Traditional Chinese medicine decoction for inhibiting invasion and metastasis of cervical cancer and preparation method thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a traditional Chinese medicine decoction for inhibiting invasion and metastasis of cervical cancer and a preparation method thereof.The traditional Chinese medicine decoction is prepared from, by weight, 20-30 parts of radix rehmanniae praeparata, 15-30 parts of radix astragali, 10-20 parts of rhizoma curcumae longae, 10-15 parts of deer-horn glue, 3-6 parts of ginger charcoal, 3-6 parts of licorice roots, 6-10 parts of mixed medicine residues and 40-80 parts of volatile oil inclusion liquid. Volatile oil of cortex cinnamomi, semen brassicae and herba ephedrae is prepared into an inclusion compound through cyclodextrin, the stability and bioavailability of a liquid preparation are improved, turmeric and astragalus membranaceus are introduced on the basis of a classic prescription Yanghe decoction to form multi-target cooperation, cellulase and pectinase are adopted for enzymolysis auxiliary extraction, the dissolution rate of effective components is increased, and the bioavailability of the liquid preparation is improved. The prepared traditional Chinese medicine decoction can effectively inhibit proliferation, migration and invasion of cervical cancer cells under the hypoxic condition, and the action mechanism of the traditional Chinese medicine decoction is closely related to down-regulation of an HIF-1alpha / CXCL6 signal axis.

Description

A traditional Chinese medicine decoction for inhibiting the invasion and metastasis of cervical cancer and its preparation method Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation technology, specifically relating to a traditional Chinese medicine decoction for inhibiting the invasion and metastasis of cervical cancer and its preparation method. Background Technology

[0002] Cervical cancer is one of the most common malignant tumors of the female reproductive system, and its highly invasive and metastatic characteristics are the main reasons for poor prognosis. In the hypoxic microenvironment within solid tumors, the stability of hypoxia-inducible factor HIF-1α is enhanced, which in turn transcribes and activates a series of downstream target genes, such as the chemokine CXCL6, to jointly promote the proliferation, migration, invasion, and angiogenesis of tumor cells, forming a positive feedback loop of malignant progression.

[0003] In Traditional Chinese Medicine (TCM) theory, cervical cancer is often classified as Yin carbuncle, its pathogenesis mainly involving Yang deficiency and blood depletion, cold stagnation and phlegm accumulation. Yanghe Decoction, a classic formula for treating Yin carbuncle, has the effects of warming Yang and replenishing blood, dispelling cold and relieving stagnation, and has shown potential in inhibiting tumor growth in modern research. However, the original formula of classic Yanghe Decoction mainly focuses on warming Yang and dispelling cold, lacking active pharmaceutical ingredients that directly regulate the HIF-1α / CXCL6 signaling axis, thus limiting its effectiveness in specifically blocking tumor invasion and metastasis mechanisms.

[0004] In terms of the preparation process of traditional Chinese medicine decoctions, due to the dense structure of plant cell walls, conventional decoction methods are insufficient to break down these cell walls. This results in low dissolution rates of macromolecular immunomodulatory components such as Astragalus polysaccharides and Rehmannia glutinosa polysaccharides, leading to an unstable pharmacodynamic basis. Furthermore, volatile oil components in herbs such as cinnamon and ephedra are easily lost through volatilization during heating or storage, while fat-soluble active ingredients such as curcumin have extremely low solubility in aqueous decoctions and are prone to oil-water separation. This directly results in low content of effective components, poor formulation stability, and low bioavailability in traditional Chinese medicine decoctions, preventing them from fully realizing their intended clinical efficacy.

[0005] Therefore, based on the traditional effects of Yanghe Decoction in warming yang and dispelling cold, through formulation optimization and process innovation, a traditional Chinese medicine preparation has been developed that can specifically regulate the hypoxic microenvironment of tumors, inhibit the invasion and metastasis of cervical cancer, and has stable and controllable quality. It has important clinical significance and application value. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a traditional Chinese medicine decoction for inhibiting the invasion and metastasis of cervical cancer and its preparation method. Based on the classic formula Yanghe Decoction, this decoction innovatively incorporates turmeric and astragalus to form a multi-target synergistic mechanism. In the preparation process, enzymatic hydrolysis-assisted extraction and cyclodextrin inclusion technology are used to significantly improve the extraction rate of effective components, the chemical stability of the preparation, and its bioavailability.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a traditional Chinese medicine decoction for inhibiting the invasion and metastasis of cervical cancer. The decoction comprises the following ingredients in parts by weight: 20-30 parts of Rehmannia glutinosa, 15-30 parts of Astragalus membranaceus, 10-20 parts of Curcuma longa, 10-15 parts of deer antler glue, 3-6 parts of charred ginger, 3-6 parts of Glycyrrhiza uralensis, 6-10 parts of mixed dregs, and 40-80 parts of volatile oil encapsulation solution.

[0009] Furthermore, the volatile oil encapsulation liquid comprises the following raw materials: cinnamon, white mustard seed, ephedra and hydroxypropyl-β-cyclodextrin, wherein the mass ratio of cinnamon, white mustard seed and ephedra is 3-6:3-6:2-5:10-20.

[0010] Furthermore, the mixed residue is an intermediate product in the preparation process of volatile oil encapsulation solution.

[0011] Furthermore, the preparation method of the volatile oil inclusion solution is as follows:

[0012] S1: Weigh 3-6 parts of cinnamon, 3-6 parts of white mustard seed and 2-5 parts of ephedra, crush them separately and sieve them to obtain mixed volatile oil medicinal materials. Extract them by steam distillation, collect the distillate, let it stand to separate the layers, separate the upper oil phase to obtain mixed volatile oil, filter the distillation residue to obtain mixed medicinal residue.

[0013] S2: Hydroxypropyl-β-cyclodextrin was prepared into a saturated aqueous solution to obtain the inclusion carrier solution;

[0014] S3: Slowly add the mixed volatile oil to the inclusion carrier liquid, and stir evenly under ultrasonic assistance to obtain the volatile oil inclusion liquid.

[0015] This invention also provides a method for preparing a traditional Chinese medicine decoction to inhibit the invasion and metastasis of cervical cancer, specifically including the following steps:

[0016] Step 1: Weigh 20-30 parts of Rehmannia glutinosa, 3-6 parts of Glycyrrhiza uralensis, 10-20 parts of Curcuma longa and 15-30 parts of Astragalus membranaceus, crush and sieve them separately and mix them evenly. Combine them with the mixed residue prepared in S1, add purified water and mix evenly to obtain the extraction mixture. Adjust the pH of the extraction mixture to 5.0-6.0, add cellulase and pectinase and carry out enzymatic hydrolysis to obtain the enzymatic hydrolysis mixture.

[0017] Step 2: Weigh 3-6 parts of ginger charcoal, add it to the enzymatic hydrolysis mixture and decoct together, filter to obtain the first decoction and the dregs, add purified water to the dregs again, decoct a second time, filter to obtain the second decoction, combine the first decoction and the second decoction to obtain the decoction.

[0018] Step 3: While stirring, slowly add the deer antler glue to the decoction. After it is evenly dispersed, concentrate it under reduced pressure to a relative density of 1.05-1.10. Add 95% ethanol by volume to make the alcohol content reach 65%. Let it stand to precipitate, collect the supernatant, filter, and concentrate it again under reduced pressure until there is no alcohol smell to obtain the concentrated medicine.

[0019] Step 4: Add the concentrated medicinal liquid to the volatile oil encapsulation liquid, add purified water to the specified volume, stir evenly to obtain a semi-finished decoction, filter, fill and sterilize to obtain a traditional Chinese medicine decoction that inhibits the invasion and metastasis of cervical cancer.

[0020] Furthermore, the deer antler glue is in the form of a liquid deer antler glue obtained through melting treatment.

[0021] Furthermore, the amount of cellulase added is 0.5%-1.0% of the total weight of Rehmannia glutinosa, Glycyrrhiza uralensis, Curcuma longa and Astragalus membranaceus.

[0022] Furthermore, the amount of pectinase added is 0.5%-1.0% of the total weight of Rehmannia glutinosa, Glycyrrhiza uralensis, Curcuma longa and Astragalus membranaceus.

[0023] The beneficial effects achieved by this invention are as follows:

[0024] This invention, based on the classic Yanghe Decoction formula, adds turmeric and astragalus, and systematically optimizes the proportions of each herb to achieve synergistic effects, significantly enhancing the formula's activity against cervical cancer invasion and metastasis. Curcumin in turmeric has been shown to specifically target and inhibit the transcriptional activity of hypoxia-inducible factor HIF-1α, blocking its regulatory effect on downstream CXCL6; astragalus polysaccharides in astragalus can significantly regulate the tumor immune microenvironment and inhibit the PI3K / Akt signaling pathway. These two ingredients, combined with the original warming and blood-tonifying, cold-dispersing and stagnation-clearing herbs in Yanghe Decoction, form a multi-synergistic mechanism targeting the HIF-1α / CXCL6 axis, enhancing immune regulation, and warming and dispersing cold, significantly enhancing the overall pharmacological activity of the formula in inhibiting cervical cancer invasion and metastasis.

[0025] This invention introduces cellulase and pectinase for enzymatic hydrolysis-assisted extraction of medicinal materials such as Rehmannia glutinosa, Astragalus membranaceus, Curcuma longa, and Glycyrrhiza uralensis. This method effectively disrupts the plant cell wall structure, significantly improving the dissolution rate of macromolecular active ingredients such as Astragalus polysaccharides, curcumin, and Rehmannia glutinosa polysaccharides, thus ensuring the material basis for the enhanced immunomodulatory and antitumor activities in the decoction. Simultaneously, hydroxypropyl-β-cyclodextrin inclusion complex technology is used for the volatile oil components of Cinnamomum cassia, Sinapis alba, and Ephedra sinica. The hydrophobic cavity of hydroxypropyl-β-cyclodextrin can convert lipid-soluble components into water-soluble inclusion complexes, successfully solving the problem of volatile oil's volatility and poor water solubility. It also significantly improves its chemical stability and bioavailability in liquid formulations, ensuring the efficacy of the active ingredients remains highly efficient and stable during storage and use.

[0026] The traditional Chinese medicine decoction designed in this invention introduces Astragalus membranaceus and Curcuma longa, and works in conjunction with Rehmannia glutinosa, deer antler glue and other warming and blood-tonifying herbs to achieve highly efficient intervention in the invasion and metastasis process of cervical cancer. The use of enzymatic extraction and inclusion technology significantly improves the pharmacodynamic material basis and clinical treatment effect of the product, providing a new and effective solution for clinical treatment. Attached Figure Description

[0027] Figure 1 shows the cell scratch test results of the traditional Chinese medicine decoction prepared in Example 2;

[0028] Figure 2 shows the statistical data of the cell scratch test of the traditional Chinese medicine decoction prepared in Example 2;

[0029] Figure 3 shows the Transwell migration and invasion ability determination of the traditional Chinese medicine decoction prepared in Example 2;

[0030] Figure 4 shows the Western Blot results of the traditional Chinese medicine decoction prepared in Example 2. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0033] Unless otherwise specified, all methods used in the following examples are conventional. Unless otherwise specified, all raw materials used in the following examples are newly purchased materials from the market. The Chinese medicinal herbs used, including Rehmannia glutinosa (processed), Astragalus membranaceus, Curcuma longa, deer antler glue, cinnamon, white mustard seed, charred ginger, Ephedra sinica, and licorice, are all standard processed medicinal materials according to the Chinese Pharmacopoeia. The hydroxypropyl-β-cyclodextrin is pharmaceutical excipient grade, with an average degree of substitution of 0.8, moisture content ≤10.0%, and heavy metal content ≤10 ppm. The cellulase is pharmaceutical excipient grade, with an enzyme activity of not less than 10,000 U / g. The pectinase is pharmaceutical excipient grade, with an enzyme activity of not less than 5,000 U / g.

[0034] Example 1: This example provides a traditional Chinese medicine decoction for inhibiting the invasion and metastasis of cervical cancer. The decoction includes the following ingredients in parts by weight: 20 parts Rehmannia glutinosa, 15 parts Astragalus membranaceus, 10 parts Curcuma longa, 10 parts Deer antler glue, 4 parts Ginger charcoal, 3 parts Glycyrrhiza uralensis, 0.4 parts Cellulase, 0.4 parts Pectinase, 6 parts Mixed dregs, and 40 parts Volatile Oil Encapsulation Solution.

[0035] The volatile oil encapsulation solution comprises the following raw materials in parts by weight: 4 parts cinnamon, 4 parts white mustard seed, 2 parts ephedra, and 12 parts hydroxypropyl-β-cyclodextrin. Its preparation method is as follows:

[0036] S1: Weigh 4 parts cinnamon, 4 parts white mustard seed and 2 parts ephedra, crush them separately and pass them through a 60-mesh sieve, mix them evenly to obtain mixed volatile oil medicinal materials, put them into a round bottom flask, add 8 times the amount of purified water (based on the total weight of the medicinal materials), extract by steam distillation for 4 hours, collect the distillate, let it stand at 4℃ for 12 hours, collect the upper oil phase to obtain mixed volatile oil, filter the distillation residue in the round bottom flask to obtain mixed medicinal residue;

[0037] S2: Weigh 12 portions of hydroxypropyl-β-cyclodextrin and add them to purified water at a mass-volume ratio of 1 g: 2 mL. Stir in a 50°C water bath until completely dissolved, and cool to room temperature to obtain the inclusion carrier solution.

[0038] S3: Slowly add the mixed volatile oil to the inclusion carrier liquid and stir with ultrasonic assistance at 50℃ and 250 W for 1.5 hours to obtain the volatile oil inclusion liquid.

[0039] This embodiment also provides a method for preparing a traditional Chinese medicine decoction to inhibit the invasion and metastasis of cervical cancer, specifically including the following steps:

[0040] Step 1: Weigh 20 parts of Rehmannia glutinosa, 3 parts of Glycyrrhiza uralensis, 10 parts of Curcuma longa, and 15 parts of Astragalus membranaceus. Crush them separately and pass them through a 40-mesh sieve. Mix them evenly and combine them with the mixed residue prepared in S1. Add purified water to make the total liquid-to-material ratio 10:1. Mix evenly to obtain the extraction mixture. Adjust the pH of the extraction mixture to 5.2, add 0.4 parts of cellulase and 0.4 parts of pectinase, and enzymatically hydrolyze in a 50℃ water bath for 2 hours to obtain the enzymatic hydrolysis mixture.

[0041] Step 2: Weigh 4 parts of ginger charcoal and add them to the enzymatic hydrolysis mixture. Boil twice. Boil for 1.5 hours for the first time, filter, and obtain the first decoction and the dregs. Add purified water to the dregs again until the total weight of the dregs is 8 times. Boil for 1 hour for the second time, filter, and obtain the second filtrate. Combine the first decoction and the second decoction to obtain the decoction.

[0042] Step 3: Weigh 10 parts of deer antler glue and slowly add it to the decoction while stirring at 55℃ and 400 rpm. Continue stirring for 25 minutes, then concentrate the solution under reduced pressure at 60℃ and -0.09 MPa to a relative density of 1.08 (60℃). While stirring, slowly add 95% ethanol to make the alcohol content reach 65%. Mix well and let stand at 4℃ for 24 hours. Collect the supernatant, filter, and concentrate it again under reduced pressure at 60℃ and -0.09 MPa until there is no alcohol odor to obtain the concentrated medicine solution.

[0043] Step 4: After the concentrated medicinal liquid cools to below 40℃, add it to the volatile oil inclusion solution, add purified water to the specified volume, stir evenly to obtain a semi-finished decoction, filter it through a 0.22 μm sterile filter membrane, fill it into a clean glass bottle, and autoclave it at 121℃ for 25 minutes to obtain a traditional Chinese medicine decoction that inhibits the invasion and metastasis of cervical cancer.

[0044] Example 2: This example provides a traditional Chinese medicine decoction for inhibiting the invasion and metastasis of cervical cancer. The decoction includes the following ingredients in parts by weight: 25 parts Rehmannia glutinosa, 20 parts Astragalus membranaceus, 15 parts Curcuma longa, 12 parts Deer antler glue, 5 parts Ginger charcoal, 5 parts Glycyrrhiza uralensis, 0.5 parts Cellulase, 0.5 parts Pectinase, 8 parts Mixed dregs, and 55 parts Volatile Oil Encapsulation Solution.

[0045] The volatile oil encapsulation solution comprises the following raw materials in parts by weight: 5 parts cinnamon, 5 parts white mustard seed, 3 parts ephedra, and 15 parts hydroxypropyl-β-cyclodextrin. Its preparation method is as follows:

[0046] S1: Weigh 5 parts cinnamon, 5 parts white mustard seed and 3 parts ephedra, crush them separately and pass them through a 60-mesh sieve, mix them evenly to obtain mixed volatile oil medicinal materials, put them into a round bottom flask, add 8 times the amount of purified water (based on the total weight of the medicinal materials), extract by steam distillation for 4 hours, collect the distillate, let it stand at 4℃ for 12 hours, collect the upper oil phase to obtain mixed volatile oil, filter the distillation residue in the round bottom flask to obtain mixed medicinal residue;

[0047] S2: Weigh 15 parts of hydroxypropyl-β-cyclodextrin and add it to purified water at a mass-volume ratio of 1 g: 2 mL. Stir in a 50°C water bath until completely dissolved. After cooling to room temperature, the inclusion carrier solution is obtained.

[0048] S3: Slowly add the mixed volatile oil to the inclusion carrier liquid and stir with ultrasonic assistance at 50℃ and 250 W for 1.5 hours to obtain the volatile oil inclusion liquid.

[0049] This embodiment also provides a method for preparing a traditional Chinese medicine decoction to inhibit the invasion and metastasis of cervical cancer, specifically including the following steps:

[0050] Step 1: Weigh 25 parts of Rehmannia glutinosa, 5 parts of Glycyrrhiza uralensis, 15 parts of Curcuma longa, and 20 parts of Astragalus membranaceus. Crush them separately and pass them through a 40-mesh sieve. Mix them evenly and combine them with the mixed residue prepared in S1. Add purified water to make the total liquid-to-material ratio 10:1. Mix evenly to obtain the extraction mixture. Adjust the pH of the extraction mixture to 5.2, add 0.5 parts of cellulase and 0.5 parts of pectinase, and enzymatically hydrolyze in a 50℃ water bath for 2 hours to obtain the enzymatic hydrolysis mixture.

[0051] Step 2: Weigh 5 parts of ginger charcoal and add them to the enzymatic hydrolysis mixture. Boil twice. Boil for 1.5 hours for the first time, filter, and obtain the first decoction and the dregs. Add purified water to the dregs again until the total weight of the dregs is 8 times. Boil for 1 hour for the second time, filter, and obtain the second filtrate. Combine the first decoction and the second decoction to obtain the decoction.

[0052] Step 3: Weigh 12 parts of deer antler glue and slowly add it to the decoction while stirring at 55℃ and 400 rpm. Continue stirring for 25 minutes, then concentrate the solution under reduced pressure at 60℃ and -0.09 MPa to a relative density of 1.08 (60℃). While stirring, slowly add 95% ethanol to make the alcohol content reach 65%. Mix well and let stand at 4℃ for 24 hours. Collect the supernatant, filter, and concentrate it again under reduced pressure at 60℃ and -0.09 MPa until there is no alcohol odor to obtain the concentrated medicine solution.

[0053] Step 4: After the concentrated medicinal liquid cools to below 40℃, add it to the volatile oil inclusion solution, add purified water to the specified volume, stir evenly to obtain a semi-finished decoction, filter it through a 0.22 μm sterile filter membrane, fill it into a clean glass bottle, and autoclave it at 121℃ for 25 minutes to obtain a traditional Chinese medicine decoction that inhibits the invasion and metastasis of cervical cancer.

[0054] Example 3: This example provides a traditional Chinese medicine decoction for inhibiting the invasion and metastasis of cervical cancer. The decoction includes the following ingredients in parts by weight: 30 parts Rehmannia glutinosa, 25 parts Astragalus membranaceus, 20 parts Curcuma longa, 15 parts Deer antler glue, 6 parts Ginger charcoal, 6 parts Glycyrrhiza uralensis, 0.6 parts Cellulase, 0.6 parts Pectinase, 10 parts Mixed dregs, and 75 parts Volatile Oil Encapsulation Solution.

[0055] The volatile oil encapsulation solution comprises the following raw materials in parts by weight: 6 parts cinnamon, 6 parts white mustard seed, 5 parts ephedra, and 20 parts hydroxypropyl-β-cyclodextrin. Its preparation method is as follows:

[0056] S1: Weigh 6 parts of cinnamon, 6 parts of white mustard seed and 5 parts of ephedra, crush them separately and pass them through a 60-mesh sieve, mix them evenly to obtain mixed volatile oil medicinal materials, put them into a round bottom flask, add 8 times the amount of purified water (based on the total weight of the medicinal materials), extract by steam distillation for 4 hours, collect the distillate, let it stand at 4℃ for 12 hours, collect the upper oil phase to obtain mixed volatile oil, filter the distillation residue in the round bottom flask to obtain mixed medicinal residue;

[0057] S2: Weigh 20 parts of hydroxypropyl-β-cyclodextrin and add it to purified water at a mass-volume ratio of 1 g: 2 mL. Stir in a 50°C water bath until completely dissolved. After cooling to room temperature, the inclusion carrier solution is obtained.

[0058] S3: Slowly add the mixed volatile oil to the inclusion carrier liquid and stir with ultrasonic assistance at 50℃ and 250 W for 1.5 hours to obtain the volatile oil inclusion liquid.

[0059] This embodiment also provides a method for preparing a traditional Chinese medicine decoction to inhibit the invasion and metastasis of cervical cancer, specifically including the following steps:

[0060] Step 1: Weigh 30 parts of Rehmannia glutinosa, 6 parts of Glycyrrhiza uralensis, 20 parts of Curcuma longa, and 25 parts of Astragalus membranaceus. Crush them separately and pass them through a 40-mesh sieve. Mix them evenly and combine them with the mixed residue prepared in S1. Add purified water to make the total liquid-to-material ratio 10:1. Mix evenly to obtain the extraction mixture. Adjust the pH of the extraction mixture to 5.3, add 0.6 parts of cellulase and 0.6 parts of pectinase, and enzymatically hydrolyze in a 50℃ water bath for 2 hours to obtain the enzymatic hydrolysate.

[0061] Step 2: Weigh 6 parts of ginger charcoal and add them to the enzymatic hydrolysis mixture. Boil twice. Boil for 1.5 hours for the first time, filter, and obtain the first decoction and the dregs. Add purified water to the dregs again until the total weight of the dregs is 8 times. Boil for 1 hour for the second time, filter, and obtain the second filtrate. Combine the first decoction and the second decoction to obtain the decoction.

[0062] Step 3: Weigh 15 parts of deer antler glue and slowly add it to the decoction while stirring at 55℃ and 400 rpm. Continue stirring for 25 minutes, then concentrate the solution under reduced pressure at 60℃ and -0.09 MPa to a relative density of 1.08 (60℃). While stirring, slowly add 95% ethanol to make the alcohol content reach 65%. Mix well and let stand at 4℃ for 24 hours. Collect the supernatant, filter, and concentrate again under reduced pressure at 60℃ and -0.09 MPa until there is no alcohol odor to obtain the concentrated medicine solution.

[0063] Step 4: After the concentrated medicinal liquid cools to below 40℃, add it to the volatile oil inclusion solution, add purified water to the specified volume, stir evenly to obtain a semi-finished decoction, filter it through a 0.22 μm sterile filter membrane, fill it into a clean glass bottle, and autoclave it at 121℃ for 25 minutes to obtain a traditional Chinese medicine decoction that inhibits the invasion and metastasis of cervical cancer.

[0064] The difference between Comparative Example 1 and Example 2 is that turmeric and astragalus were not added, and the original formula of the classic Yanghe Decoction was used. The rest of the parts are the same as Example 2.

[0065] The difference between Comparative Example 2 and Example 2 is that the enzymatic hydrolysis process was not used; the rest of the process is the same as in Example 2.

[0066] The difference between Comparative Example 3 and Example 2 is that the cyclodextrin inclusion process was not used; the rest is the same as Example 2.

[0067] The difference between Comparative Example 4 and Example 2 is that the enzymatic hydrolysis and inclusion process was not used; the rest is the same as Example 2.

[0068] HeLa cell proliferation activity test

[0069] After digesting and centrifuging HeLa cells in the logarithmic growth phase, the cell concentration was adjusted to 5 × 10⁻⁶. 3 Cells were seeded at a rate of 100 μL per well in 96-well plates, with 6 replicates per group. A blank control group (culture medium only), a normoxic control group (no drug intervention, normoxic culture), a hypoxic control group (no drug intervention, hypoxic culture), and each drug-treated experimental group were also included. After cell adhesion, the experimental groups were stimulated with drugs, while the control groups were treated with culture medium containing 10% drug-free serum. Subsequently, all experimental groups and the hypoxic control group were transferred to a hypoxic incubator at 1% O2, 5% CO2, and 37°C, while the normoxic control group remained in an incubator at 20% O2, 5% CO2, and 37°C. After 48 hours of drug treatment, 10 μL of CCK-8 solution was added to each well in the dark and incubated for another 2 hours. The absorbance (OD value) of each well was measured at 450 nm using an ELISA reader. The cell proliferation rate of each group was calculated as follows: proliferation rate (%) = (OD value of experimental group - OD value of blank control group / OD value of hypoxia control group - OD value of blank control group) × 100%. The experimental results are shown in Table 1.

[0070] Table 1 Results of cell proliferation activity test

[0071]

[0072] Cell scratch assay

[0073] HeLa cells in logarithmic growth phase were seeded into 6-well plates. Once the cells reached over 90% confluence, a straight scratch was made on each well using a sterile pipette tip. After washing with PBS, the culture medium was replaced. Experimental setup: Hypoxia model group (drug-free culture medium) and Example 2 group. All groups were cultured in a hypoxic incubator with 1% O2 and 5% CO2. At 0 hours and 24 hours after scratching, images were taken of the same scratch location under a microscope. The scratch width was measured using ImageJ software, and the scratch healing rate (%) was calculated using the formula: [(initial scratch width - 24-hour scratch width) / initial scratch width] × 100%. The experimental results are shown in Figures 1 and 2.

[0074] Transwell migration and invasion experiments

[0075] Transwell migration assay: Logarithmic growth phase HeLa cells were harvested, digested with trypsin, and then the concentration was adjusted to 5 × 10⁶ cells in serum-free DMEM medium. 5 Cells were added at a density of 100 μL / mL to the upper chamber of the Transwell chamber. In Example 2, serum-free medium containing the drug was added, while in the hypoxia control group, serum-free medium without the drug was added. 600 µL of DMEM medium containing 20% ​​fetal bovine serum was added to the lower chamber. Both Example 2 and the hypoxia control group were incubated at 1% O2, 5% CO2, and 37°C for 24 hours. Afterward, cells that had not penetrated the membrane in the upper chamber were wiped off, fixed with 4% paraformaldehyde, stained with crystal violet, and five fields of view were randomly selected under a microscope to count cells that had penetrated the membrane. The experiment was repeated three times.

[0076] Transwell invasion assay: Matrigel was diluted with serum-free medium at a ratio of 1:8 in advance, and 50 μL was placed in the upper chamber of each Transwell chamber and incubated at 37°C for 4 hours to solidify. Subsequent cell preparation, grouping intervention, culture conditions and counting methods were the same as those for the migration assay, except that the culture time was extended to 36 hours. The experimental results are shown in Figure 3.

[0077] Western Blot Experiment

[0078] HeLa cells were collected after 48 hours of hypoxia (1% O2) and drug intervention. Total protein was extracted from each group (hypoxia model group and Example 2 group), and the protein concentration of each sample was determined using a BCA protein quantification kit. 20 μg of total protein was subjected to SDS-PAGE, followed by transfer to a PVDF membrane. After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with primary antibodies HIF-1α, CXCL6, Ki67, and the internal control GAPDH antibody. After washing, the membrane was incubated with the corresponding secondary antibodies at room temperature for 1 hour. Developing was performed using an ECL Plus chemiluminescence kit, and the imaging results are shown in Figure 4.

[0079] Results Analysis

[0080] Table 1 shows that the traditional Chinese medicine decoctions prepared in Examples 1-3 all exhibited inhibitory effects on hypoxia-induced proliferation, with HeLa cell proliferation activity all below 75%. Among them, the group in Example 2 had the strongest anti-proliferative activity, at 34.2%. The anti-proliferative activity of the traditional Chinese medicine decoctions prepared in Comparative Example 1 (without turmeric and astragalus), Comparative Example 2 (without enzymatic hydrolysis), Comparative Example 3 (without cyclodextrin inclusion process), and Comparative Example 4 (without enzymatic hydrolysis and inclusion process) all decreased.

[0081] Figures 1 and 2 show that, 24 hours after drug intervention, the scratch healing rate in the hypoxic model group reached 58.3%, indicating that hypoxia effectively promoted cell migration. Compared with the model group, the Example 2 group showed a significant migration inhibition effect, with a healing rate of 23.7%.

[0082] Figure 3 shows that the hypoxia model group exhibited active migration ability, with a cell count of 157 per field of view. The Example 2 group significantly inhibited this process, reducing the number of migrating cells to 55 per field of view, with an inhibition rate of 64.9%. In the invasion experiment, the hypoxia model group had a higher number of invading cells, reaching 123 per field of view. The Example 2 group showed stronger inhibitory efficacy, sharply reducing the number of invading cells to 39 per field of view, with an inhibition rate of 68.3%.

[0083] Figure 4 shows that the traditional Chinese medicine decoction prepared in Example 2 can significantly downregulate the expression of HIF-1α, CXCL6 and ki67 proteins in HeLa cells under hypoxic conditions and inhibit the activation of the HIF-1α / CXCL6 signaling axis.

[0084] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0085] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine decoction for inhibiting the invasion and metastasis of cervical cancer, characterized in that, The herbal decoction comprises the following ingredients in parts by weight: 20-30 parts Rehmannia glutinosa (processed), 15-30 parts Astragalus membranaceus, 10-20 parts Curcuma longa, 10-15 parts Deer antler glue, 3-6 parts Ginger charcoal, 3-6 parts Glycyrrhiza uralensis, 6-10 parts mixed dregs, and 40-80 parts volatile oil inclusion solution; the volatile oil inclusion solution comprises the following ingredients: Cinnamomum cassia, Sinapis alba, Ephedra sinica, and hydroxypropyl-β-cyclodextrin; the mixed dregs are intermediate products in the preparation process of the volatile oil inclusion solution, and the preparation method of the volatile oil inclusion solution is as follows: S1: Cinnamomum cassia, Sinapis alba, and Ephedra sinica are pulverized, distilled, and separated to obtain mixed volatile oil; the distillation residue is filtered to obtain mixed dregs; S2: Hydroxypropyl-β-cyclodextrin is dissolved to obtain inclusion carrier liquid; S3: The mixed volatile oil is added dropwise to the inclusion carrier liquid to obtain the volatile oil inclusion solution.

2. The traditional Chinese medicine decoction for inhibiting the invasion and metastasis of cervical cancer according to claim 1, characterized in that, The mass ratio of cinnamon, white mustard seed, ephedra and hydroxypropyl-β-cyclodextrin is 3-6:3-6:2-5:10-20.

3. A method for preparing a traditional Chinese medicine decoction for inhibiting the invasion and metastasis of cervical cancer according to any one of claims 1-2, characterized in that, The specific steps are as follows: Step 1: Crush Rehmannia glutinosa, Glycyrrhiza uralensis, Curcuma longa, and Astragalus membranaceus, combine them with the mixed residue to obtain an extract mixture, add cellulase and pectinase to it to obtain an enzymatic hydrolysis mixture; Step 2: Add charred ginger to the enzymatic hydrolysis mixture, decoct and filter to obtain a decoction; Step 3: Add deer antler glue to the decoction, concentrate, precipitate with alcohol, filter, and concentrate again to obtain a concentrated medicinal liquid; Step 4: Add the concentrated medicinal liquid to the volatile oil encapsulation liquid, filter, fill and sterilize to obtain a traditional Chinese medicine decoction that inhibits the invasion and metastasis of cervical cancer.

4. The method for preparing a traditional Chinese medicine decoction for inhibiting the invasion and metastasis of cervical cancer according to claim 3, characterized in that, The deer antler glue is in the form of a liquid deer antler glue that has been melted and processed.

5. The method for preparing a traditional Chinese medicine decoction for inhibiting the invasion and metastasis of cervical cancer according to claim 3, characterized in that, The amount of cellulase added is 0.5%-1.0% of the total weight of Rehmannia glutinosa, licorice, turmeric and Astragalus membranaceus; the amount of pectinase added is 0.5%-1.0% of the total weight of Rehmannia glutinosa, licorice, turmeric and Astragalus membranaceus.

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