Northwest licorice isoflavone nanocrystal and preparation method and application thereof, and antitumor drug

Abstract

The present application relates to the technical field of biological medicine, in particular to a kind of northwest licorice isoflavone nanocrystal and its preparation method and application, antitumor drug.The northwest licorice isoflavone used in the present application has significant inhibitory activity on breast cancer, lung cancer and liver cancer cells, and its IC 50 It reaches nanomolar level, more than 5 times higher than soybean isoflavone.For its poor solubility, low bioavailability and drug-making defects, the present application prepares northwest licorice isoflavone nanocrystal by anti-solvent precipitation combined with high-pressure homogenization technology, the particle size is controlled below 200nm, no crystal precipitates within 7 days storage period.In vitro dissolution rate is significantly improved, cell uptake and internalization efficiency are enhanced.In vivo pharmacodynamic evaluation shows that the antitumor efficacy of northwest licorice isoflavone nanocrystal is comparable to that of doxorubicin, while the raw material is only moderately inhibited;Biological safety of tumor-bearing mouse model is better than that of doxorubicin, and the northwest licorice isoflavone nanocrystal has significant clinical transformation potential.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a type of Northwest licorice isoflavone nanocrystals, its preparation method and application, and antitumor drugs. Background Technology

[0002] Tumors are new growths formed under the long-term influence of various tumorigenic factors, resulting from alterations in the genetic material of local tissue cells and abnormal gene expression. These growths, characterized by "autonomous" excessive growth, produce progeny cells through a genetic process, and are essentially genetic abnormalities. Traditional cancer treatments primarily include surgery, radiotherapy, and chemotherapy. However, surgery and radiotherapy are ineffective in treating metastatic lesions, while chemotherapy generally suffers from low bioavailability, significant toxic side effects, poor targeting, and a high risk of multidrug resistance. Therefore, developing safe and highly effective targeted anti-tumor drugs remains a major need in the field of drug development.

[0003] Natural products, due to their wide availability, structural diversity, multi-target and multi-pathway action, and relatively low toxicity, have become an important source for anti-tumor drug development. Modern research indicates that approximately 50% of clinical anti-tumor drugs are derived directly or indirectly from natural products, encompassing a variety of chemical components such as alkaloids, polysaccharides, polyphenols, and terpenes. Isoflavones, as an important class of plant secondary metabolites, possess broad pharmacological activities. Soy isoflavones are representative members of the isoflavone family, and early clinical trials have been conducted targeting solid tumors such as breast cancer, endometrial cancer, colon cancer, and rectal cancer.

[0004] However, current research on isoflavones in the field of antitumor activity still has many shortcomings, specifically: First, the application of isoflavones with specific structures in the antitumor activity of breast cancer, lung cancer, and liver cancer remains a blank. Existing research mainly focuses on exploring the antitumor activity of common isoflavone monomers such as soy isoflavones and daidzein, while there are no reports or technological insights to date regarding the antitumor activity, mechanism of action, and uses of licorice isoflavones in these three cancers. Second, the known antitumor activity intensity and selectivity of isoflavones are insufficient. Taking soy isoflavones as an example, although they have entered the early clinical research stage, preclinical and clinical research data indicate that the antitumor activity of soy isoflavones is not ideal, and there is generally a half-maximal inhibitory concentration (IC50). 50The high concentration of certain substances and limited inhibitory effects on tumor cell growth make it difficult to meet the clinical demand for highly effective anti-tumor drugs. Third, isoflavones generally suffer from severe drug-like defects. Most isoflavones, such as soy isoflavones, have inherent defects such as poor solubility, low oral absorption, rapid in vivo metabolism, and insufficient bioavailability, which seriously restrict their clinical translation and formulation development. In summary, there is an urgent need in this field to develop a novel anti-tumor drug that can effectively treat breast cancer, lung cancer, and / or liver cancer, and possesses significant activity, high selectivity, and good drug-like properties. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide Northwest Glycyrrhiza Isoflavone Nanocrystals, their preparation method and applications, and anti-tumor drugs. This invention is the first to discover the excellent activity of the specific isoflavone compound Northwest Glycyrrhiza Isoflavone in the treatment of breast cancer, lung cancer and liver cancer, and to address its poor solubility and low bioavailability in drug formulation, a systematic breakthrough has been achieved through nanocrystal formulation technology.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of licorice isoflavones in the preparation of antitumor drugs, wherein the tumor includes one or more of breast cancer, lung cancer, and liver cancer.

[0007] This invention provides a method for preparing licorice isoflavone nanocrystals, comprising the following steps: Northwest licorice isoflavones were dissolved in an organic solvent to obtain an organic phase; The organic phase was mixed with an aqueous phase containing a stabilizer and subjected to high-pressure homogenization to obtain Northwest licorice isoflavone nanocrystals.

[0008] Preferably, the mass ratio of the licorice isoflavones to the stabilizer is 1:0.001~0.01.

[0009] Preferably, the concentration of the organic phase is 4~20 g / L; The organic solvent includes one or more of the following: alcohol solvents, ether solvents, ketone solvents, ester solvents, sulfur-containing compound solvents, and amide solvents.

[0010] Preferably, the alcohols include methanol and / or ethanol; The ether solvents include tetrahydrofuran and / or methyl tert-butyl ether; The ketone solvent includes acetone; The ester solvent includes ethyl acetate; The sulfur-containing compound includes dimethyl sulfoxide; The amide solvents include dimethylformamide.

[0011] Preferably, the stabilizer comprises one or more of sodium dodecyl sulfate, poloxamer, povidone, hydroxypropyl methylcellulose, carboxymethyl cellulose, and F127.

[0012] Preferably, the high-pressure homogenization process involves a pressure of 500~3000 bar, 5~20 cycles, and a temperature of -5~60℃.

[0013] The present invention also provides Northwest Glycyrrhiza Isoflavone Nanocrystals prepared by the preparation method described above, wherein the Northwest Glycyrrhiza Isoflavone Nanocrystals have an average particle size of 50~300nm, a polydispersity index of 0.1~0.25, and an absolute value of Zeta potential of -5~25mV.

[0014] The present invention also provides the application of the Northwest Glycyrrhiza Isoflavone Nanocrystals described in the above technical solution in the preparation of antitumor drugs, wherein the tumor includes one or more of breast cancer, lung cancer and liver cancer.

[0015] The present invention also provides an antitumor drug comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises Northwest licorice isoflavone raw material and / or Northwest licorice isoflavone nanocrystals as described in the above technical solution; the antitumor drug comprises a drug for treating one or more tumors including breast cancer, lung cancer and liver cancer.

[0016] Existing technologies (such as "Research Progress on the Preparation and Application of Flavonoid Nanocrystals") summarize general preparation strategies for flavonoid nanocrystals (such as NanoEdge technology, i.e., antisolvent precipitation combined with high-pressure homogenization, and the addition of stabilizers such as sodium dodecyl sulfate (SDS) or D-α-tocopherol polyethylene glycol 1000 succinate (TPGS)). However, they neither address the preparation of flavonoid nanocrystals from galangal, nor disclose any use of this type of compound in the treatment of specific tumors. This invention is the first to discover a novel use of a specific structural isoflavone compound—galangal—in the treatment of specific tumors (breast cancer, lung cancer, liver cancer). Furthermore, addressing the physicochemical properties of this compound, nanocrystal formulation technology overcomes its drug-like defects of poor solubility and low bioavailability.

[0017] Compared with the prior art, the advantages of the present invention are reflected in the following three aspects: Compound activity advantages. The compound involved in this invention, Northwest Glycyrrhiza Isoflavone, itself possesses superior in vitro antitumor activity. In vitro cytotoxicity experiments (CCK-8 assay) show that Northwest Glycyrrhiza Isoflavone has an IC50 value against specific tumor cells. 50 It reaches the nanomolar level and has more than 5 times the activity of known isoflavone control compounds (such as soy isoflavones).

[0018] Formulation advantages. The Northwest Glycyrrhiza Isoflavone Nanocrystals prepared by this invention significantly improve the solubility, stability, and cellular uptake efficiency of the drug. In vitro dissolution rate is significantly enhanced; cellular uptake experiments show that these Northwest Glycyrrhiza Isoflavone Nanocrystals can be effectively internalized by tumor cells, significantly improving the drug's cell penetration and internalization efficiency, and effectively improving the intracellular accumulation behavior of the drug.

[0019] Advantages in in vivo efficacy and safety. In in vivo antitumor pharmacodynamic evaluation, the antitumor efficacy of the Northwest Glycyrrhiza Isoflavone Nanocrystals prepared in this invention was comparable to that of the positive control, doxorubicin, while the un-nanocrystalized Northwest Glycyrrhiza Isoflavone raw material only showed a moderate inhibitory effect. Live and dead staining experiments showed that Northwest Glycyrrhiza Isoflavone Nanocrystals significantly enhanced the proportion of late apoptosis and cell death in tumor cells, which is closely related to the improvement of intracellular drug accumulation by Northwest Glycyrrhiza Isoflavone Nanocrystals. Regarding safety, in tumor-bearing mouse models, the Northwest Glycyrrhiza Isoflavone Nanocrystal group showed stable body weight, no pathological damage to major organs, and no abnormalities in hematological and liver / kidney function indicators, demonstrating significantly better biosafety than the doxorubicin group.

[0020] Furthermore, this invention innovatively optimizes the nanocrystal preparation process and stabilizer system for the physicochemical properties of licorice isoflavones, rather than simply applying the commonly used antisolvent precipitation combined with high-pressure homogenization and SDS stabilizers in existing technologies. In existing technologies, when preparing flavonoid nanocrystals using NanoEdge technology, the type and amount of stabilizer mainly affect the physicochemical stability of the suspension during storage. However, this invention, through innovative design of the stabilizer system, not only significantly improves the storage stability of the nanocrystals over a certain period, but more importantly, it significantly improves the in vivo release behavior and tumor targeting of the drug. Specifically, this invention precisely controls the average particle size of Northwest Glycyrrhiza Isoflavone nanocrystals to below 200 nm by precisely controlling key parameters such as stabilizer type, stabilizer dosage, homogenization pressure, and number of cycles. Through systematic screening of various stabilizers (SDS, TPGS, hydroxypropyl methylcellulose, polyvinylpyrrolidone, etc.), a preferred stabilizer combination matching Northwest Glycyrrhiza Isoflavone was identified, ensuring no crystal precipitation in the Northwest Glycyrrhiza Isoflavone nanocrystal suspension during a 7-day storage period. This is significantly superior to the shortcomings of conventional stabilizers in existing technologies, which are unable to effectively inhibit the aggregation and crystal growth of nanocrystals of this type of compound. The comprehensive advantages brought about by the above process improvements are: the in vitro dissolution rate of Northwest Glycyrrhiza Isoflavone nanocrystals is significantly improved compared to the raw drug, which is beneficial to drug absorption; after nano-sizing, the in vivo antitumor effect of Northwest Glycyrrhiza Isoflavone is further enhanced compared to the raw drug, with tumor-suppressing efficacy comparable to doxorubicin; at the same time, the Northwest Glycyrrhiza Isoflavone nanocrystal formulation does not increase additional toxicity while improving efficacy, and no abnormalities were observed in the body weight and major organ function indicators of tumor-bearing mice.

[0021] This invention represents a systematic breakthrough, from the discovery of the therapeutic effects of licorice isoflavones on specific tumors, to the innovation of nanocrystalline formulations, and then to application verification. It demonstrates comprehensive advantages in terms of formulation stability, anti-tumor efficacy, and safety, and has significant potential for clinical translation. Attached Figure Description

[0022] Figure 1 The images show the preparation and characterization of Northwest Glycyrrhiza Isoflavone Nanocrystals. A is a schematic diagram of the preparation process; B is a particle size distribution diagram; C is the zeta potential measurement result; D is a stability study diagram; E is the Fourier Transmission Infrared (FTIR) spectrum of the Northwest Glycyrrhiza Isoflavone Nanocrystals and the active pharmaceutical ingredient; F is a Transmission Electron Microscopy (TEM) image; and G is a comparison of the X-ray Diffraction (XRD) spectra of the Northwest Glycyrrhiza Isoflavone Active Pharmaceutical Ingredient and the Nanocrystals. Figure 2 Different isoflavone samples (soy isoflavones, licorice isoflavones, Northwest licorice isoflavone raw materials, Northwest licorice isoflavone nanocrystals) and doxorubicin hydrochloride media were compared for 4T1 cells. 50 ) Measurement results graph; Figure 3 Figure 1 shows the results of 4T1 cell live / dead staining experiments on Northwest Glycyrrhiza Isoflavone Nanocrystals and Northwest Glycyrrhiza Isoflavone Raw Materials. Figure 4 Figure 4T1 cell uptake results of Northwest Glycyrrhiza Isoflavone Nanocrystals and Northwest Glycyrrhiza Isoflavone Raw Material. Figure 5 The figures show the 4T1 cell uptake results of Northwest Glycyrrhiza Isoflavone Nanocrystals and Northwest Glycyrrhiza Isoflavone Raw Materials. A is a schematic diagram of the 4T1 cell seeding, administration, and material collection process; B shows the tumor volume change curves of each group of mice over time; C shows the mouse body weight change curves of each group over time; D shows the tumor weight statistics of each group at the experimental endpoint; E shows the actual images of the dissected tumors of each group; F shows the Ki-67 and YBX-1 immunohistochemical staining results of the tumor tissues of each group; and G shows the HE staining results of the tumor tissues of each group. Figure 6 The figure shows the effects of Northwest Glycyrrhiza Isoflavone Nanocrystals and Northwest Glycyrrhiza Isoflavone Raw Materials on the morphology of major organs, blood routine tests, and liver and kidney function indicators of mice. In the figure, A represents the HE staining results of major organs of mice in each group; B represents the blood routine test results of mice in each group; and C represents the test results of liver and kidney function-related biochemical indicators of mice in each group. Detailed Implementation

[0023] This invention provides the application of Northwest Glycyrrhiza Isoflavones in the preparation of antitumor drugs, wherein the tumor includes one or more of breast cancer, lung cancer, and liver cancer. In this invention, the Northwest Glycyrrhiza Isoflavones (structural formula shown in Formula I) comprises Northwest Glycyrrhiza Isoflavone raw material and / or Northwest Glycyrrhiza Isoflavone nanocrystals.

[0024] Formula I.

[0025] This invention provides a method for preparing licorice isoflavone nanocrystals, comprising the following steps: Northwest licorice isoflavones were dissolved in an organic solvent to obtain an organic phase; The organic phase was mixed with an aqueous phase containing a stabilizer and subjected to high-pressure homogenization to obtain Northwest licorice isoflavone nanocrystals.

[0026] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0027] In this invention, the organic solvent may include one or more of alcohol solvents, ether solvents, ketone solvents, ester solvents, sulfur-containing compound solvents, and amide solvents; the alcohol may include methanol and / or ethanol; the ether solvent may include tetrahydrofuran and / or methyl tert-butyl ether; the ketone solvent may include acetone; the ester solvent may include ethyl acetate; the sulfur-containing compound may include dimethyl sulfoxide (DMSO); and the amide solvent may include dimethylformamide. In this invention, the concentration of the organic phase may be 4-20 g / L, or 6-18 g / L, or further 8-16 g / L, specifically 10 g / L, 12 g / L, or 14 g / L.

[0028] In this invention, the stabilizer may include one or more of sodium dodecyl sulfate (SDS), poloxamer, povidone, hydroxypropyl methylcellulose, carboxymethyl cellulose, and F127; the poloxamer may include poloxamer 188. In this invention, the solvent in the aqueous phase is water; the concentration of the stabilizer in the aqueous phase may be 0.1~1 w / v%, or 0.5~1 w / v%, specifically 0.1 w / v%, 0.2 w / v%, 0.3 w / v%, 0.4 w / v%, 0.5 w / v%, 0.6 w / v%, 0.7 w / v%, 0.8 w / v%, 0.9 w / v%, or 1 w / v.

[0029] In this invention, the mass ratio of the licorice isoflavones to the stabilizer can be 1:0.001~0.01, or 1:0.005~0.01, specifically 1:0.001, 1:0.002, 1:0.003, 1:0.004, 1:0.005, 1:0.006, 1:0.007, 1:0.008, 1:0.009 or 1:0.01.

[0030] In this invention, the pressure of the high-pressure homogenization process can be 500~3000 bar, or 1000~2500 bar, or even 1500~2000 bar; the number of cycles of the high-pressure homogenization process can be 5~20 times, or 8~18 times, or even 10~15 times; the temperature of the high-pressure homogenization process can be -5~30℃, or -5~20℃, or even 5~10℃, or specifically 0℃, 4℃, 10℃ or 15℃; the high-pressure homogenizer used for the high-pressure homogenization process can be a PandaPlus 2000 (GEA).

[0031] After completing the high-pressure homogenization treatment, the present invention may further include: performing solid-liquid separation on the system after high-pressure homogenization, washing the obtained solid component with water to obtain licorice isoflavone nanocrystals. In this invention, the solid-liquid separation may include centrifugation, the centrifugation speed may be 5000~20000 rpm, or 10000~15000 rpm, specifically 12000 rpm; the centrifugation time may be 1~20 min, or 5~15 min, specifically 10 min. In this invention, the water washing may be deionized water washing, and the number of water washings may be 1~3 times, specifically 2 times. In this invention, the Northwest Glycyrrhiza Isoflavone Nanocrystals can be dispersed in a solvent for storage; the solvent may include water or phosphate buffered saline (PBS), the pH of the PBS may be 5-8, or 6-7.8, or further 7-7.6, or specifically 7.4; the concentration of the PBS may be 1-200 mM (mmol / L), or 5-100 mM, or further 10-50 mM, or specifically 10 mM.

[0032] In this invention, the washing process further includes dialysis of the washed nanocrystals to obtain Northwest licorice isoflavone nanocrystals. In this invention, the dialysis may include purified water dialysis; the molecular weight cutoff of the dialysis bag used for dialysis may be 500-3000 Da, or 1000-3000 Da, or further 1500-2500 Da, specifically 2000 Da; the dialysis time may be 8-24 hours, or 10-20 hours, specifically 12 hours, 14 hours, 16 hours, or 18 hours; during the dialysis process, the purified water is replaced every 2-6 hours (or 3-5 hours, specifically 4 hours).

[0033] This invention also provides Northwest Glycyrrhiza Isoflavone Nanocrystals prepared by the method described in the above technical solution. The average particle size of the Northwest Glycyrrhiza Isoflavone Nanocrystals is 50-300 nm, and can also be 100-250 nm, or further 150-200 nm; the polydispersity index (PDI) of the Northwest Glycyrrhiza Isoflavone Nanocrystals is 0.1-0.25, and can also be 0.15-2; the absolute value of the Zeta potential of the Northwest Glycyrrhiza Isoflavone Nanocrystals is -5-25 mV, and can also be 0-20 mV, or further 5-15 mV, or specifically 10 mV. The saturated solubility of the Northwest Glycyrrhiza Isoflavone Nanocrystals prepared by this invention is 10-500 times higher than that of the Northwest Glycyrrhiza Isoflavone raw material.

[0034] The present invention also provides the application of the Northwest Glycyrrhiza Isoflavone Nanocrystals described in the above technical solution in the preparation of antitumor drugs, wherein the tumor includes one or more of breast cancer, lung cancer and liver cancer.

[0035] The present invention also provides an antitumor drug comprising an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient comprises Northwest licorice isoflavone raw material and / or Northwest licorice isoflavone nanocrystals as described in the above technical solution; the antitumor drug comprises a drug for treating one or more tumors including breast cancer, lung cancer and liver cancer.

[0036] This invention is the first to discover the excellent activity of Northwest Glycyrrhiza Isoflavone Nanocrystals in the treatment of specific tumors (breast cancer, lung cancer, and liver cancer), and significantly improves the solubility and bioavailability of the compound through nanocrystal formulation technology. It provides a novel technical solution to solve the insufficient anti-tumor activity and formulation defects of isoflavones in the prior art, and has significant clinical application value.

[0037] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] In the following examples, the high-pressure homogenizer is a PandaPlus 2000, GEA.

[0039] Human tumor cell lines (such as MCF-7, A549, HepG2) and non-tumorigenic cell lines (such as MCF-10A) were purchased from the American Type Culture Collection (ATCC).

[0040] In the following examples, the Northwest Glycyrrhiza Isoflavone API was purchased from Beterenkang.

[0041] Example 1 Preparation and characterization of Northwest Glycyrrhisoflavone Nanocrystals (denoted as Glycyrrhisoflavone-NC).

[0042] according to Figure 1 The process flow diagram shown in Figure A illustrates the preparation of Northwest Glycyrrhiza Isoflavone Nanocrystals using an antisolvent precipitation-high pressure homogenization method. The steps are as follows: 20 mg of Northwest Glycyrrhiza Isoflavone raw material is dissolved in 2 mL of dimethyl sulfoxide (DMSO) to obtain an organic phase (Northwest Glycyrrhiza Isoflavone Solution). Under magnetic stirring at 1000 rpm, this organic phase is rapidly injected into 20 mL of a 1 w / v% poloxamer 188 aqueous solution. The resulting pre-suspension is homogenized for 15 cycles using a high-pressure homogenizer at 15 ± 1 °C and 1500 bar. The resulting nanocrystal suspension is centrifuged at 12000 rpm for 10 min, washed twice with deionized water to remove free stabilizer and DMSO, and dialyzed against purified water in a dialysis bag (molecular weight cutoff of 1000 Da) for 6 h to remove the organic solvent. The resulting precipitate is resuspended in phosphate-buffered saline (PBS, pH=7.4) for later use, yielding a uniformly dispersed Northwest Glycyrrhiza Isoflavone Nanocrystal suspension (concentration 10 mg / mL).

[0043] Particle size, polydispersity index (PDI), and zeta potential were determined using a Zetasizer Nano ZS (Malvern Instruments) dynamic light scattering (DLS). Morphology was observed using transmission electron microscopy (TEM, Hitachi HT7800) after 2% uranium acetate negative staining. Crystal state was analyzed using powder X-ray diffraction (PXRD, Bruker D8 Advance) and differential scanning calorimetry (DSC, TA Instruments Q2000). Physical stability was assessed by monitoring changes in particle size and PDI of the suspension stored at 4°C for 30 days. Experimental results are shown below. Figure 1 Data are expressed as mean ± SD.

[0044] DLS results showed that the isoflavones (NPs) from Northwest Glycyrrhiza uralensis exhibited a unimodal particle size distribution, with no obvious large particle aggregation and uniform dispersion. Figure 1 (B). Zeta potential results show that the nanocrystal surface carries a negative charge ( Figure 1 (C). After storage at 4℃ for 7 days, the particle size and PDI of the Northwest Glycyrrhiza Isoflavone Nanoparticle Suspension did not show a significant increase or drastic fluctuation. Figure 1 The presence of TEM (dielectric density) indicates that the formulation exhibits good physical stability under low-temperature storage conditions, effectively preventing particle aggregation, sedimentation, or crystal growth. TEM morphology observations further validated the formation of nanocrystals. FTIR (Flat Fiber Optic Resonance) images... Figure 1The TEM results show that the characteristic absorption peak positions of the two are basically the same, indicating that the preparation of nanocrystals did not change the main chemical structure of glycyrrhizin isoflavones. Compared with the raw material, the characteristic peak absorption intensity and peak shape of the Northwest glycyrrhizin isoflavone nanocrystals are significantly different, and significant inverted peaks appear in the spectrum. This is mainly due to the light scattering effect caused by the reduction of nanocrystal particle size and the baseline fluctuation caused by the sample preparation method. The infrared spectrum results confirm that the Northwest glycyrrhizin isoflavone nanocrystals were successfully prepared. Figure 1 The XRD results showed that after negative staining with 2% uranium acetate, the NPs of Northwest Glycyrrhiza Isoflavones were distributed in nanoscale particles with relatively regular spherical morphology and good particle dispersion. Furthermore, the particle size observed under TEM was generally slightly smaller than the hydrated particle size measured by DLS. This is because DLS detects the hydrated diameter of the particles in solution, while TEM reflects the actual morphology in the dry state. XRD results were used to further analyze the crystal state of the drug after nano-sizing. In the XRD results, the Northwest Glycyrrhiza Isoflavone raw material showed obvious crystal diffraction peaks, while the Northwest Glycyrrhiza Isoflavone NPs still retained some characteristic diffraction peaks, but the peak intensity and shape changed. Figure 1 The presence of G indicates that the nano-sizing process did not completely destroy the drug crystal structure, but may have reduced the crystallinity.

[0045] Test Example 1 Cell culture: Human tumor cell lines (MCF-7, A549, HepG2) and non-tumorigenic cell line (MCF-10A). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin / streptomycin in a humidified incubator at 37°C and 5% CO2. All cell lines were routinely tested for mycoplasma contamination, and cells up to passage 20 were used.

[0046] 1. Evaluation of the in vitro antitumor activity of licorice isoflavones from Northwest China Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo) kit. Cells were counted at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells / well in 96-well plates. After 24 h of culture, cells were treated with serially diluted Northwest Glycyrrhiza Isoflavone raw material or Northwest Glycyrrhiza Isoflavone nanocrystals for 48 h. CCK-8 reagent was added to each well, and cells were incubated for 2 h. The absorbance at 450 nm was measured using a BioTek Synergy H1 microplate reader. The half-maximal inhibitory concentration (IC50) was calculated using a nonlinear regression model (log(inhibitor) vs. normalized response) in GraphPad Prism 9.0 software. 50 ).

[0047] Experimental groups: Doxorubicin hydrochloride group, Northwest licorice isoflavone group, Northwest licorice isoflavone nanocrystal group, soy isoflavone group, and licorice isoflavone group. Drug dissolution method: Dissolved in 1 mL of DMSO, diluted to the prescribed concentration using culture medium, with concentrations of 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, and 160 μg / mL, respectively.

[0048] Depend on Figure 2 The results showed that the IC50 of the soy isoflavone group was... 50 The concentration was 23.92 μg / mL, indicating a certain inhibitory effect on the proliferation of these cells. The IC50 of glycyrrhizin isoflavones... 50 The concentration was 9.335 μg / mL, which significantly enhanced the inhibitory effect on cell proliferation compared to soybean isoflavones. The Northwest licorice isoflavone group exhibited a good dose-dependent inhibitory effect, with an IC50 concentration of 9.335 μg / mL. 50 The concentration was 9.335 μg / mL. The Northwest Glycyrrhiza Isoflavone Nanocrystals group exhibited a better dose-dependent inhibitory effect, with an IC50 concentration of 9.335 μg / mL. 50 The concentration was 8.331 μg / mL. Doxorubicin hydrochloride induced increased cell death at relatively low concentrations, with an IC50 value of 8.331 μg / mL. 50 The concentration was 5.252 μg / mL, exhibiting a strong inhibitory effect on the proliferation of these cells. Compared with doxorubicin hydrochloride, the IC50 of Northwest Glycyrrhiza Isoflavone Nanocrystals was significantly higher. 50 The concentration required for 50% inhibition of the effect by Northwest licorice isoflavone nanocrystals is slightly higher, under the same treatment time, and its cytotoxicity is slightly lower than that of doxorubicin hydrochloride. Doxorubicin hydrochloride, as a commonly used chemotherapy drug in clinical practice, usually has a strong cytotoxic effect, but its non-specific toxic side effects are also an important factor limiting its application. Although the IC50 of Northwest licorice isoflavone nanocrystals is slightly higher... 50 While exhibiting higher activity than doxorubicin hydrochloride, it still demonstrated clear concentration-dependent anti-proliferative activity, indicating that the Northwest Glycyrrhiza isoflavone nanocrystal formulation can effectively exert the drug's cytotoxic effects. Furthermore, the Northwest Glycyrrhiza isoflavone nanocrystal system may offer potential advantages for subsequent delivery and in vivo antitumor applications by improving drug dispersibility, stability, and cellular uptake behavior. Therefore, Northwest Glycyrrhiza isoflavone nanocrystals hold significant research value for further studies on apoptosis detection, cellular uptake, and in vivo efficacy evaluation.

[0049] 2. Live and dead staining imaging Apoptosis was detected using the Annexin V-FITC / PI apoptosis detection kit (BD Biosciences). Cells were analyzed using their respective IC50 values. 50Cells were treated with concentrated Northwest Glycyrrhiza Isoflavone raw material or Northwest Glycyrrhiza Isoflavone nanocrystals for 24 h. Cells were then collected, washed with PBS, and stained with Annexin V-FITC and propidium iodide (PI) according to the manufacturer's instructions. Stained cells were immediately analyzed using confocal staining, and data were processed using FlowJo V10 software.

[0050] Annexin V-FITC can bind to phosphatidylserine residues that have everted in early apoptotic cells, exhibiting green fluorescence; PI can enter late apoptotic or necrotic cells with impaired cell membrane integrity, producing red fluorescence upon binding to nucleic acids. Therefore, enhanced green fluorescence usually indicates increased early apoptosis, while increased red fluorescence or yellow / orange superimposed signal indicates elevated levels of late apoptosis or cell death. From confocal imaging results ( Figure 3 As can be seen, the control group cells mainly exhibited green fluorescence signals, while the PI red fluorescence was weak and fewer in number, indicating that the untreated cells were in good overall condition and their cell membrane integrity was basically maintained. The number of PI-positive cells in the Northwest Glycyrrhiza Isoflavone solution group increased compared to the control group, and some red and yellow / orange superimposed signals were visible in the Merge plot, indicating that the Northwest Glycyrrhiza Isoflavone raw material could induce a certain degree of apoptosis or cell death. In contrast, the Northwest Glycyrrhiza Isoflavone nanocrystal group showed a significantly enhanced red PI fluorescence signal, and a significant increase in red and yellow / orange positive cells in the Merge plot, indicating that the cell membrane integrity was more severely damaged after treatment with Northwest Glycyrrhiza Isoflavone nanocrystals, and the proportion of late-stage apoptotic / dead cells increased. These results indicate that both Northwest Glycyrrhiza Isoflavone raw material and Northwest Glycyrrhiza Isoflavone nanocrystals can induce apoptosis, but the nanocrystal formulation has a more significant pro-apoptotic / pro-cell death effect. This may be related to the fact that nanocrystals improve drug dispersibility, enhance cell contact and uptake, and increase intracellular drug accumulation.

[0051] 3. Cellular uptake research Experimental groups: Northwest Glycyrrhiza Isoflavone Raw Material Group and Northwest Glycyrrhiza Isoflavone Nanocrystal Group.

[0052] Qualitative uptake: 100 μL of Northwest Glycyrrhiza Isoflavone Nanocrystals and Northwest Glycyrrhiza Isoflavone Raw Material were incubated with 10 μL of Coumarin-6 for 1 h, followed by dialyzing in a dialysis bag (molecular weight cutoff of 1000 Da) for 6 h to obtain Coumarin-6 labeled Northwest Glycyrrhiza Isoflavone and Northwest Glycyrrhiza Isoflavone Nanocrystals. 4T1 cells were co-incubated with Coumarin-6 labeled Northwest Glycyrrhiza Isoflavone and Northwest Glycyrrhiza Isoflavone Nanocrystals for 1 h, 2 h, and 4 h. Cells were then washed, fixed with paraformaldehyde solution (4%, wt / vol), and cell nuclei were stained with DAPI. Images were acquired using a confocal laser scanning microscope (CLSM, Leica TCS SP8). Quantitative analysis: After trypsin digestion and washing, the mean fluorescence intensity (MFI) of the similarly treated cells was measured by flow cytometry.

[0053] Figure 4 Confocal imaging results showed that almost no green fluorescence signal was observed in the control group. After co-incubation with coumarin-6-labeled glycyrrhiza isoflavone nanocrystals, the intracellular green fluorescence signal gradually increased, mainly distributed in the cytoplasm and around the nucleus. As the incubation time was extended from 1 h to 2 h and 4 h, the green fluorescence intensity increased significantly. In the Merge plot, the green signal highly overlapped with the blue area around the nucleus, suggesting that the glycyrrhiza isoflavone nanocrystals can be effectively taken up by cells and exhibit obvious time-dependent uptake characteristics.

[0054] 4. Evaluation of in vivo antitumor efficacy and safety Female BALB / c nude mice (5-6 weeks old) were subcutaneously inoculated with 4T1 cells until the tumor volume reached approximately 100 mm. 3 At the time, the subjects were randomly divided into four groups (n=6): (1) solvent control group (physiological saline), (2) Northwest licorice isoflavone solution group (raw material), (3) Northwest licorice isoflavone nanocrystal group, and (4) positive control group (doxorubicin). The subjects were administered via tail vein on days 0, 3, 6, 9, and 12 after inoculation, and were sacrificed on day 14 for sampling. The subjects were administered intravenously every 3 days for a total of 5 times. The tumor volume and body weight were measured every other day. At the end of the experiment, the tumors and major organs were collected for histopathological (H&E staining) and immunohistochemical (Ki-67, TUNEL) analysis. In terms of safety, serum biochemical indicators (ALT, AST, BUN, CRE) and hematological parameters were analyzed, and H&E staining sections of organs such as the heart, liver, spleen, lungs, and kidneys were examined.

[0055] The results of the drug efficacy are as follows Figure 5 As shown, Figure 5 In section A, the drug efficacy experimental protocol is used. Figure 5 Figure B shows the tumor volume results at different time points during the drug administration process. The tumor volume growth of the Northwest Glycyrrhiza Isoflavone Nanocrystal group was significantly inhibited, while the solution group only showed moderate inhibition. Figure 5 In the middle C, the mouse body weight results were shown at different time points during the drug administration process. The body weight of mice in the Northwest Glycyrrhiza Isoflavone Nanocrystal group did not decrease significantly. Figure 5 The scatter plot (D) represents the statistical distribution of tumor weight at the experimental endpoint (day 14). The results showed that the average tumor weight in the Northwest Glycyrrhiza Isoflavone Nanocrystal group was approximately 0.21 g, significantly lower than that in the solvent control group (0.87 g, P < 0.001) and the Northwest Glycyrrhiza Isoflavone Solution group (0.58 g, P < 0.01), and showed no statistically significant difference compared to the doxorubicin hydrochloride group (0.20 g) (P > 0.05). This further confirms that the Northwest Glycyrrhiza Isoflavone Nanocrystals possess tumor-suppressing efficacy comparable to the positive control drug. This indicates that the nanocrystal formulation has a stronger tumor-suppressing ability in vivo. Figure 5The image in Figure E shows a visual representation of the tumors in each group. Both the Northwest Glycyrrhiza Isoflavone Solution and the Northwest Glycyrrhiza Isoflavone Nanocrystals showed good tumor inhibition, with the Northwest Glycyrrhiza Isoflavone Nanocrystals exhibiting stronger inhibitory effects. Figure 5 The results of immunohistochemistry of tumor tissue are shown in Figure F. The expression levels of Ki67 and YBX-1 proteins were reduced in the Northwest Glycyrrhiza Isoflavone Nanocrystals, indicating that they have a good tumor-suppressive effect. Figure 5 The results of H&E staining of tumor tissue in the middle G also show the tumor inhibitory effect of the Northwest Glycyrrhiza Isoflavone Nanocrystals.

[0056] H&E staining results of major organs as follows Figure 6 As shown in Figure A, H&E staining was performed on major organs such as the heart, liver, spleen, lungs, and kidneys for observation. In the blank control group, the tissue structures of all organs were basically intact, with no obvious pathological damage. Compared with the solvent control group, no obvious abnormal changes such as inflammatory cell infiltration, tissue necrosis, hemorrhage, edema, or structural destruction were observed in any of the major organs in both the Northwest Glycyrrhiza Isoflavone Solution Group and the Northwest Glycyrrhiza Isoflavone Nanocrystal Group. Specifically, the myocardial fibers were arranged relatively regularly, with no obvious myocardial rupture or degeneration; the hepatic cords in the liver tissue were clearly arranged, and the hepatocyte morphology was basically normal, with no obvious hepatocyte necrosis or fatty degeneration; the red and white pulp structures of the spleen tissue were distinguishable, with no obvious abnormal proliferation or atrophy; the alveolar structure was intact, with no obvious alveolar wall thickening, inflammatory exudation, or hemorrhage; the glomeruli and renal tubules were relatively intact, with no obvious renal tubular epithelial cell degeneration or necrosis. These results indicate that Northwest Glycyrrhiza Isoflavone Nanocrystals, after repeated intravenous administration, did not cause significant histopathological damage to major organs and have good tissue compatibility. Data are expressed as mean ± SD.

[0057] Further testing of peripheral blood routine indicators was conducted to evaluate the effects of Northwest Glycyrrhiza Isoflavone Nanocrystals and Raw Materials on the hematopoietic system and immune status. Figure 6 The results of the B-cell assay showed that the white blood cell, red blood cell, hemoglobin, platelet, and related red blood cell parameters in each treatment group remained relatively stable. No significant abnormal fluctuations were observed in the Northwest Glycyrrhiza Isoflavone Nanocrystal group compared to the model group or the control group. These results indicate that administration of Northwest Glycyrrhiza Isoflavone Nanocrystals did not cause significant bone marrow suppression, anemia, or thrombocytopenia, or other hematological toxicities.

[0058] Further evaluation of liver and kidney function safety, Figure 6 The results showed that ALT and AST levels in the Northwest Glycyrrhiza Isoflavone Nanocrystal group did not show significant abnormal increases, suggesting that it did not cause significant hepatocellular damage or abnormal liver function. Meanwhile, BUN and CRE levels remained within the relatively normal range, indicating that the Northwest Glycyrrhiza Isoflavone Nanocrystal did not cause significant renal impairment.

[0059] Therefore, no obvious pathological changes were observed in the liver and kidney tissues of the Northwest Glycyrrhiza Isoflavone Nanocrystal group, and no significant liver and kidney toxicity was observed in the serum biochemical indicators, indicating that the Northwest Glycyrrhiza Isoflavone Nanocrystal has good biosafety in vivo.

[0060] Statistical analysis in animal experiments: Data are expressed as mean ± standard deviation (SD) of at least three independent experiments. Student's t-test (for comparisons between two groups) or one-way ANOVA followed by Tukey's post-hoc test (for comparisons between multiple groups) was used for statistical significance analysis using GraphPad Prism 9.0 software. P < 0.05 was considered statistically significant. P<0.05, P<0.01, P<0.001).

[0061] 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. The application of licorice isoflavones in the preparation of antitumor drugs, wherein the tumor includes one or more of breast cancer, lung cancer, and liver cancer.

2. A method for preparing Northwest licorice isoflavone nanocrystals, characterized in that, Includes the following steps: Northwest licorice isoflavones were dissolved in an organic solvent to obtain an organic phase; The organic phase was mixed with an aqueous phase containing a stabilizer and subjected to high-pressure homogenization to obtain Northwest licorice isoflavone nanocrystals.

3. The preparation method according to claim 2, characterized in that, The mass ratio of the licorice isoflavones and stabilizer is 1:0.001~0.

01.

4. The preparation method according to claim 2, characterized in that, The concentration of the organic phase is 4~20 g / L; The organic solvent includes one or more of the following: alcohol solvents, ether solvents, ketone solvents, ester solvents, sulfur-containing compound solvents, and amide solvents.

5. The preparation method according to claim 4, characterized in that, The alcohols include methanol and / or ethanol; The ether solvents include tetrahydrofuran and / or methyl tert-butyl ether; The ketone solvent includes acetone; The ester solvent includes ethyl acetate; The sulfur-containing compound includes dimethyl sulfoxide; The amide solvents include dimethylformamide.

6. The preparation method according to claim 2, characterized in that, The stabilizer includes one or more of sodium dodecyl sulfate, poloxamer, povidone, hydroxypropyl methylcellulose, carboxymethyl cellulose, and F127.

7. The preparation method according to any one of claims 2 to 6, characterized in that, The high-pressure homogenization process involves a pressure of 500~3000 bar, 5~20 cycles, and a temperature of -5~60℃.

8. The licorice isoflavone nanocrystals prepared by the method according to any one of claims 2 to 7, characterized in that, The average particle size of the Northwest Glycyrrhiza Isoflavone Nanocrystals is 50~300nm, the polydispersity index is 0.1~0.25, and the absolute value of the Zeta potential is -5~25mV.

9. The use of the Northwest Glycyrrhiza Isoflavone Nanocrystals according to claim 8 in the preparation of antitumor drugs, wherein the tumor includes one or more of breast cancer, lung cancer, and liver cancer.

10. An antitumor drug, characterized in that, It includes an active ingredient and pharmaceutically acceptable excipients, wherein the active ingredient includes Northwest Glycyrrhiza Isoflavone API and / or Northwest Glycyrrhiza Isoflavone Nanocrystals as described in claim 8; the antitumor drug includes drugs for one or more tumors including breast cancer, lung cancer and liver cancer.

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