucose, which then participates in subsequent metabolic processes to provide energy for the body. Hexokinases are mainly found in insulin-sensitive tissues such as adipose tissue, skeletal muscle, and cardiac muscle. One of their functions is to directly regulate apoptosis, and they are considered potential targets for preventing organelle damage and maintaining post-apoptotic cell viability. Hexokinases are key enzymes affecting glycolysis and inducing apoptosis. Due to their high expression in cancer cells, hexokinases have become biomarkers for cancer cell development and prognosis, as well as targets for anti-cancer research. Therefore, hexokinases inhibitors hold promise as novel anti-cancer drugs to interfere with tumor metabolic processes and achieve clinical treatment of cancer.

[0003] 2-Deoxy-D-glucose (2-DG) is a hexokinase inhibitor that has been studied for its use in cancer treatment (see: Shao Xia, Wang Ting. Research progress on the antitumor mechanism of 2-deoxy-D-glucose and its combined use in cancer treatment [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 its short half-life limits its use as a monotherapy in clinical practice.

[0004] Therefore, developing new hexokinase inhibitors is of great significance for enriching tumor treatment methods and improving tumor treatment outcomes. Summary of the Invention

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

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

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

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

[0009]

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

[0011] This invention provides a method for preparing the above-mentioned glycyrrhizic acid metal complex, comprising the following steps:

[0012] Glycyrrhizic acid, metal salt, and organic solvent are mixed, and the pH of the resulting mixture is adjusted to alkaline. A complexation reaction is then carried out to obtain a glycyrrhizic acid-metal complex.

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

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

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

[0016] The complexation reaction takes 10–450 min.

[0017] This invention provides the application of the above-mentioned glycyrrhizic acid metal complex in the preparation of hexokinase inhibitors.

[0018] This invention provides the application of the above-mentioned glycyrrhizic acid metal complex in the preparation of antitumor drugs.

[0019] Preferably, the antitumor drug includes one or more of the following: anti-lung cancer drugs, anti-pancreatic cancer drugs, anti-breast cancer drugs, and anti-melanoma drugs.

[0020] This invention provides the application of the above-mentioned glycyrrhizic acid metal complex in the preparation of anti-inflammatory drugs.

[0021] This invention provides a glycyrrhizic acid metal complex comprising 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, possesses various pharmacological effects such as antitumor and anti-inflammatory properties. In antitumor studies, glycyrrhizic acid requires a high concentration to achieve its antitumor effect, and its solubility in water is poor. This invention constructs a hexokinase inhibitor by metal complexing glycyrrhizic acid with cobalt, nickel, or rubidium. The results of the examples show that, compared with the NS group, the glycyrrhizic acid metal complex provided by this invention exhibits a significant inhibitory effect on hexokinase and demonstrates good antiproliferative activity against 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). Furthermore, the glycyrrhizic acid metal complex provided by this invention enhances the inhibitory activity of glycyrrhizic acid against OH free radicals and exhibits stronger anti-inflammatory activity compared to glycyrrhizic acid.

[0022] This invention provides a method for preparing the above-mentioned glycyrrhizic acid metal complex. This method is simple to operate, low in cost, and easy to achieve industrial-scale mass production. Attached Figure Description

[0023] Figure 1 The mass spectrum of CoCl2(Gly) is shown.

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

[0025] Figure 3 The mass spectrum of RbNO3(Gly) is shown.

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

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

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

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

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

[0031] Figure 9 The results of different compounds scavenging free radicals;

[0032] Figure 10The results are from an in vivo mouse ear swelling experiment. Detailed Implementation

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

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

[0035]

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

[0037] This invention provides a method for preparing the above-mentioned glycyrrhizic acid metal complex, comprising the following steps:

[0038] Glycyrrhizic acid, metal salt, and organic solvent are mixed, and the pH of the resulting mixture is adjusted to alkaline. A complexation reaction is then carried out to obtain a glycyrrhizic acid-metal complex.

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

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

[0041] In this invention, the organic solvent is preferably one or more of methanol, ethanol, and dioxane. This invention does not have specific requirements regarding the amount of the organic solvent used, as long as it is sufficient to completely dissolve the glycyrrhizic acid and the metal salt. This invention also does not have specific requirements regarding the mixing method; any mixing method well-known to those skilled in the art can be used.

[0042] In this invention, the alkaline reagent used to adjust the pH of the resulting mixture to alkalinity is preferably an aqueous solution of sodium hydroxide, and the concentration of the sodium hydroxide aqueous solution is preferably 1 mmol / L. In this invention, the alkaline pH is preferably 8.

[0043] In this invention, the complexation reaction is carried out under ultrasonic conditions, preferably with a power of 1000–2500 W. The temperature of the complexation reaction is preferably 20–25 degrees Celsius, and the reaction 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 does not have any special limitations on the process of the reduced pressure concentration, and any process known to those skilled in the art can be used.

[0045] This invention provides the application of the above-mentioned glycyrrhizic acid metal complex in the preparation of hexokinase inhibitors.

[0046] This invention provides the application of the above-mentioned glycyrrhizic acid metal complex in the preparation of antitumor drugs. In this invention, the antitumor drug preferably includes one or more of the following: anti-lung cancer drugs, anti-pancreatic cancer drugs, anti-breast cancer drugs, and anti-melanoma drugs.

[0047] This invention provides the application of the above-mentioned glycyrrhizic acid metal complex in the preparation of anti-inflammatory drugs. In this invention, the preferred dosage of the glycyrrhizic acid metal complex in the preparation of the anti-inflammatory drug is 0.1 mg / kg.

[0048] The following examples illustrate the glycyrrhizic acid metal complexes provided by the present invention, their preparation methods, and their applications in the preparation of antitumor and anti-inflammatory drugs. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0049] In the following examples, the hexokinase (HK) activity assay kit was purchased from Beijing Solarbio Science & 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 Kaiji Biotechnology Co., Ltd.

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

[0054] Example 1

[0055] Preparation of compound CoCl2(Gly):

[0056]

[0057] 16.01 mg (0.12 mmol) of CoCl2 and 104.8 mg (0.12 mmol) of glycyrrhizic acid (Gly) were weighed and dissolved in methanol. The pH was adjusted to 8 with 1 mmol / L sodium hydroxide aqueous solution, and the mixture was sonicated for 20 min to obtain a glycyrrhizic acid-CoCl2 complex, denoted as CoCl2(Gly). Anhydrous CoCl2 showed a significant color change after reacting with glycyrrhizic acid at pH 8, changing from light brown to purple. After the reaction was complete, the mixture was evaporated to dryness. The mass spectrum of the obtained CoCl2(Gly) is shown below. Figure 1 As shown. ESI + -MS(m / e):889.72[M+H] + Mp 270.4~271.6℃; [α] 25 D = +22.3 (c 0.5, H2O).

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

[0059] Example 2

[0060] Preparation of compound NiCl2(Gly):

[0061]

[0062] 8.08 mg (0.06 mmol) of NiCl2 and 51.56 mg (0.06 mmol) of Gly were weighed, dissolved in methanol, and the pH was adjusted to 8 with 1 mmol / L sodium hydroxide aqueous solution. The mixture was sonicated for 20 min to obtain a glycyrrhizic acid-NiCl2 complex, denoted as NiCl2(Gly). Anhydrous NiCl2 showed a significant color change after reacting with glycyrrhizic acid at pH 8, changing from light brown to green. The mixture was then evaporated to dryness after the reaction. The mass spectrum of the obtained NiCl2(Gly) is shown below. 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 complexed with one molecule of nickel chloride.

[0064] Example 3

[0065] Preparation of compound RbNO3(Gly):

[0066]

[0067] 28.2 mg (0.19 mmol) of RbNO3 and 162.2 mg (0.20 mmol) of Gly were weighed, dissolved in methanol, and the pH was adjusted to 8 with 1 mmol / L sodium hydroxide aqueous solution. The mixture was sonicated for 20 min to obtain a glycyrrhizic acid-RbNO3 complex, denoted as RbNO3(Gly). The color of anhydrous RbNO3 changed from colorless to yellow after reacting with glycyrrhizic acid at pH 8. The mixture was then evaporated to dryness after the reaction was complete. The mass spectrum of the obtained RbNO3(Gly) is shown below. Figure 3 As shown. ESI - -MS(m / e):931.16[MH] - Mp 267.9~268.4℃; [α] 25 D = +28.0 (c 0.5, H2O).

[0068] Depend on Figure 3 It can be inferred that two molecules of glycyrrhizic acid 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 compound CoCl2(Gly) is as follows: Figure 5 As shown in the figure. It can be seen that CoCl2(Gly) and compound Gly have different performance at 1700 cm⁻¹. -1 The absorption caused the shift. Therefore, it was determined that the metal formed a complex with the carbonyl group of glycyrrhizic acid.

[0072] The infrared spectrum of compound NiCl2(Gly) is as follows: Figure 6 As shown, it can be seen that NiCl2(Gly) and compound Gly have different performance at 1710 cm⁻¹. -1 The absorption shift caused by the metal indicates that the metal has formed a complex with the carbonyl group at that location.

[0073] The infrared spectrum of compound RbNO3(Gly) is as follows: Figure 7 As shown, it can be seen that RbNO3(Gly) and compound Gly have different performance at 1700 cm⁻¹. -1 The shift caused by absorption proves the formation of complex bonds.

[0074] (2) Nanostructure characterization

[0075] A glass bottle containing a solution was illuminated with a laser pointer (λ = 650 nm), with water used as a control group. The Tyndall effect was observed in each group. The Tyndall effect test results before and after laser irradiation are as follows: 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 The aqueous solutions of M all exhibited a significant Tyndall effect, indicating that the complexes can self-assemble into nanosystems.

[0076] (3) Particle size and potential measurement

[0077] CoCl2(Gly), NiCl2(Gly), and RbNO3(Gly) were prepared using pure water to form 10 -5 The particle size and zeta potential of the aqueous solution of M were measured using a laser nanoparticle size analyzer and DTS (Nano) software at 25°C. 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 sample 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 shown in Table 1, the glycyrrhizic acid metal complexes provided by this invention have a particle size range of 177–250 nm, and exhibit low and stable PDI values. The NiCl2(Gly) was found to have a maximum Zeta potential of -52.2 ± 8.03, demonstrating excellent nanoscale 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. Then, 85 μL of a mixed solution was added, consisting of 100 mM TrisHCl (pH 8.0), 5 mM MgCl2, 200 mM glucose, 0.8 mM ATP, and 1 mM oxidized coenzyme I (NAD). + 0.25 Units of glucose-6-phosphate dehydrogenase (G6P-DH) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (IN0010). The microplate reader was preheated to 37°C. The absorbance was measured at 340 nm every 1 minute for a total of 5 minutes. The IC50 was calculated based on the rate of change of absorbance. 50 .

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

[0085] Table 2. In vitro inhibitory effects of different compounds on hexokinase (n=3, IC50, 1000 mg / kg / dL) 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, the inhibitory activity of NiCl2(Gly) was significantly different from that of glycyrrhizic acid (p<0.01), indicating that the activity of NiCl2(Gly) was increased compared to that of Gly.

[0088] Test Example 2

[0089] In vitro tumor cell anti-proliferation experiment:

[0090] Weigh out 0.1 mmol of each of Gly, CoCl2 (Gly), NiCl2 (Gly), and RbNO3 (Gly), dissolve them in 100 μL of DMSO, and add 9.9 mL of PBS solution to prepare a 10 mM solution. Add 25 μL of this solution to 100 μL of culture medium for cell culture, resulting in a final concentration of 2.5 mM. Dilute according to preliminary experimental results to establish a 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. All four cell types were adherent cells.

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

[0093] Cell resuscitation: Cells frozen in liquid nitrogen were rapidly thawed in a 37°C water bath. The cell suspension was aspirated in a biosafety cabinet and transferred to a centrifuge tube. 5 mL of complete culture medium was added, and the tube was centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and fresh complete culture medium was added. The cells were then transferred to a T25 cell culture flask containing 5 mL of fresh culture medium and cultured in a 37°C, 5% CO2 incubator for 24 h. Cell growth was observed, and the cells were passaged according to the cell number.

[0094] Cell passage: Cells with a healing rate of over 90% can be passaged. Discard the old culture medium, wash with 2 mL of PBS buffer, add 3 mL of trypsin (KGL2101-100, purchased from Jiangsu Kaiji Biotechnology Co., Ltd.), and incubate at 37℃ and 5% CO2 for 3-5 minutes until most cells are in a spherical floating state. Add 3 mL of fresh complete culture medium to stop digestion, gently blow up the cells with a pipette, transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 3 minutes, discard the supernatant, add fresh complete culture medium, transfer to a T25 cell culture flask, and incubate at 37℃ and 5% CO2 for 24 hours. Observe cell growth.

[0095] Seeding: Take adherent cells in the logarithmic growth phase, discard the old culture medium in the culture flask, add 2 mL of PBS buffer to wash, add 3-5 mL of trypsin to the culture flask, and place it in an incubator at 37℃ and 5% CO2 for 3-5 minutes to digest until most cells are in a spherical floating state. Gently blow them up with a pipette and transfer the cell suspension to a centrifuge tube. Set the centrifuge to 1000 rpm and centrifuge for 3 minutes. Remove the supernatant and then add 2 mL of fresh complete culture medium to the tube. Mix well with a sterilized pipette tip and then count the cells.

[0096] The seeding density was 50,000 cells / mL. 100 μL was aspirated from each well and incubated in a 37°C, 5% CO2 cell culture incubator. After 12 hours of culture, the cell adhesion was observed under a microscope. Once all cells had adhered, 25 μL of the prepared test sample was added. Each test sample was prepared in 6 wells, with negative control wells and blank control wells. No drug was administered to the negative control wells; only an equal volume of PBS was added. The blank control wells contained no cells; only culture medium and an equal volume of PBS were added. The wells were incubated in a 37°C, 5% CO2 cell culture incubator for 24 hours. After 24 hours, 25 μL of prepared MTT solution (5 mg / mL) was added to each well, and the plate was incubated for another 4 hours. The culture was then terminated. The 96-well plate was removed, the culture medium was aspirated from the wells, and 150 μL of DMSO was added to each well. The plate was then shaken for 15 minutes on a plate shaker to fully dissolve the formazan crystals in the DMSO. Finally, the absorbance of each well was measured using a multi-mode microplate reader with wavelengths set to 490 nm and 570 nm, and the cell inhibition rate was calculated. This was repeated three times, and the average value was taken.

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

[0098] This 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. The IC50 was calculated using GraphPadPrism8 by fitting a linear regression curve of logarithmic concentration versus cell viability percentage. 50 The results are shown in Table 3.

[0099] Table 3. Antiproliferative effects of compounds on cancer cells (IC50) 50 Mean±SD,mM,n=18)

[0100] Test sample 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] Experimental results showed that both CoCl2 (Gly) and NiCl2 (Gly) could inhibit the proliferation of A549, SW480, MCF-7, and SK-Mel-28 cells, with an IC50 concentration of 100%. 50 The values ​​ranged from 0.42 to 0.82 mM. Compared with glycyrrhizic acid, it exhibited enhanced antiproliferative activity.

[0102] Test Example 3

[0103] Hexokinase activity assay:

[0104] Glycyrrhizic acid, CoCl2(Gly), NiCl2(Gly), and RbNO3(Gly) were used as test samples. The concentrations of CoCl2(Gly) and NiCl2(Gly) were determined based on the measured IC50 values. 50 The drugs were administered at a concentration of 2.5 mM, with glycyrrhizic acid and RbNO3(Gly) administered at 2.5 mM. The normal saline (NS) group received only an equal volume of PBS, while the control group received only deionized water. The experimental method is as follows:

[0105] The cells were seeded in 1 mL plates containing 100,000 cells per well and cultured in a cell culture incubator at 37°C and 5% CO2. After 12 h of culture, the cells adhered and 250 μL of the prepared test sample was added. Each test sample was prepared in 3 wells and a control well was set up. No drug was administered to the control wells, only PBS was added. After culturing in a cell culture incubator at 37°C and 5% CO2 for 48 h, the cells were collected and the cell count was calculated. The assay was performed according to the instructions on the hexokinase activity assay kit (Solepro, China). Collected cells were added to Extraction Buffer 1 at a ratio of 5 million cells: 1 mL, and the cells were lysed using a non-contact DNA disruptor (power: 22.5 kW, time: 3 s working, 7 s interval, total time 10 min). The cells were centrifuged at 8000 g, 4℃ for 10 min, and the supernatant was collected and placed on ice for testing. A mixture was prepared according to the sample volume using Reagent 2A: Reagent 2B: Reagent 2C: Reagent 2D = 100 μL: 500 μL: 150 μL: 150 μL (5T), and prepared fresh before use. Half the prescribed volume was used during the test. The microplate reader was preheated to 37℃, and the absorbance (A1) was measured at 340 nm for 20 s, and the absorbance (A2) was measured after 5 min and 20 s. The inhibition ratio of the tested sample to hexokinase in cells was calculated compared to the control group (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 represents the total number of bacteria or cells, in the tens of thousands.

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

[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: *, p<0.05 compared to the Gly group.

[0114] It can be seen that in MCF-7 cells, RbNO3(Gly) and CoCl2(Gly) exhibited inhibitory effects on hexokinase, with significant differences compared to the NS group. In SW480 and A549 cells, all three metal complexes showed significant inhibitory effects on hexokinase compared to the NS group.

[0115] Test Example 4

[0116] Free radical scavenging experiment:

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

[0118] OH radical determination method: Pipette 5 μL of ferrous sulfate solution into a 0.5 mL EP tube, add 5 μL of DMPO aqueous solution, 5 μL of blank solvent / sample, and 5 μL of 1% hydrogen peroxide aqueous solution. Vortex mix, siphon the sample using a capillary tube, seal with adhesive tape, place in a paramagnetic tube, and test the sample while maintaining the reaction time for 2 min. Repeat six times and obtain the average peak height.

[0119] The scavenging effects of different compounds on free radicals are as follows: Figure 9 As shown in the figure, glycyrrhizic acid did not show a significant inhibitory effect on OH free radicals compared with the blank group. However, the metal complexes (RbNO3(Gly), CoCl2(Gly), and NiCl2(Gly)) showed significant inhibitory effects compared with the blank control group. Among them, the CoCl2(Gly) and NiCl2(Gly) groups showed significant differences compared with glycyrrhizic acid. Therefore, CoCl2 and NiCl2 modification improved the inhibitory activity of glycyrrhizic acid on OH free radicals.

[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 ears of mice. Under its stimulating effect, local capillary congestion is caused, increasing the permeability of the mouse ear and increasing the infiltration of 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±5g, were selected as experimental animals. They were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed at the Laboratory 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 2.39 mg of NiCl2 (Gly) were added to 8 mL of 0.1% sodium carboxymethyl cellulose aqueous solution to prepare suspensions.

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

[0126] The experimental model was established as follows:

[0127] After administering the drug via gavage at a volume of 20g / 0.5mL for 0.5h, apply 20μL of xylene solution evenly to both sides of the right ear of the mouse. After depriving the mouse of water for 2h, collect blood and sacrifice the mouse. Remove both ears, align them, punch holes, weigh the mice, and calculate the swelling rate according to the following formula.

[0128]

[0129] Results of in vivo mouse ear swelling experiment as follows Figure 10 As shown (n=6). Figure 10 In the middle, 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] As can be seen, compared with the NS group, the swelling rates of the Gly, RbNO3(Gly), and CoCl2(Gly) groups were statistically different after administration (P<0.05). Compared with the glycyrrhizic acid Gly group, the CoCl2(Gly) group had a smaller swelling rate, which was statistically different (P<0.05), showing stronger anti-inflammatory activity.

[0131] In summary, this invention synthesized and identified three target compounds, RbNO3(Gly), CoCl2(Gly), and NiCl2(Gly), using glycyrrhizic acid as a modifying compound. The target compounds were characterized by infrared and ultraviolet spectroscopy, confirming their correct structures and exhibiting good nanoscale properties. The antiproliferative activity and hexokinase inhibitory activity of the target compounds against A549, SW480, MCF-7, and SK-Mel-28 cells were evaluated using the MTT assay. CoCl2(Gly) and NiCl2(Gly) showed improved antiproliferative activity against all four cell types, but in A549 (0.38±0.04U / 10⁻⁶), the activity was significantly lower. 4 cell), SW480 (0.25±0.09U / 10) 4cell), MCF-7 (0.762±0.22U / 10) 4 The drug showed inhibitory activity against hexokinase in A549 cells, and was superior to glycyrrhizic acid (1.16±0.17U / 10⁻⁶). 4 Inhibitory effects on hexokinase (cells). CoCl2 (Gly) showed inhibitory effects on hexokinase in SW480 and MCF-7, while NiCl2 (Gly) showed inhibitory effects on hexokinase in MCF-7 (1.18±0.09U / 10). 4 (cell) showed an inhibitory effect on hexokinase.

[0132] Inflammation is closely related to the occurrence, development, and treatment of tumors. In a 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 anti-inflammatory activities. Among them, CoCl2 (Gly) and glycyrrhizic acid showed significant differences.

[0133] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of a glycyrrhizin metal complex in the preparation of an antitumor drug, the glycyrrhizin metal complex having a structure shown in Formula 1 or Formula 2: Formula 1 Formula 2; the antitumor drug being an anti-pancreatic cancer drug. Formula 1; Formula 2; The particle size of the glycyrrhizin metal complex is 177-250 nm.

2. Use according to claim 1, characterized in that, The preparation method of the glycyrrhizin metal complex comprises the following steps:

3. Use according to claim 1, characterized in that, mixing glycyrrhizin, a metal salt and an organic solvent, adjusting the pH value of the obtained mixture to alkaline, and performing a complexation reaction to obtain the glycyrrhizin metal complex. The molar ratio of the glycyrrhizin to the metal salt is 1: (1.0-1.5).

4. Use according to claim 3, characterized in that, The alkaline pH value is 8.

5. Use according to claim 3, characterized in that, The complexation reaction is performed under ultrasonic conditions.

6. Use according to claim 3 or 5, characterized in that, The complexation reaction time is 10-45 min. ​

Images