Icaritin and tripterine composition as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to an icaritin and tripterine composition as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing an icaritin solution and a tripterine solution, adding water, carrying out ultrasonic treatment, dialyzing, and collecting the residual solution, thereby obtaining the icaritin and tripterine compound. The tripterine (CEL) and the icaritin (ICT) are prepared into the self-assembled nano-drug, the preparation process is simpler, the drug delivery efficiency is higher, and the tripterine (CEL) and the icaritin (ICT) have a remarkable synergistic effect when being applied to treatment of liver cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a composition of icariin and tripterine, a preparation method and application thereof. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is the most common type of liver cancer, and the main causes of HCC include HBV, HCV, alcohol-related liver disease and metabolic dysfunction-related fatty liver disease. The treatment of liver cancer, including the application of targeted drugs, immunotherapy and comprehensive treatment strategies, significantly prolongs the survival of patients at each stage. However, some HCC patients are diagnosed at an advanced stage and have lost the opportunity for radical treatment such as surgery and liver transplantation. Therefore, it is extremely critical to find an effective treatment for advanced HCC.

[0003] At present, significant progress has been made in the field of systemic therapy for advanced hepatocellular carcinoma, especially with the widespread use of tyrosine kinase inhibitors (such as sorafenib and lenvatinib) and immune checkpoint inhibitors (such as atezolizumab and bevacizumab), the overall survival and quality of life of patients have been improved to some extent. However, there are still many severe challenges in clinical practice. First, hepatocellular carcinoma has high heterogeneity and is prone to drug resistance. There are significant differences in driver genes among patients, and multiple pathogenic factors promote the development of HCC through different mechanisms, which leads to the easy occurrence of primary and secondary drug resistance when using single drug treatment, especially single targeted drug. Second, existing targeted and immune drugs and their combination regimens are often accompanied by significant side effects, such as hypertension, thrombosis, immune hepatitis and hand-foot skin reactions, etc. These adverse reactions not only limit the application of drugs, but also can seriously affect the quality of life of patients. More notably, different causes shape the immunosuppressive microenvironment of tumors through their own unique mechanisms. For example, in HBV-related liver cancer, the continuous stimulation of HBV viral antigens (such as HBsAg) can lead to the exhaustion of effector CD8+ T cells, while recruiting regulatory T cells (Treg), regulatory B cells (Breg) and granulocyte-like myeloid-derived suppressor cells (gMDSC), etc., forming a tolerogenic immune microenvironment. This complex immunosuppressive state significantly weakens the efficacy of immunotherapy drugs. Therefore, developing new multi-target synergistic, efficient and low-toxicity drugs is still an important direction for future research.

[0004] Traditional Chinese medicine (TCM) has shown unique therapeutic advantages in the field of cancer treatment, with its multi-pathway, multi-target, small side effects and significant efficacy, etc. providing an important supplement to modern tumor treatment. From the molecular mechanism, traditional Chinese medicine can exert anti-tumor effects through multiple dimensional action pathways, including inducing tumor cell apoptosis, inhibiting tumor cell proliferation and cycle arrest, inducing tumor immunogenic death, inhibiting angiogenesis and remodeling the immune microenvironment, etc. Therefore, in-depth study of the anti-tumor efficacy of traditional Chinese medicine has important clinical value.

[0005] Celastrol, a pentacyclic triterpenoid derived from the traditional medicinal plant Tripterygium hookeri (T. hookeri) Hook. f., is considered to have special potential as a cancer treatment drug. It has a wide range of pharmacological activities, including significant anti-inflammatory, anti-obesity, and anti-tumor effects. Multiple lines of evidence show that Celastrol exhibits significant proliferation-inhibiting activity against a variety of tumor cell lines. Icaritin is a bioactive compound derived from the traditional Chinese medicine Epimedium brevicornum Maxim. Icaritin has a variety of biological activities, not only directly killing tumors by inducing cell death and autophagy, but also regulating the tumor immune microenvironment and promoting anti-tumor immune responses, showing a wide range of anti-tumor effects in various solid tumors, including colorectal cancer, prostate cancer, and urothelial cancer. Some basic and clinical studies have shown that Icaritin has good biological safety in patients with advanced HCC, significantly improving survival. In 2022, Icaritin soft capsules were approved by the National Medical Products Administration (NMPA) for the immunotherapy of advanced HCC. Tripterygium wilfordii Celastrol, a pentacyclic triterpenoid derived from the traditional medicinal plant Tripterygium hookeri (T. hookeri) Hook. f., is considered to have special potential as a cancer treatment drug. It has a wide range of pharmacological activities, including significant anti-inflammatory, anti-obesity, and anti-tumor effects. Multiple lines of evidence show that Celastrol exhibits significant proliferation-inhibiting activity against a variety of tumor cell lines. Icaritin is a bioactive compound derived from the traditional Chinese medicine Epimedium brevicornum Maxim. Icaritin has a variety of biological activities, not only directly killing tumors by inducing cell death and autophagy, but also regulating the tumor immune microenvironment and promoting anti-tumor immune responses, showing a wide range of anti-tumor effects in various solid tumors, including colorectal cancer, prostate cancer, and urothelial cancer. Some basic and clinical studies have shown that Icaritin has good biological safety in patients with advanced HCC, significantly improving survival. In 2022, Icaritin soft capsules were approved by the National Medical Products Administration (NMPA) for the immunotherapy of advanced HCC. Epimedium brevicornu

[0006] Although Icaritin soft capsules have been used in the immunotherapy of advanced hepatocellular carcinoma and have shown certain therapeutic potential in clinical practice, their application still has many limitations. First, Icaritin has poor water solubility and exhibits low intestinal mucosal and cell membrane permeability in vivo, severely limiting its absorption efficiency. At the same time, Icaritin undergoes significant first-pass effect when passing through the liver after oral administration, resulting in a large amount of drug being metabolized and inactivated, thus making the oral bioavailability extremely low. In addition, Icaritin lacks an active targeting mechanism, and it is easily recognized by the reticuloendothelial system (RES) and rapidly cleared in vivo, resulting in short blood circulation time, limited tumor tissue accumulation, and difficulty in achieving effective therapeutic concentrations in the lesion area, with overall low drug delivery efficiency. Clinical treatment of advanced HCC usually employs a combination drug strategy, such as the combined use of immunotherapy with anti-angiogenic drugs, chemotherapy drugs, or other targeted preparations. However, existing preparations often fail to achieve the simultaneous arrival of two or more drugs at the tumor site in terms of time and space.

[0007] ​Nanoparticle drug delivery systems (nano-DDS) have been extensively studied in the field of cancer therapeutic delivery. Nano-DDS can prolong drug circulation time and achieve tumor-specific distribution by enhancing permeability and retention (EPR). Furthermore, specific surface functionalization can enable nano-DDS to respond specifically to the physiological microenvironment of the tumor site, such as excessive enzyme production, hypoxia, redox reactions, and acidic conditions. Pure drug self-delivery nanosystems (PDANS), assembled from single or multiple pure drug molecules, have attracted considerable attention due to their simpler fabrication processes and higher drug delivery efficiency. PDANS are composed entirely of pharmacologically active compounds, further reducing carrier-related biotoxicity. Compared to traditional nanocrystal delivery systems, PDANS offer advantages such as high drug loading efficiency and simple fabrication processes.

[0008] Therefore, this paper proposes a nanomedicine that self-assembles icariin and tripterygium wilfordii, and deeply analyzes the relevant mechanism of action of the key pharmacological components of this drug in the process of renal fibrosis, which can provide a material basis and transformational value for the clinical treatment of liver cancer. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a composition of icariin and tripterygium wilfordii, its preparation method, and its application.

[0010] To achieve the above-mentioned objectives of this invention, the specific technical solution adopted by this invention is as follows: A method for preparing a composition of icariin and tripterygium wilfordii includes the following steps: The icariin (ICT) solution and triptolide (CEL) solution were mixed, water was added, the mixture was sonicated, dialyzed, and the remaining solution was collected to obtain the icariin and triptolide composition (CAI).

[0011] Preferably, the solvent for both the epimedium solution and the triptolide solution is dimethyl sulfoxide (DMSO).

[0012] Preferably, the mass concentration of both the epimedium solution and the triptolide solution is 8-12 mg / mL, and more preferably 10 mg / mL.

[0013] Preferably, the volume ratio of the epimedium solution to the triptolide solution is 1-3:5, more preferably 3:5.

[0014] Preferably, the amount of water added is 15-20 times the volume of the triptolide solution, and more preferably 18 times.

[0015] Preferably, the ultrasonic power is 35-45 kHz and the ultrasonic duration is 5-10 min.

[0016] Preferably, the molecular weight cutoff for dialysis is 3000-5000 Da, and more preferably 3500 Da.

[0017] More preferably, dialysis is performed using dialysis tubing.

[0018] The present invention also relates to a composition of icariin and tripterygium wilfordii obtained by the above preparation method.

[0019] The present invention also relates to the use of the icariin and tripterygium oleoresin composition prepared by the above preparation method in the preparation of a drug for treating liver cancer.

[0020] Preferably, the drug further comprises a pharmaceutically acceptable carrier selected from one or more of emulsifiers, fillers, binders, wetting agents, disintegrants, absorption enhancers, flavoring agents, coloring agents, and solubilizers.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) In this invention, triptolide (CEL) and icariin (ICT) are prepared into self-assembled nanomedicines, which have a simpler preparation process and higher drug delivery efficiency.

[0022] (2) The self-assembled nanomedicines of triptolide and icariin in this invention are applied to the treatment of liver cancer, and there is a significant synergistic effect between the two. Attached Figure Description

[0023] Figure 1 This is a hydrated particle size distribution diagram of CAI; Figure 2 This is a graph showing the changes in particle size and PDI of CAI over seven days; Figure 3 This is a transmission electron microscope image of CAI; Figure 4 These are the infrared spectra of CEL, ICT, and CAI. Figure 5 These are the UV-Vis absorption spectra of CAI and CEL in water and CAI in DMSO solution; Figure 6 These are the UV-Vis absorption spectra of CAI, 0.2% SDS solution, and CAI in 0.2% SDS solution; Figure 7 These are the UV-Vis absorption spectra of CAI, 0.5M NaCl solution of CAI, and 1M NaCl solution of CAI. Figure 8 This is a graph showing the changes in body weight of mice treated with PBS, CAI, ICT, and CEL. Figure 9This graph shows the changes in tumor volume in mice treated with PBS, CAI, ICT, and CEL. Compared to the PBS group, & P<0.05, && P<0.01; compared with the CAI group, # P<0.05, ## P<0.01; Figure 10 This is a graph showing the average tumor weight of mice in each group on day 15 after drug administration. Compared to the PBS group, && P<0.01, &&& P<0.001; compared with the CAI group, ## P<0.01, ### P<0.001; Figure 11 These are images of isolated tumor tissues from various groups. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0025] Example 1 The preparation method of the icariin and tripterygium extract composition (CAI) includes the following steps: (1) Prepare a stock solution of triptolide (CEL) and icariin (ICT) with DMSO to a concentration of 10 mg / mL; (2) Mix CEL (50 μL) and ICT (30 μL) in ultrapure water (900 μL), sonicate at 40 kHz for 5 min, transfer the solution into a dialysis tube (MWCO 3500 Da) for dialysis for 12 h, collect the remaining solution, and obtain the icariin and tripterygium oleoresin composition (CAI).

[0026] Example 2 The preparation method of the icariin and tripterygium extract composition (CAI) includes the following steps: (1) Prepare a stock solution of triptolide (CEL) and icariin (ICT) with DMSO to a concentration of 10 mg / mL; (2) Mix CEL (50 μL) and ICT (20 μL) in ultrapure water (900 μL), sonicate at 35 kHz for 10 min, transfer the solution into a dialysis tube (MWCO 3500 Da) for dialysis for 12 h, and collect the remaining solution to obtain the solution.

[0027] Example 3 The preparation method of the icariin and tripterygium extract composition (CAI) includes the following steps: (1) Prepare a stock solution of triptolide (CEL) and icariin (ICT) with DMSO to a concentration of 10 mg / mL; (2) Mix CEL (50 μL) and ICT (10 μL) in ultrapure water (900 μL), sonicate at 40 kHz for 15 min, transfer the solution into a dialysis tube (MWCO 3500 Da) for dialysis for 12 h, and collect the remaining solution to obtain the solution.

[0028] Effect test The following tests were performed on the icariin and tripterygium oleoresin composition (CAI) prepared in Example 1.

[0029] Test Example 1: Particle Size and Stability Test Under ultrasonic conditions (40 kHz, 5 min), the CAI in Example 1 was diluted 10 times with ultrapure water, and 1 mL was taken into a particle size cup. Dynamic light scattering (DLS) analysis was performed using a Malvern particle size analyzer to obtain the particle size and dispersity index (PDI) of the CAI. The stability of the CAI was observed by continuous measurement for 7 days.

[0030] The results are as follows Figure 1 As shown, the dynamic light scattering hydrated particle size of the combination of epimedium and tripterygium (CAI) is approximately 160.72 nm, and the PDI is 0.109.

[0031] Stability results are as follows Figure 2 As shown, the PDI and particle size of CAI did not change significantly over 7 days, indicating that CAI has good stability in ultrapure water.

[0032] Test Example 2: Transmission Electron Microscopy Test Using a pipette, take 10 μL of CAI diluted solution (the CAI in Example 1 was diluted 20 times with ultrapure water), add it dropwise onto a 100-mesh copper grid, allow it to air dry, and then observe the morphology of the CAI using TEM. Figure 3 As shown in the TEM image, CAI consists of uniformly dispersed spherical nanoparticles.

[0033] Test Example 3: Fourier Transform Infrared Spectroscopy Test The CAI prepared in Example 1 was freeze-dried. The CEL, ICT, and CAI were detected using total reflectance infrared spectroscopy (ATR) at 4000-600 cm⁻¹. -1 The infrared spectrum.

[0034] The results are as follows Figure 4 As shown, infrared spectral analysis reveals that ICT and CAI are at 1147 cm⁻¹. -1 The area exhibits a significant characteristic absorption peak for ether bonds, while CEL and CAI show a peak at 1704 cm⁻¹. -1 The presence of a characteristic absorption peak for the C=O bond of the carboxyl group nearby indicates that CAI contains both CEL and ICT components. Furthermore, CAI exhibits an absorption peak at 3309 cm⁻¹. -1 The absorption peaks in the vicinity show significant changes, which may be due to the conversion of free OH groups into bound OH groups during the self-assembly process.

[0035] Test Example 4 UV-vis Characterization Solution preparation: The preparation of the following CEL stock solution and ICT stock solution is the same as step (1) in Example 1.

[0036] CAI solution: Take 50 μL of CEL stock solution and 30 μL of ICT stock solution, and mix them with 900 μL of ultrapure water to obtain the solution. CEL aqueous solution: Take 50 μL of CEL stock solution and mix it with 900 μL of ultrapure water to obtain the solution; CAI's DMSO solution: Take 50 μL of CEL stock solution and 30 μL of ICT stock solution, and mix them with 900 μL of DMSO to obtain the solution; 0.2% sodium dodecyl sulfate (SDS) solution: Dissolve 2g SDS in 1000mL of water to obtain the solution. The 0.2% SDS solution for CAI is prepared by mixing 50 μL of CEL stock solution and 30 μL of ICT stock solution with 900 μL of 0.2% SDS solution. CAI 0.5M NaCl solution: Take 50μL CEL stock solution and 30μL ICT stock solution, and mix with 900μL 0.5M NaCl solution to obtain the solution; CAI 1M NaCl solution: Take 50μL CEL stock solution and 30μL ICT stock solution, and mix with 900μL 1M NaCl solution to obtain the solution.

[0037] UV-vis was used to detect the ultraviolet spectra in different solutions, with a detection wavelength range of 300-800 nm. The results are as follows: Figure 5 to Figure 7 As shown.

[0038] Compared to water, CAI exhibits a significant blue shift in DMSO. Figure 5 This indicates that Ca2+ decomposes in DMSO, suggesting the presence of π-π stacking interactions. Subsequently, the addition of sodium dodecyl sulfate (SDS) to the Ca2+ aqueous solution resulted in a significant blue shift in the absorption peak, indicating that intermolecular hydrophobic interactions are involved in the self-assembly process.Figure 6 However, when CAI was dispersed in sodium chloride solutions of different concentrations, its characteristic absorption peaks did not change significantly, indicating the absence of hydrogen bond interactions. Figure 7 This is consistent with the results of infrared spectroscopy.

[0039] Test Example 5: In vivo tumor suppression test To detect antitumor behavior in vivo, H22 cells resuspended in PBS were subcutaneously injected into female BALB / c mice (purchased from Guangzhou Ruige Biotechnology Co., Ltd., 5 mice per group, 20 mice in total) at a concentration of 1.5 × 10⁻⁶. 6 (each tumor). When the tumor volume reaches approximately 50 mm... 3 Mice were randomly divided into four groups and administered PBS (pH 7.2), CEL (1.875 mg / kg), ICT (1.125 mg / kg), and CAI (3 mg / kg) via tail vein on days 1, 3, 4, 6, 8, 9, and 11. Tumors were collected on day 15. During treatment, tumor size and body weight were measured every two days using calipers and a balance. After treatment, all mice were euthanized, and tumors were collected for photography and weighing.

[0040] according to Figure 8 As shown in the weight change graph, compared with the PBS group, there was no significant change in weight after ICT administration, while weight decreased to some extent after CAI and CEL administration, but recovered significantly after discontinuation of administration.

[0041] according to Figure 9 It was observed that during the entire treatment period, tumor growth was rapid in the PBS and ICT groups, while it was slightly slower in the CEL and CAI groups, with the CAI group exhibiting the strongest tumor-suppressive effect. Furthermore, at the end of treatment, all tumor-bearing mice were sacrificed, and the tumor weight results showed that CAI had a significant anti-tumor effect. Figure 10 The actual tumor size and tumor volume curves of different groups were basically consistent. Figure 11 ).

[0042] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a composition of icariin and tripterygium wilfordii, characterized in that, Includes the following steps: Mix the epimedium solution and tripterygium wilfordii solution, add water, sonicate, dialyze, and collect the remaining solution to obtain the final product.

2. The preparation method according to claim 1, characterized in that, The solvent for both the epimedium solution and the triptolide solution is dimethyl sulfoxide.

3. The preparation method according to claim 1, characterized in that, The mass concentrations of the epimedium solution and the triptolide solution were both 8-12 mg / mL.

4. The preparation method according to claim 1, characterized in that, The volume ratio of the epimedium solution to the triptolide solution is 1-3:

5.

5. The preparation method according to claim 1, characterized in that, The amount of water added is 15-20 times the volume of the triptolide solution.

6. The preparation method according to claim 1, characterized in that, The ultrasonic power is 35-45kHz, and the ultrasonic time is 5-10min.

7. The preparation method according to claim 1, characterized in that, The molecular weight cutoff for dialysis is 3000-5000 Da.

8. A composition of icariin and tripterygium wilfordii prepared by the preparation method according to any one of claims 1-7.

9. The use of a composition of icariin and tripterygium wilfordii obtained by the preparation method according to any one of claims 1-7 in the preparation of a medicament for treating liver cancer.

10. The application according to claim 9, characterized in that, The drug further comprises a pharmaceutically acceptable carrier selected from one or more emulsifiers, fillers, binders, humectants, disintegrants, absorption enhancers, flavoring agents, colorants, and solubilizers.

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