Use of a safflower-echinacea drug pair composition in the preparation of a medicament for treating hepatocellular carcinoma and / or inhibiting liver fibrosis

Abstract

This invention discloses the application of a safflower-blue safflower herb pair composition in the preparation of drugs for treating hepatocellular carcinoma and / or inhibiting liver fibrosis; wherein the safflower-blue safflower herb pair composition is composed of total flavonoids from safflower and total flavonoids from blue safflower, with a mass ratio of (1-3):1. Experiments have shown that the safflower-blue safflower herb pair composition can treat hepatocellular carcinoma and / or inhibit liver fibrosis, and its efficacy is significantly higher than that of total flavonoids from safflower or blue safflower alone. This invention has significant theoretical value and clinical significance.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to the application of the safflower-blue safflower herb pair composition in the preparation of drugs for treating hepatocellular carcinoma and / or inhibiting liver fibrosis. Background Technology

[0002] Liver fibrosis is a key pathological step in the progression of various chronic liver diseases (such as viral hepatitis, alcoholic liver disease, and non-alcoholic steatohepatitis) to cirrhosis and even hepatocellular carcinoma (HCC). Essentially, it is a dynamic process of excessive deposition of extracellular matrix (especially collagen) during repeated liver damage and repair. If the progression of liver fibrosis is not effectively curbed, it will eventually lead to liver failure or HCC. Currently, there are no universally recognized specific chemical drugs or biological products that can reverse liver fibrosis. Treatment mainly focuses on etiological control (such as antiviral therapy) and symptomatic support, which have limitations in efficacy, high relapse rates after drug discontinuation, and significant side effects with long-term use. As one of the most common malignant tumors worldwide, HCC treatment also faces significant challenges, especially for patients in the middle and late stages. Existing chemotherapy drugs have drawbacks such as strong drug resistance, high systemic toxicity, and poor patient tolerance.

[0003] safflower( Carthamus tinctorius L. is a classic herb for promoting blood circulation and removing blood stasis. Modern pharmacological studies have shown that it has effects such as improving microcirculation, anti-inflammation, anti-oxidation, and anticoagulation. Bluebell (L.) ScabiosacomosaFisch . exRoem Safflower (.etSchult.) is a commonly used medicine in Mongolian medicine, possessing effects such as clearing heat, detoxifying, and promoting blood circulation. To date, there are no research reports on the use of safflower and safflower in combination for the treatment of hepatocellular carcinoma or the inhibition of liver fibrosis. Summary of the Invention

[0004] The purpose of this invention is to provide a method for treating hepatocellular carcinoma and / or inhibiting liver fibrosis.

[0005] This invention first protects a safflower-sappanflower anther composition. The safflower-sappanflower anther composition may include total flavonoids from safflower and total flavonoids from sappanflower.

[0006] The safflower-sappanflower herb pair composition may specifically consist of total flavonoids from safflower and total flavonoids from sappanflower.

[0007] In any of the above-mentioned safflower-spotted safflower herb pair compositions, the mass ratio of total safflower flavonoids to total spotted safflower flavonoids can be (1-3):1 (e.g., (1-2):1, (2-3):1, (1-1.5):1, (1.5-1.6):1, (1.6-2):1, 1:1, 1.6:1, 1.5:1, 2:1 or 3:1).

[0008] In any of the above-described safflower-spotted flower herb pair compositions, the mass ratio of total safflower flavonoids to total spotted flower flavonoids can specifically be 2:1, 1.6:1, or 1.5:1.

[0009] The preparation method of any of the above-mentioned safflower-blue safflower herb pair composition is as follows: mix (1-3) parts by weight (such as (1-2) parts by weight, (2-3) parts by weight, 1 part by weight, 2 parts by weight or 3 parts by weight) of safflower total flavonoid powder and 1 part by weight of blue safflower total flavonoid powder to obtain the safflower-blue safflower herb pair composition.

[0010] The preparation method of the total flavonoid powder of *Sedum morganianum* described above can be an alcohol extraction of *Sedum morganianum* medicinal material.

[0011] The specific preparation method of the total flavonoid powder of *Sedum morganianum* mentioned above can be as follows:

[0012] (s1) The sappan flower herb was pulverized, then an ethanol aqueous solution was added, and the mixture was refluxed for extraction. The filtrate was concentrated to obtain sappan flower extract. (s2) The extract of Sedum sarmentosum was adsorbed with resin (such as AB-8 macroporous resin), then eluted with ethanol aqueous solution, the filtrate was collected and concentrated and dried to obtain total flavonoid powder of Sedum sarmentosum.

[0013] The preparation method of the total flavonoid powder of safflower described above can be the alcohol extraction of safflower medicinal material.

[0014] The specific preparation method of any of the above-mentioned safflower total flavonoid powders can be as follows: (t1) The safflower herb was pulverized, then an ethanol aqueous solution was added, and the mixture was refluxed for extraction. The filtrate was concentrated to obtain the safflower extract. (t2) The safflower extract was adsorbed with resin (such as AB-8 macroporous resin), then eluted with an aqueous ethanol solution, the filtrate was collected and concentrated and dried to obtain total safflower flavonoid powder.

[0015] The use of any of the above-described safflower-blue clover herb pairs in the preparation of drugs for inhibiting liver fibrosis is also within the scope of protection of this invention.

[0016] In the above applications, the inhibition of liver fibrosis can manifest as at least one of A1)-A3): A1) Reduces the accumulation of fibrous septa and / or collagen fibers in liver tissue; A2) Reduce the content of hydroxyproline in liver tissue; A3) Reduce the levels of liver function indicators and liver fibrosis indicators in serum.

[0017] Furthermore, the liver function indicators may include ALT, AST, and / or ALP. The liver fibrosis indicators may include PⅢP and / or IV-C.

[0018] The use of any of the above-described safflower-blue clover herb pairs in the preparation of medicaments for the prevention and / or treatment of hepatocellular carcinoma is also within the scope of protection of this invention.

[0019] In the above applications, the prevention and / or treatment of hepatocellular carcinoma may manifest as at least one of B1)-B3): B1) Inhibits the proliferation of liver cancer cells; B2) Inhibits the migration of liver cancer cells; B3) Delays the progression of hepatocellular carcinoma.

[0020] Furthermore, the delay in the progression of hepatocellular carcinoma can manifest as at least one of C1)-C4): C1) Delays weight gain during the progression of hepatocellular carcinoma; C2) Reduces the accumulation of fibrous septa and / or collagen fibers in liver tissue; C3) Reduce inflammatory cell infiltration and / or fibrous septa in the portal area; C4) causes hepatocytes to arrange themselves in a regular pattern.

[0021] The liver cancer cells mentioned above can specifically be Huh-7 cells.

[0022] Any of the drugs described above can be administered via intravenous injection or oral administration. Intravenous injection may be intraperitoneal or intravenous.

[0023] Based on Masson staining results and hydroxyproline content detection results of liver tissue from a mouse model of liver fibrosis, as well as the detection results of serum liver function indicators (such as ALT, AST, ALP) and liver fibrosis indicators (such as PⅢP, IV-C), the inventors of this application found that the safflower-blue safflower herb pair composition prepared in this invention can significantly inhibit liver fibrosis, and its efficacy is significantly higher than that of total flavonoids from safflower or total flavonoids from blue safflower. Furthermore, it was found that the safflower-blue safflower herb pair composition can treat hepatocellular carcinoma in a dose-dependent manner; the treatment of hepatocellular carcinoma manifests as inhibition of the proliferation and migration of hepatocellular carcinoma cells (such as Huh-7 cells) and delay in the progression of hepatocellular carcinoma.

[0024] Therefore, the safflower-blue safflower herb combination provided by the present invention can treat hepatocellular carcinoma and / or inhibit liver fibrosis, and has important theoretical value and clinical significance. Attached Figure Description

[0025] Figure 1 Masson staining micrographs (100×) of partial mouse liver tissue from each group in Example 2.

[0026] Figure 2The above are the statistical results of collagen area in the liver tissue of mice in each group in Example 2 (n=3); among them, for P <0.05, for P <0.01, for P <0.001, for P <0.0001.

[0027] Figure 3 The statistical results of hydroxyproline content in the liver tissues of mice in each group in Example 2 (n=6) are shown; among them, for P <0.05, for P <0.001, for P <0.0001.

[0028] Figure 4 The results show the detection levels of liver function indicators and liver fibrosis indicators in the serum of mice in each group in Example 2 (n=6); among them, for P <0.05, for P <0.01, for P <0.0001.

[0029] Figure 5 The statistical results of the body weight of mice in each group in Example 3 (n=10).

[0030] Figure 6 HE staining images (100×) of partial mouse liver tissue from each group in Example 3.

[0031] Figure 7 Masson staining micrographs (400×) of partial mouse liver tissue from each group in Example 3.

[0032] Figure 8 To detect the ability of the safflower-blue safflower anther pair combination to promote the proliferation of Huh-7 cells.

[0033] Figure 9 To detect the ability of the safflower-blue safflower anther pair to promote the migration of Huh-7 cells.

[0034] Figure 10 To detect the effect of the safflower-blue safflower anther pair composition on the expression level of metastasis-related proteins in Huh-7 cells. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0037] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0038] The blue safflower and safflower in the following examples are commercially available medicinal materials.

[0039] Example 1: Preparation of Safflower-Syzygium aromaticum anther pair composition I. Component Identification of Bluebell and Safflower 1. Soak 0.3g of bluebell flowers in 50% (v / v) methanol aqueous solution for 3-4 hours, then extract by ultrasonication at 40℃ for 40 minutes, centrifuge at 4℃ and 12000r / min for 10 minutes, and collect the supernatant.

[0040] 2. Take 10 μL of the supernatant collected in step 1 and perform HPLC-MS analysis. The chromatographic column was a SHIMADZU InerSustain C18 (2 mm × 100 mm, 2 μm); column temperature was 35℃; flow rate was 0.3 mL / min. The mobile phase consisted of acetonitrile (A) and 0.1% acetic acid (B); gradient elution was performed, with the following elution program: 0-10 min, 5%-50% A; 10-13 min, 50%-95% A; 13-14 min, 95%-5% A; 14-15 min, 5% A.

[0041] 3. After completing step 2, the extracted mass spectrometry peaks were first compared using the MassBank (https: / / mona.fiehnlab.ucdavis.edu / ), ReSpect (https: / / spectra.psc.riken.jp / menta.cgi / respect / index), and GNPS (https: / / gnps.ucsd.edu / ) databases to infer the possible active ingredients of *Sedum aizoon* and preliminarily determine their molecular formulas. Then, the SwissADME database (http: / / www.swissadme.ch / ) was used to preliminarily screen the above-mentioned active ingredients of *Sedum aizoon* according to the Lipinsk five principles. Finally, the active ingredients of *Sedum aizoon* were further screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) (https: / / old.tcmsp-e.com / tcmsp.php) with oral bioavailability (OB) > 30% and drug-likeness (DL) ≥ 0.18 as screening criteria. The resulting information on the active ingredients of *Sedum aizoon* is shown in Table 1.

[0042] 4. Using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), the chemical components of safflower were retrieved using "safflower" as the keyword. Then, the oral bioavailability (OB) > 30% and drug-likeness (DL) ≥ 0.18 were used as screening criteria to obtain the active ingredient information of safflower shown in Table 1.

[0043] Table 1

[0044] The above results indicate that both safflower and safflower contain a large number of active ingredients, including lignins, flavonoids, alkaloids, and phytosterols.

[0045] II. Preparation of total flavonoid powder from Sappanflower and total flavonoid powder from Safflower 1. Preparation of total flavonoid powder from Sedum aizoon (1) The sappan flower herb was pulverized to obtain sappan flower powder. Then, 10 parts by volume of 70% (v / v) ethanol aqueous solution were added to 1 part by mass of sappan flower powder, and the mixture was refluxed for 2 hours. After filtration, filtrate 1 and residue 1 were obtained. Residue 1 was refluxed for another 1 hour and then filtered to obtain filtrate 2 and residue 2.

[0046] (2) After completing step (1), combine filtrate 1 and filtrate 2, and then concentrate to obtain blue sappan extract.

[0047] (3) After completing step (2), the extract of Sedum sarmentosum is first suspended in water, then adsorbed with AB-8 macroporous resin, and then eluted with 95% (v / v) ethanol aqueous solution. The filtrate is collected, concentrated and dried to obtain total flavonoid powder of Sedum sarmentosum.

[0048] 2. Preparation of Safflower Total Flavonoid Powder (1) The safflower herb was pulverized to obtain safflower powder. Then, 10 parts by volume of 80% (v / v) ethanol aqueous solution were added to 1 part by mass of safflower powder, and the mixture was refluxed for 2 hours. After filtration, filtrate a and residue a were obtained. The residue a was refluxed for another 1 hour and then filtered to obtain filtrate b and residue b.

[0049] (2) After completing step (1), combine filtrate a and filtrate b, then concentrate to obtain safflower extract.

[0050] (3) After completing step (2), the safflower extract is first suspended in water, then adsorbed with AB-8 macroporous resin, and then eluted with 95% (v / v) ethanol aqueous solution. The filtrate is collected, concentrated and dried to obtain safflower total flavonoid powder.

[0051] III. Preparation of Safflower-Syzygium aromaticum herb pair composition Two parts by mass of safflower total flavonoid powder and one part by mass of safflower total flavonoid powder were mixed to obtain a safflower-safflower herb pair composition. That is, in the safflower-safflower herb pair composition, the mass ratio of safflower total flavonoid powder to safflower total flavonoid powder is 2:1.

[0052] Application of the safflower-blue safflower herb pair prepared in Example 2 and Example 1 in inhibiting liver fibrosis 1. Fifty male Kunming mice, aged 6-8 weeks and weighing approximately 18-20 g, were acclimatized for one week in an SPF-grade environment. During this period, the room temperature was maintained at 20±2℃, humidity at 45%-55%, with alternating day and night light, and free access to food and water. Afterward, they were randomly divided into five groups: a control group, a model group, a drug pair group, a safflower group, and a blue safflower group, with 10 mice in each group. The following treatments were administered to each group: Control group: 0.5% CMC-Na solution was administered by gavage daily at a dose of 10 μL / g body weight for 8 weeks; Model group: 0.5% CMC-Na solution was administered by gavage daily at a dose of 10 μL / g body weight for 8 weeks; at the same time, 20% CCl4-peanut oil solution was administered by gavage every Tuesday and Friday from 9:00 to 11:00 AM at a dose of 10 μL / g body weight for 8 weeks. The drug pair group: The safflower-blue peony drug pair composition solution (safflower-blue peony drug pair composition as solute, 0.5% CMC-Na solution as solvent) was administered by gavage daily at a dose of 120 mg / kg body weight for 8 weeks; at the same time, 20% CCl4-peanut oil solution was administered by gavage every Tuesday and Friday from 9:00 to 11:00 AM at a dose of 10 μL / g body weight for 8 weeks. Safflower group: Safflower total flavonoids solution (sole is safflower total flavonoids powder, solvent is 0.5% CMC-Na solution) was administered by gavage daily at a dose of 150 mg / kg body weight for 8 weeks; at the same time, 20% CCl4-peanut oil solution was administered by gavage every Tuesday and Friday from 9:00 to 11:00 AM at a dose of 10 μL / g body weight for 8 weeks. The sappanwood group: The mice were administered a total flavonoid solution of sappanwood (sole consisting of total flavonoid powder of sappanwood and 0.5% CMC-Na solution) by gavage daily at a dose of 100 mg / kg body weight for 8 weeks. At the same time, the mice were administered a 20% CCl4-peanut oil solution by gavage every Tuesday and Friday from 9:00 to 11:00 AM at a dose of 10 μL / g (body weight of the mouse) for 8 weeks.

[0053] The model group, drug pair group, safflower group, and blue safflower group all used 20% CCl4-peanut oil solution to construct mouse models of liver fibrosis.

[0054] 2. After completing step 1, fasting but water restriction for 12 hours. Weigh and record the weight 1-2 hours after the last administration the next day. The administration of each group is as follows: the blank group and the model group are still administered 0.5% CMC-Na solution by gavage. The drug pair group is administered safflower-spotted safflower drug pair combination solution by gavage. The safflower group is administered safflower total flavonoid solution by gavage. The spotted safflower group is administered spotted safflower total flavonoid solution by gavage.

[0055] 3. After completing step 2, the mouse was euthanized by spinal dislocation. The limbs were quickly fixed, the abdomen was fully exposed, and the area was disinfected with alcohol spray. The surrounding soft tissue was dissected and separated to obtain intact liver tissue. The liver tissue was photographed and weighed again for recording. Blood samples were collected at the same time, and serum was separated for later use.

[0056] 4. Masson staining was performed on the liver tissues obtained in step 3, and then ImageJ software was used to count the area of ​​the blue region in the stained sections.

[0057] Masson staining micrographs of liver tissues from each group are shown below. Figure 1Masson staining results showed that in the model group, a large number of blue collagen fibers were deposited around the portal areas in the liver tissue, hepatocytes were aggregated, and the fibrous septa were significantly increased. Compared with the model group, the fibrous septa and collagen fiber accumulation were significantly reduced in the sclerotium group, safflower group, and herb pair group.

[0058] The statistical results of collagen deposition area (blue area) for each group are shown in the figure. Figure 2 The results showed that, compared with the control group, the collagen deposition area in the model group was significantly increased; compared with the model group, the collagen deposition area in the safflower group, safflower group, and herb pair group was significantly reduced, and the reduction in collagen deposition area in the herb pair group was greater.

[0059] 5. Detect the hydroxyproline (HYP) content in the liver tissues obtained in step 3. Hydroxyproline is a characteristic indicator of collagen related to the extracellular matrix (ECM).

[0060] Test results are shown Figure 3 The results showed that the hydroxyproline content in the model group was significantly increased compared with the control group. Compared with the model group, the hydroxyproline content in the safflower group, safflower group, and herb pair group was significantly decreased, with the decrease in hydroxyproline content in the herb pair group being more pronounced.

[0061] 6. Detect the levels of liver function indicators (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP)) and liver fibrosis indicators (type III procollagen amino-terminal peptide (PⅢP), type IV collagen (IV-C)) in the serum obtained in step 3.

[0062] Test results are shown Figure 4 The results showed that, compared with the blank group, the serum liver function indicators (such as ALT, AST, ALP) and liver fibrosis indicators (such as PⅢP, IV-C) in the model group were significantly increased; compared with the model group, the liver function indicators (such as ALT, AST, ALP) and liver fibrosis indicators (such as PⅢP, IV-C) in the safflower group, safflower group and drug pair group were significantly decreased, and the degree of decrease in the above indicators was more obvious in the drug pair group.

[0063] In summary, the results of Masson staining of liver tissue and detection of hydroxyproline content in mouse models of liver fibrosis, as well as the detection of liver function indicators (such as ALT, AST, ALP) and liver fibrosis indicators (such as PⅢP, IV-C) in serum, indicate that the safflower-blue safflower herb pair composition prepared in Example 1 can significantly inhibit liver fibrosis, and its efficacy is significantly higher than that of total flavonoids from safflower or total flavonoids from blue safflower.

[0064] Example 3: Application of the safflower-blue safflower herb combination in the prevention and treatment of hepatocellular carcinoma. I. In vivo experiments 1. One hundred 3-week-old C57BL / 6J mice were housed in an SPF-grade environment. During the housing period, the room temperature was maintained at 20±2℃, humidity at 45%-55%, with alternating day and night light, and free access to food and water. They were then randomly divided into five groups: a control group, a model group, a low-dose drug pair group, a high-dose drug pair group, and an obeticholic acid group, with 20 mice in each group. The following treatments were administered to each group: Control group (or blank group): fed with a standard diet for 27 weeks; Model group: First, diethylnitrosamine (DEN) was injected intraperitoneally at a dose of 25 mg / kg body weight, and the animals were fed a regular diet for 3 to 6 weeks. Then, they were fed a fructose-rich, high-fat diet (HFD) for 24 weeks; at the same time, diethylnitrosamine was injected intraperitoneally at a dose of 25 mg / kg body weight in the 8th and 12th weeks of feeding. Low-dose group of the drug pair: First, diethylnitrosamine (DEN) was injected intraperitoneally at a dose of 25 mg / kg body weight, and the animals were fed a regular diet for 3 to 6 weeks. Then, they were fed a high-fat diet (HFD) with fructose for 24 weeks. At the same time, diethylnitrosamine was injected intraperitoneally at a dose of 25 mg / kg body weight in the 8th and 12th weeks of feeding. Starting from the 8th week of feeding, the safflower-blue peony drug pair composition solution (the solute was the safflower-blue peony drug pair composition prepared in Example 1, and the solvent was 0.5% CMC-Na solution) was administered by gavage once a day at a dose of 100 mg / kg body weight. High-dose group of the drug pair: First, diethylnitrosamine (DEN) was injected intraperitoneally at a dose of 25 mg / kg body weight, and the animals were fed a regular diet for 3 to 6 weeks. Then, they were fed a high-fat diet (HFD) with fructose for 24 weeks. At the same time, diethylnitrosamine was injected intraperitoneally at a dose of 25 mg / kg body weight in the 8th and 12th weeks of feeding. Starting from the 8th week of feeding, the safflower-blue peony drug pair composition solution (the solute was the safflower-blue peony drug pair composition prepared in Example 1, and the solvent was 0.5% CMC-Na solution) was administered by gavage once a day at a dose of 200 mg / kg body weight. Obeticholic acid group (or positive control group): First, diethylnitrosamine (DEN) was injected intraperitoneally at a dose of 25 mg / kg body weight, and the animals were fed a regular diet for 3 to 6 weeks. Then, they were fed a fructose-rich, high-fat diet (HFD) for 24 weeks. At the same time, diethylnitrosamine was injected intraperitoneally at a dose of 25 mg / kg body weight in weeks 8 and 12 of feeding. Starting from week 8 of feeding, obeticholic acid aqueous solution was administered once daily by gavage at a dose of 15 mg / kg body weight.

[0065] The model group, the low-dose drug pair group, the high-dose drug pair group, and the obeticholic acid group all used diethylnitrosamine to induce the construction of a mouse model of hepatocellular carcinoma.

[0066] 2. During step 1, record the weight and condition of the mice weekly and analyze the general situation during the feeding process (such as activity, mental state, and weight changes).

[0067] The weight statistics of mice in each group during step 1 are shown in the figure. Figure 5 The results showed that, compared with the model group, both the low-dose and high-dose drug pairs could delay the weight gain of mice in the progression of hepatocellular carcinoma.

[0068] 3. After completing step 1, pathological examination of the liver tissue of each group of mice was performed. Specifically, the mice in each group were euthanized by spinal dislocation, their limbs were quickly fixed, their abdomens were fully exposed, and the area was disinfected with alcohol spray. The surrounding soft tissues were dissected and dissected to obtain intact liver tissue. The liver tissue was then fixed with 4% paraformaldehyde, and paraffin sections (4μm) and frozen sections (8μm) were prepared. Finally, HE staining and Masson staining were performed, and the staining was observed and photographed under a microscope. The area of ​​the blue region in the stained sections was statistically analyzed using ImageJ software.

[0069] HE staining results are shown in […]. Figure 6 The results showed that the model group exhibited abnormal hepatocyte structure, disordered hepatic cord arrangement, hepatocyte necrosis, increased lipid vacuoles, and a large number of inflammatory cells accumulating around the portal area. After administration of the low-dose and high-dose drug pairs, the swelling was reduced, the inflammatory cell infiltration and fibrous septa in the portal area decreased, and the hepatocyte arrangement became more regular. Furthermore, the improvement in the high-dose drug pair group was significantly greater than that in the low-dose drug pair group.

[0070] Masson staining results are shown in […]. Figure 7 The results showed that in the liver tissue of the model group, a large number of blue collagen fibers were deposited around the portal areas, hepatocytes aggregated, and the fibrous septa were significantly increased. Administration of the low-dose and high-dose drug pairs significantly reduced the accumulation of fibrous septa and collagen fibers, with the high-dose drug pair showing a significantly greater reduction than the low-dose drug pair.

[0071] The above results indicate that, compared with the model group, both the low-dose and high-dose drug pairs can delay the progression of hepatocellular carcinoma. Therefore, the safflower-blue clover drug pair composition prepared in Example 1 can prevent hepatocellular carcinoma.

[0072] II. In vitro experiments Huh-7 cells are a product of the Shanghai Cell Bank, Chinese Academy of Sciences, catalog number SCSP-526. Transwell chambers are a product of BD Biosciences, Inc. (USA), catalog number 353097. The BCA protein assay kit is a product of Solarbio Science & Technology Co., Ltd. (Beijing), catalog number PC0020.

[0073] 1. Preparation of Safflower-Sappanflower Anther Pair Composition A and Safflower-Sappanflower Anther Pair Composition B 1.6 parts by weight of safflower total flavonoid powder and 1 part by weight of safflower total flavonoid powder were mixed to obtain safflower-safflower herb pair composition A. That is, in safflower-safflower herb pair composition A, the mass ratio of safflower total flavonoid powder to safflower total flavonoid powder is 1.6:1.

[0074] 1.5 parts by weight of safflower total flavonoid powder and 1 part by weight of safflower total flavonoid powder were mixed to obtain safflower-safflower herb pair composition B. That is, in safflower-safflower herb pair composition B, the mass ratio of safflower total flavonoid powder to safflower total flavonoid powder is 1.5:1.

[0075] 2. The proliferative capacity of safflower-sappanflower anther pair A and safflower-sappanflower anther pair B on Huh-7 cells was detected by colony formation assay. (1) Collect Huh-7 cells in the logarithmic growth phase and blow them into single cells.

[0076] (2) After completing step (1), use 3×10 3 Cell / well density: Huh-7 cells were seeded into 6-well culture plates and cultured for one week in fresh complete culture medium (Gibco product, catalog number 12430-047).

[0077] (3) After completing step (2), treat with safflower-blue potted flower anther combination solution or 0.5% CMC-Na solution (i.e. blank group) for 24 hours, then replace with fresh complete culture medium and place in an incubator to continue culturing for 7-10 days.

[0078] The safflower-blue violet herb pair composition solution is either safflower-blue violet herb pair composition A solution (high dose group of the herb pair) or safflower-blue violet herb pair composition B solution (low dose group of the herb pair).

[0079] The solute in the safflower-blue safflower anther pair composition A solution is safflower-blue safflower anther pair composition A, and the solvent is 0.5% CMC-Na solution; the dosage is 98 μg / ml.

[0080] The solute in the safflower-blue safflower anther pair composition B solution is safflower-blue safflower anther pair composition B, and the solvent is 0.5% CMC-Na solution; the dosage is 50 μg / ml.

[0081] (4) After completing step (3), take the 6-well culture plate, gently rinse with PBS, fix with 4% paraformaldehyde for 15-20 min, stain with 0.1% crystal violet, rinse slowly and repeatedly with water, and air dry. Take pictures and count under a microscope.

[0082] Test results are shown Figure 8 (A shows the staining results of cell clones, and B shows the statistical results of cell clone counts). The results indicate that both safflower-blue violet herb pair composition A and safflower-blue violet herb pair composition B can inhibit the proliferation of Huh-7 cells, and the inhibitory ability of the high-dose group of the herb pair is significantly higher than that of the low-dose group. That is, the inhibitory ability of safflower-blue violet herb pair composition A on the proliferation of Huh-7 cells is significantly higher than that of safflower-blue violet herb pair composition B.

[0083] 3. The migration ability of safflower-blue safflower anther pair A and safflower-blue safflower anther pair B on Huh-7 cells was detected by Transwell assay. The porous membrane in the Transwell cell (8 µm, for 24-well plates) is a polycarbonate membrane with micropores.

[0084] (1) Collect Huh-7 cells in the logarithmic growth phase and use (2-4)×10 5 Cells were added to complete culture medium without FBS at a density of cells / mL to prepare a cell suspension.

[0085] (2) After completing step (1), add 200 µL of cell suspension to the upper chamber of the Transwell chamber and add 600 µL of complete culture medium containing 10% (v / v) FBS to the lower chamber; then place the Transwell chamber in a cell culture incubator and incubate at 37°C and 5% CO2 for 24 hours. After that, discard the culture medium in the upper chamber and add complete culture medium (i.e. blank group) or complete culture medium containing safflower-blue potpourri anther pair composition, and continue to incubate for 24 hours.

[0086] The complete culture medium containing the safflower-blue clover herb pair composition is either the complete culture medium containing safflower-blue clover herb pair composition A (i.e., the high-dose group of the herb pair, with a dosage of 98 μg / ml) or the complete culture medium containing safflower-blue clover herb pair composition B (i.e., the low-dose group of the herb pair, with a dosage of 50 μg / ml).

[0087] (3) After completing step (2), carefully remove the upper chamber, aspirate the culture medium, wipe away the cells on the upper layer of the filter membrane with a cotton swab, wash once with PBS, fix with 4% paraformaldehyde for 5 min, stain with 0.5% crystal violet, wash twice with ultrapure water to remove excess dye, and let stand to air dry. Finally, observe the cells in the lower layer of the filter membrane under a microscope, randomly select different fields of view to count and calculate the average value.

[0088] Test results are shown Figure 9 (A represents staining results, and B represents the number of migrating cells.) The results showed that both safflower-blue violet herb pair composition A and safflower-blue violet herb pair composition B could inhibit the migration of Huh-7 cells, and the inhibitory ability of the high-dose group of the herb pair was significantly higher than that of the low-dose group. That is, the inhibitory ability of safflower-blue violet herb pair composition A on the migration of Huh-7 cells was significantly higher than that of safflower-blue violet herb pair composition B.

[0089] 4. Western blot analysis of the expression levels of Huh-7 cell transfer-related proteins by safflower-blue violet herb pair composition A and safflower-blue violet herb pair composition B. (1) Take Huh-7 cells that have completed step 3 and have migrated, extract total protein, i.e. total cell protein; then measure the protein concentration in total cell protein using the BCA protein concentration assay kit.

[0090] (2) After completing step (1), the total cell protein was detected by Western blot; the E-cadherin antibody used to detect E-cadherin protein, the N-cadherin antibody used to detect N-cadherin protein, the Vimentin antibody used to detect Vimentin protein, and the GAPDH antibody used to detect GAPDH protein were all products of Wuhan Sanying Biotechnology Co., Ltd.

[0091] Test results are shown Figure 10 (E-Cad is E-cadherin protein, N-Cad is N-cadherin protein, and Vim is Vimentin protein). The results showed that safflower-blue clover anther pair composition A and safflower-blue clover anther pair composition B could upregulate the expression level of the transfer marker protein E-cadherin and downregulate the expression levels of the transfer marker proteins N-cadherin and Vimentin.

[0092] The results showed that the safflower-blue clover herb pair composition A and safflower-blue clover herb pair composition B could treat hepatocellular carcinoma in a dose-dependent manner; the treatment of hepatocellular carcinoma was manifested by inhibiting the proliferation and migration of hepatocellular carcinoma cells (such as Huh-7 cells).

[0093] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A safflower-sappanflower herb pair composition, comprising total flavonoids from safflower and total flavonoids from sappanflower.

2. The safflower-blue safflower anther composition according to claim 1, characterized in that: The safflower-sappanflower herb pair composition consists of total flavonoids from safflower and total flavonoids from sappanflower.

3. The safflower-blue safflower anther composition according to claim 1 or 2, characterized in that: In the safflower-spotted flower herb pair composition, the mass ratio of total safflower flavonoids to total spotted flower flavonoids is (1-3):

1.

4. The safflower-blue safflower anther composition according to claim 3, characterized in that: In the safflower-spotted safflower herb pair composition, the mass ratio of total safflower flavonoids to total spotted safflower flavonoids is 2:1, 1.6:1, or 1.5:

1.

5. The use of the safflower-blue clover herb pair composition according to any one of claims 1 to 4 in the preparation of a medicament for inhibiting liver fibrosis.

6. The application according to claim 5, characterized in that: The inhibition of liver fibrosis is manifested in at least one of A1)-A3): A1) Reduces the accumulation of fibrous septa and / or collagen fibers in liver tissue; A2) Reduce the content of hydroxyproline in liver tissue; A3) Reduce the levels of liver function indicators and liver fibrosis indicators in serum.

7. The application according to claim 6, characterized in that: The liver function indicators include ALT, AST and / or ALP; The liver fibrosis markers include PⅢP and / or IV-C.

8. The use of the safflower-blue clover herb pair composition according to any one of claims 1 to 4 in the preparation of a medicament for the prevention and / or treatment of hepatocellular carcinoma.

9. The application according to claim 8, characterized in that: The prevention and / or treatment of hepatocellular carcinoma is manifested in at least one of B1)-B3): B1) Inhibits the proliferation of liver cancer cells; B2) Inhibits the migration of liver cancer cells; B3) Delays the progression of hepatocellular carcinoma.

10. The application according to claim 9, characterized in that: The delay in the progression of hepatocellular carcinoma is manifested in at least one of C1)-C4): C1) Delays weight gain during the progression of hepatocellular carcinoma; C2) Reduces the accumulation of fibrous septa and / or collagen fibers in liver tissue; C3) Reduce inflammatory cell infiltration and / or fibrous septa in the portal area; C4) causes hepatocytes to arrange themselves in a regular pattern.

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