Pharmaceutical composition for reversing pancreatic cancer gemcitabine resistance and application thereof

Abstract

The invention discloses a pharmaceutical composition for reversing pancreatic cancer gemcitabine resistance. The pharmaceutical composition is prepared from active ingredients such as a radix sophorae flavescentis extract, a poria cocos extract, emodin, glucosamine, gambogic acid, tranilast and aspirin. The preparation method of the sophora flavescens extract and the poria cocos extract comprises the steps of raw material pretreatment, dynamic ultrasonic-microwave synergistic extraction, membrane separation and nanofiltration, high-efficiency countercurrent chromatography purification, vacuum low-temperature concentration, drying and the like. The pharmaceutical composition can be prepared into a plurality of pharmaceutical preparations such as tablets, capsules, granules, cream, injections and the like. The invention further provides a preparation method of the oral tablet for reversing pancreatic cancer gemcitabine drug resistance, and the pharmaceutical composition has a remarkable treatment effect on a drug resistance mechanism formed by up-regulating expression of SREBP1 by histone lactylation mediated PKM2 through an AKT / mTOR pathway. The pharmaceutical composition provided by the invention provides a new treatment choice for pancreatic cancer patients, and has an important clinical application value.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals, and specifically discloses a pharmaceutical composition for reversing gemcitabine resistance of pancreatic cancer and application thereof. Background Art

[0002] Pancreatic cancer is a highly malignant digestive system tumor with an increasing incidence rate year by year worldwide. Due to the special location of the pancreas and the hidden nature of the disease, most patients are already in the middle or late stage when diagnosed, missing the best time for surgery. Gemcitabine is currently the first-line drug for pancreatic cancer chemotherapy. Although it can prolong the patient's survival to a certain extent, it is easy to develop drug resistance after long-term use, resulting in a significant decrease in the treatment effect. This is a major problem that needs to be solved in the clinic.

[0003] The development of drug resistance involves multiple mechanisms, among which histone lactylation-mediated PKM2 upregulating the expression of SREBP1 through the AKT / mTOR pathway is an important mechanism discovered in recent years. PKM2 is a subtype of pyruvate kinase, which is often highly expressed in tumor cells and participates in regulating the energy metabolism and proliferation of tumor cells. When tumor cells develop gemcitabine resistance, histone lactylation levels increase, which in turn promotes the expression and activity of PKM2, activates SREBP1 through the AKT / mTOR signaling pathway, and reduces the sensitivity of tumor cells to gemcitabine.

[0004] In view of this drug resistance mechanism, it is particularly important to develop a pharmaceutical composition that can reverse gemcitabine resistance in pancreatic cancer. However, existing drugs still have shortcomings in reversing drug resistance, improving efficacy and reducing side effects. Therefore, it is urgent to develop a new, efficient and safe pharmaceutical composition for reversing gemcitabine resistance in pancreatic cancer to meet clinical needs. Summary of the invention

[0005] In view of the above problems, the present invention discloses a pharmaceutical composition for reversing gemcitabine resistance in pancreatic cancer and its application. These active ingredients are extracted and purified through a specific preparation process to ensure high purity and high activity of the drug. At the same time, the present invention also aims at the specific mechanism of gemcitabine resistance in pancreatic cancer, and through the optimization of scientific proportions and drug preparation technology, effectively reverses the resistance, providing a new treatment option for pancreatic cancer patients.

[0006] The purpose of the present invention is achieved through the following technical solutions.

[0007] A pharmaceutical composition for reversing gemcitabine resistance in pancreatic cancer, comprising the following active ingredients by weight:

[0008]

[0009] Furthermore, in the above-mentioned pharmaceutical composition for reversing gemcitabine resistance in pancreatic cancer, the Sophora flavescens extract is prepared by the following steps:

[0010] 1) Raw material pretreatment and selection:

[0011] Select dried Sophora flavescens roots as raw materials, and use high-pressure micro-jet ultrafine grinding technology to grind the Sophora flavescens roots into particles with a particle size of less than 10 microns;

[0012] 2) Dynamic ultrasound-microwave synergistic extraction:

[0013] The crushed Sophora flavescens particles are mixed with a 60%-80% (v / v) ethanol solution at a mass volume ratio of 1:8-1:12 g / ml to prepare 5-40 L of a mixed solution;

[0014] The mixed solution is subjected to dynamic ultrasonic-microwave synergistic extraction technology, and intermittent extraction is performed at a temperature range of 45°C to 55°C under the combined action of ultrasonic waves with a frequency of 20kHz to 40kHz and microwaves with a power of 200W to 500W, and each extraction time is 10-20min, and the extraction is performed 3 times in total to obtain an extract;

[0015] 3) Membrane separation and nanofiltration:

[0016] The extract was initially filtered through a 0.2 micron microporous membrane to remove large particle impurities; then the filtrate was further separated using a nanofiltration membrane with a molecular weight cutoff of 500 Daltons to remove small molecular impurities and residues in the solvent;

[0017] 4) High performance countercurrent chromatography purification:

[0018] The nanofiltered extract was used as a sample and injected into a high performance countercurrent chromatography system, using a n-hexane-ethanol-water system for gradient elution;

[0019] By monitoring the contents of matrine and oxymatrine in the eluate, the eluate fraction rich in target components is collected;

[0020] 5) Vacuum low temperature concentration and drying:

[0021] The collected eluate segments are vacuum-low-temperature concentrated under the conditions of a vacuum degree of not less than 0.08 MPa and a temperature not exceeding 40°C until a concentrated solution is obtained; the concentrated solution is frozen at a temperature of -40°C to -50°C using vacuum freeze-drying technology, and then sublimated and dried under the condition of a vacuum degree of not less than 0.01 MPa to obtain a high-purity Sophora flavescens extract.

[0022] Furthermore, in the above-mentioned pharmaceutical composition for reversing gemcitabine resistance in pancreatic cancer, the Poria cocos extract is prepared by the following steps:

[0023] 1) Raw material pretreatment and activation:

[0024] Dried Poria cocos rhizomes were selected as raw materials, and preliminary degreasing treatment was carried out through supercritical carbon dioxide fluid extraction technology to remove oil and volatile impurities in the raw materials and improve the subsequent extraction efficiency;

[0025] The defatted Poria cocos raw material was subjected to microwave-assisted enzymatic hydrolysis, using a specific ratio of cellulase and pectinase at 40°C to 50°C for 30 minutes to destroy the cell wall structure, and the precipitate was obtained by centrifugation;

[0026] 2) Dynamic pulsed ultrasound-microwave synergistic extraction:

[0027] The pretreated Poria raw material is mixed with a 60%-80% (v / v) ethanol-water solution at a material-liquid ratio of 1:10-1:15 g / ml;

[0028] Dynamic pulsed ultrasound-microwave synergistic extraction technology was applied, in which ultrasound was in intermittent pulse mode, with a frequency of 20kHz to 40kHz, a pulse interval of 1 second, a microwave power of 300W to 600W, and extraction at 45℃ to 55℃. The total extraction time was divided into 3 cycles, each cycle was 10 minutes, and the interval in between was 5 minutes.

[0029] 3) Multi-stage membrane separation and nanofiltration:

[0030] The extract is first passed through a 0.45 micron microfiltration membrane to remove large particle impurities.

[0031] Then, multi-stage membrane separation technology was used to gradually separate components of different molecular weights through ultrafiltration membranes with molecular weight cutoffs of 10,000 Daltons, 5,000 Daltons and 1,000 Daltons;

[0032] Finally, a nanofiltration membrane with a molecular weight cutoff of less than 500 Daltons was used for further purification to remove small molecule impurities and residual solvents to obtain a high-purity Poria cocos extract;

[0033] 4) High performance countercurrent chromatography-mass spectrometry purification:

[0034] The nanofiltered extract was used as a sample and injected into a high performance countercurrent chromatography system, and gradient elution was performed using a n-hexane-acetonitrile system;

[0035] At the same time, liquid chromatography-mass spectrometry was used to monitor the components in the eluent in real time, and the target active ingredients were identified and collected based on the mass spectrometry data: the eluent segment where polysaccharides, flavonoids and triterpenoids were located;

[0036] 5) Vacuum low temperature concentration and spray drying:

[0037] The collected target eluate segment is vacuum-concentrated at low temperature under the conditions of a vacuum degree of not less than 0.09 MPa and a temperature not exceeding 40° C. to obtain a concentrated solution;

[0038] The concentrated solution is spray-dried using spray drying technology at an inlet temperature of 120° C. to 150° C. and an outlet temperature of 70° C. to 80° C. to obtain a powdered Poria extract.

[0039] The invention also discloses a pharmaceutical preparation containing the pharmaceutical composition.

[0040] Furthermore, the above-mentioned pharmaceutical preparation is selected from one of tablets, capsules, granules, creams and injections.

[0041] The present invention discloses an oral tablet for reversing gemcitabine resistance of pancreatic cancer, which is prepared by the following steps:

[0042] a) Raw material preparation and accurate weighing:

[0043] Accurately weigh the following active ingredients by weight: 40 parts of Sophora flavescens extract, 20-40 parts of Poria cocos extract, 10-20 parts of emodin, 10-15 parts of glucosamine, 5-10 parts of gambogic acid, 5-10 parts of tranilast, and 3-6 parts of aspirin;

[0044] Meanwhile, pharmaceutical excipients are prepared, including but not limited to fillers, binders, disintegrants, lubricants and glidants;

[0045] b) Mixing and granulation:

[0046] Using efficient mixing equipment, under a sterile and controlled environment, the active ingredient is fully mixed with the pharmaceutical excipients to form a uniform mixture; using wet granulation technology, an appropriate amount of wetting agent or binder is added to the mixture to form wet granules; the wet granules are dried in a fluidized bed or oven to remove excess water to obtain dry granules;

[0047] c) Granulation and screening:

[0048] Use granulation equipment to granulate the dried granules to ensure uniform particle size; use screening equipment to remove oversized or undersized particles and any possible impurities;

[0049] d) Tableting:

[0050] The sieved particles are fed into a tablet press and tableted according to the preset tablet specifications and weight;

[0051] During tableting, the pressure, speed and tablet thickness need to be controlled to ensure uniform tablet quality and moderate hardness;

[0052] f) Quality inspection and packaging:

[0053] The compressed tablets are subjected to quality tests, including but not limited to weight variation, hardness, friability, dissolution, etc.

[0054] Furthermore, in the above-mentioned oral tablet for reversing gemcitabine resistance in pancreatic cancer, the active ingredient content in each tablet is selected from 25 mg, 50 mg or 100 mg.

[0055] Furthermore, the present invention discloses the use of the above-mentioned pharmaceutical composition in the preparation of a drug for reversing gemcitabine resistance in pancreatic cancer, characterized in that the drug resistance mechanism is formed by PKM2 mediated by histone lactylation upregulating the expression of SREBP1 through the AKT / mTOR pathway.

[0056] Compared with the existing technology, the present invention has the following advantages and beneficial effects:

[0057] 1) Reversal of drug resistance: The drug composition proposed in the present invention is precisely designed for the specific mechanism of gemcitabine resistance in pancreatic cancer, namely, histone lactylation-mediated PKM2 upregulating the expression of SREBP1 through the AKT / mTOR pathway. Through scientific proportioning and optimization of drug preparation technology, the composition can effectively reverse the drug resistance of tumor cells, restore or enhance the killing effect of gemcitabine on pancreatic cancer cells, and thus improve the therapeutic effect.

[0058] 2) Improve drug activity and purity: The present invention adopts advanced extraction and purification technologies, such as high-pressure microfluidic ultrafine grinding, dynamic ultrasound-microwave synergistic extraction, membrane separation and nanofiltration, high-efficiency countercurrent chromatography purification, etc., to ensure the high purity and high activity of active ingredients such as Sophora flavescens extract and Poria cocos extract. This not only improves the efficacy of the drug, but also reduces possible side effects.

[0059] 3) Optimization of drug preparations: The present invention provides a variety of drug preparation forms, such as tablets, capsules, granules, creams, injections, etc., to meet the medication needs of different patients. In particular, for oral tablets, the present invention describes in detail its preparation process, including raw material preparation, mixed granulation, whole granule screening, tableting, and quality inspection steps, to ensure that the tablets are uniform in quality, moderate in hardness, and easy for patients to take and carry.

[0060] 4) Broaden treatment options: The pharmaceutical composition of the present invention provides a new treatment option for pancreatic cancer patients. In the case of limited existing treatment methods and easy drug resistance, the pharmaceutical composition of the present invention is expected to become a new breakthrough in the field of pancreatic cancer treatment, prolonging the survival of patients and improving their quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Pcan-1 cell growth inhibition rate;

[0062] Figure 2 Growth inhibition rate of drug-resistant Pcan-1 cells. DETAILED DESCRIPTION

[0063] An oral tablet for reversing gemcitabine resistance in pancreatic cancer, characterized in that it is prepared by the following steps:

[0064] a) Raw material preparation and accurate weighing:

[0065] Accurately weigh the following active ingredients by weight: 40 parts of Sophora flavescens extract, 20-40 parts of Poria cocos extract, 10-20 parts of emodin, 10-15 parts of glucosamine, 5-10 parts of gambogic acid, 5-10 parts of tranilast, and 3-6 parts of aspirin;

[0066] Meanwhile, pharmaceutical excipients are prepared, including but not limited to fillers, binders, disintegrants, lubricants and glidants;

[0067] b) Mixing and granulation:

[0068] Using efficient mixing equipment, under a sterile and controlled environment, the active ingredient is fully mixed with the pharmaceutical excipients to form a uniform mixture; using wet granulation technology, an appropriate amount of wetting agent or binder is added to the mixture to form wet granules; the wet granules are dried in a fluidized bed or oven to remove excess water to obtain dry granules;

[0069] c) Granulation and screening:

[0070] Use granulation equipment to granulate the dried granules to ensure uniform particle size; use screening equipment to remove oversized or undersized particles and any possible impurities;

[0071] d) Tableting:

[0072] The sieved particles are fed into a tablet press and tableted according to the preset tablet specifications and weight;

[0073] During tableting, the pressure, speed and tablet thickness need to be controlled to ensure uniform tablet quality and moderate hardness;

[0074] f) Quality inspection and packaging:

[0075] The compressed tablets are subjected to quality tests, including but not limited to weight variation, hardness, friability, dissolution, etc.

[0076] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below through examples, but it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0077] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0078] Example 1

[0079] An oral tablet for reversing gemcitabine resistance in pancreatic cancer, characterized in that it is prepared by the following steps:

[0080] a) Raw material preparation and accurate weighing:

[0081] Accurately weigh the following active ingredients by weight: 40 parts of Sophora flavescens extract, 20 parts of Poria cocos extract, 10 parts of emodin, 10 parts of glucosamine, 5 parts of gambogic acid, 5 parts of tranilast, and 3 parts of aspirin;

[0082] Meanwhile, pharmaceutical excipients are prepared, including but not limited to fillers, binders, disintegrants, lubricants and glidants;

[0083] b) Mixing and granulation:

[0084] Using efficient mixing equipment, under a sterile and controlled environment, the active ingredient is fully mixed with the pharmaceutical excipients to form a uniform mixture; using wet granulation technology, an appropriate amount of wetting agent or binder is added to the mixture to form wet granules; the wet granules are dried in a fluidized bed or oven to remove excess water to obtain dry granules;

[0085] c) Granulation and screening:

[0086] Use granulation equipment to granulate the dried granules to ensure uniform particle size; use screening equipment to remove oversized or undersized particles and any possible impurities;

[0087] d) Tableting:

[0088] The sieved particles are fed into a tablet press and tableted according to the preset tablet specifications and weight;

[0089] During tableting, the pressure, speed and tablet thickness need to be controlled to ensure that the tablets are of uniform quality and moderate hardness;

[0090] f) Quality inspection and packaging:

[0091] The compressed tablets are subjected to quality tests, including but not limited to weight variation, hardness, friability, dissolution, etc.

[0092] The sophora flavescens extract was purchased from Hubei Haijia Biotechnology Co., Ltd. Matrine PE (concentrated extraction fully water-soluble sophora flavescens powder).

[0093] The Poria cocos extract was purchased from Shanghai Zhongfeng Biotechnology Co., Ltd., brand Indian Bread PE.

[0094] Example 2

[0095] An oral tablet for reversing gemcitabine resistance in pancreatic cancer, characterized in that it is prepared by the following steps:

[0096] a) Raw material preparation and accurate weighing:

[0097] Accurately weigh the following active ingredients by weight: 40 parts of Sophora flavescens extract, 30 parts of Poria cocos extract, 15 parts of Rhein, 12.5 parts of glucosamine, 7.5 parts of Garcinia acid, 7.5 parts of Tranilast, and 4.5 parts of aspirin;

[0098] Meanwhile, pharmaceutical excipients are prepared, including but not limited to fillers, binders, disintegrants, lubricants and glidants;

[0099] b) Mixing and granulation:

[0100] Using efficient mixing equipment, under a sterile and controlled environment, the active ingredient is fully mixed with the pharmaceutical excipients to form a uniform mixture; using wet granulation technology, an appropriate amount of wetting agent or binder is added to the mixture to form wet granules; the wet granules are dried in a fluidized bed or oven to remove excess water to obtain dry granules;

[0101] c) Granulation and screening:

[0102] Use granulation equipment to granulate the dried granules to ensure uniform particle size; use screening equipment to remove oversized or undersized particles and any possible impurities;

[0103] d) Tableting:

[0104] The sieved particles are fed into a tablet press and tableted according to the preset tablet specifications and weight;

[0105] During tableting, the pressure, speed and tablet thickness need to be controlled to ensure uniform tablet quality and moderate hardness;

[0106] f) Quality inspection and packaging:

[0107] The compressed tablets are subjected to quality tests, including but not limited to weight variation, hardness, friability, dissolution, etc.

[0108] The sophora flavescens extract was purchased from Hubei Haijia Biotechnology Co., Ltd. Matrine PE (concentrated extraction fully water-soluble sophora flavescens powder).

[0109] The Poria cocos extract was purchased from Shanghai Zhongfeng Biotechnology Co., Ltd., brand Indian Bread PE.

[0110] Example 3

[0111] An oral tablet for reversing gemcitabine resistance in pancreatic cancer, characterized in that it is prepared by the following steps:

[0112] a) Raw material preparation and accurate weighing:

[0113] Accurately weigh the following active ingredients by weight: 40 parts of Sophora flavescens extract, 40 parts of Poria cocos extract, 20 parts of Rhein, 15 parts of glucosamine, 10 parts of Garcinia acid, 10 parts of Tranilast, and 6 parts of Aspirin;

[0114] Meanwhile, pharmaceutical excipients are prepared, including but not limited to fillers, binders, disintegrants, lubricants and glidants;

[0115] b) Mixing and granulation:

[0116] Using efficient mixing equipment, under a sterile and controlled environment, the active ingredient is fully mixed with the pharmaceutical excipients to form a uniform mixture; using wet granulation technology, an appropriate amount of wetting agent or binder is added to the mixture to form wet granules; the wet granules are dried in a fluidized bed or oven to remove excess water to obtain dry granules;

[0117] c) Granulation and screening:

[0118] Use granulation equipment to granulate the dried granules to ensure uniform particle size; use screening equipment to remove oversized or undersized particles and any possible impurities;

[0119] d) Tableting:

[0120] The sieved particles are fed into a tablet press and tableted according to the preset tablet specifications and weight;

[0121] During tableting, the pressure, speed and tablet thickness need to be controlled to ensure uniform tablet quality and moderate hardness;

[0122] f) Quality inspection and packaging:

[0123] The compressed tablets are subjected to quality tests, including but not limited to weight variation, hardness, friability, dissolution, etc.

[0124] The sophora flavescens extract was purchased from Hubei Haijia Biotechnology Co., Ltd. Matrine PE (concentrated extraction fully water-soluble sophora flavescens powder).

[0125] The Poria cocos extract was purchased from Shanghai Zhongfeng Biotechnology Co., Ltd., brand Indian Bread PE.

[0126] Example 4

[0127] Same as Example 2, but the Sophora flavescens extract and Poria cocos extract in this example were prepared by the following method.

[0128] The Sophora flavescens extract is prepared by the following steps:

[0129] 1) Raw material pretreatment and selection:

[0130] Select dried Sophora flavescens roots as raw materials, and use high-pressure micro-jet ultrafine grinding technology to grind the Sophora flavescens roots into particles with a particle size of less than 10 microns;

[0131] 2) Dynamic ultrasound-microwave synergistic extraction:

[0132] The crushed Sophora flavescens particles were mixed with a 70% (v / v) ethanol solution at a mass volume ratio of 1:10 g / ml to prepare 20 L of a mixed solution;

[0133] The mixed solution was subjected to dynamic ultrasonic-microwave synergistic extraction technology, and intermittent extraction was performed at a temperature range of 50°C under the combined action of ultrasonic waves with a frequency of 30kHz and microwaves with a power of 350W. Each extraction time was 15min, and the extraction was performed 3 times in total to obtain an extract.

[0134] 3) Membrane separation and nanofiltration:

[0135] The extract was initially filtered through a 0.2 micron microporous membrane to remove large particle impurities; then the filtrate was further separated using a nanofiltration membrane with a molecular weight cutoff of 500 Daltons to remove small molecular impurities and residues in the solvent;

[0136] 4) High performance countercurrent chromatography purification:

[0137] The nanofiltered extract was used as a sample and injected into a high performance countercurrent chromatography system, using a n-hexane-ethanol-water system for gradient elution;

[0138] By monitoring the contents of matrine and oxymatrine in the eluate, the eluate fraction rich in target components is collected;

[0139] 5) Vacuum low temperature concentration and drying:

[0140] The collected eluate segments are vacuum-low-temperature concentrated under the conditions of a vacuum degree of not less than 0.08 MPa and a temperature not exceeding 40°C until a concentrated solution is obtained; the concentrated solution is frozen at a temperature of -40°C to -50°C using vacuum freeze-drying technology, and then sublimated and dried under the condition of a vacuum degree of not less than 0.01 MPa to obtain a high-purity Sophora flavescens extract.

[0141] The Poria cocos extract is prepared by the following steps:

[0142] 1) Raw material pretreatment and activation:

[0143] Dried Poria cocos rhizomes were selected as raw materials, crushed through a 100-mesh sieve, and then subjected to preliminary degreasing treatment using supercritical carbon dioxide fluid extraction technology to remove oil and volatile impurities in the raw materials and improve subsequent extraction efficiency;

[0144] The defatted Poria raw material is subjected to microwave-assisted enzymatic hydrolysis, 200 U / ml cellulase and 400 U / ml pectinase are used to prepare an excess enzyme solution, which is treated at 40° C. to 50° C. for 30 minutes, and the precipitate is collected by centrifugation to obtain the pretreated Poria raw material;

[0145] 2) Dynamic pulsed ultrasound-microwave synergistic extraction:

[0146] The pretreated Poria raw material was mixed with a 70% (v / v) ethanol-water solution at a material-liquid ratio of 1:12 g / ml;

[0147] Dynamic pulsed ultrasound-microwave synergistic extraction technology was applied, in which ultrasound was in intermittent pulse mode, with a frequency of 30kHz, a pulse interval of 1 second, a microwave power of 450W, and extraction at 50°C. The total extraction time was divided into 3 cycles, each cycle was 10 minutes, and the interval was 5 minutes.

[0148] 3) Multi-stage membrane separation and nanofiltration:

[0149] The extract is first passed through a 0.45 micron microfiltration membrane to remove large particle impurities.

[0150] Then, multi-stage membrane separation technology was used to gradually separate components of different molecular weights through ultrafiltration membranes with molecular weight cutoffs of 10,000 Daltons, 5,000 Daltons and 1,000 Daltons;

[0151] Finally, a nanofiltration membrane with a molecular weight cutoff of less than 500 Daltons was used for further purification to remove small molecule impurities and residual solvents to obtain a high-purity Poria cocos extract;

[0152] 4) High performance countercurrent chromatography-mass spectrometry purification:

[0153] The nanofiltered extract was used as a sample and injected into a high performance countercurrent chromatography system, and gradient elution was performed using a n-hexane-acetonitrile system;

[0154] At the same time, liquid chromatography-mass spectrometry was used to monitor the components in the eluent in real time, and the target active ingredients were identified and collected based on the mass spectrometry data: the eluent segment where polysaccharides, flavonoids and triterpenoids were located;

[0155] 5) Vacuum low temperature concentration and spray drying:

[0156] The collected target eluate segment is vacuum-concentrated at low temperature under the conditions of a vacuum degree of not less than 0.09 MPa and a temperature not exceeding 40° C. to obtain a concentrated solution;

[0157] The concentrated solution was spray-dried by using spray drying technology at an inlet temperature of 120°C-150°C and an outlet temperature of 70°C-80°C to obtain a powdered Poria extract.

[0158] Comparative Example 1

[0159] The other ingredients are the same as those in Example 2 except that the Sophora flavescens extract is not included.

[0160] Comparative Example 2

[0161] The other parts are the same as those in Example 2 except that the Poria cocos extract is not included.

[0162] Comparative Example 3

[0163] The other ingredients are the same as those in Example 2 except that the extract of Sophora flavescens and the extract of Poria cocos are not included.

[0164] Test Case

[0165] The compositions of Examples 1-4 and Comparative Examples 1-3 (excluding pharmaceutical excipients, based on rheumatoid artemisinin, with an applied concentration of 10 μg / mL rheumatoid artemisinin), as well as Comparative Example 4 (10 μM gemcitabine) and Comparative Example 5 (20 μM gemcitabine) were tested by MTT colorimetry and applied in the cell culture medium of Pcan-1 cells or drug-resistant Panc-1 cells.

[0166] Pcan-1 cells or drug-resistant Panc-1 cells were seeded at a concentration of 1×10 5 The concentration of cells / mL was inoculated in a 96-well plate (100 μL / well), and blank zero wells (wells for culture medium plating) and control wells treated with phosphate buffer were set up at the same time. 5% CO 2 , cultured overnight at 37°C, and treated with the combination and gemcitabine. Five replicate wells were set for each sample, and the culture was carried out at 5% CO 2After incubation at 37°C for 72 hours, carefully aspirate the culture medium and add 100 μL of MTT solution (5 mg / mL, i.e., 0.5% MTT) prepared in the culture medium to each well; after continuing to culture for 1 hour, carefully aspirate the culture medium in the well. Add 100 μL of DMSO to each well and shake it on a shaker at a low speed for 10 minutes to fully dissolve the crystals; use an ELISA reader to measure the absorbance of each well at 570 nm to calculate the inhibition rate of the drug on cell growth.

[0167] The results are shown in Tables 1, 2 and Figure 1 , Figure 2 .

[0168] Table 1 Pcan-1 cell growth inhibition rate

[0169]

[0170] Table 2 Growth inhibition rate of drug-resistant Pcan-1 cells

[0171]

[0172]

[0173] From the data in Table 1 and Table 2, it can be seen that the pharmaceutical composition of the present invention can effectively reverse the drug resistance of gemcitabine and produce good growth inhibition on drug-resistant Pcan-1 cells. In particular, when it contains both Sophora flavescens extract and Poria cocos extract, it can synergistically increase the growth inhibition on drug-resistant Pcan-1 cells, and has good pharmaceutical prospects.

[0174] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Therefore, based on the innovative concept of the present invention, changes and modifications to the embodiments described herein, or equivalent structural or equivalent process transformations made using the contents of the specification and drawings of the present invention, directly or indirectly applying the above technical solutions to other related technical fields are all included in the protection scope of the patent of the present invention.

Claims

1. A pharmaceutical composition for reversing gemcitabine resistance in pancreatic cancer, characterized in that: The active ingredients include the following by weight:

2. A pharmaceutical composition for reversing gemcitabine resistance in pancreatic cancer according to claim 1, characterized in that: The Sophora flavescens extract is prepared by the following steps: 1) Raw material pretreatment and selection: Select dried Sophora flavescens roots as raw materials, and use high-pressure micro-jet ultrafine grinding technology to grind the Sophora flavescens roots into particles with a particle size of less than 10 microns; 2) Dynamic ultrasound-microwave synergistic extraction: The crushed Sophora flavescens particles are mixed with a 60%-80% (v / v) ethanol solution at a mass volume ratio of 1:8-1:12 g / ml to prepare 5-40 L of a mixed solution; The mixed solution is subjected to dynamic ultrasonic-microwave synergistic extraction technology, and intermittent extraction is performed at a temperature range of 45°C to 55°C under the combined action of ultrasonic waves with a frequency of 20kHz to 40kHz and microwaves with a power of 200W to 500W, and each extraction time is 10-20min, and the extraction is performed 3 times in total to obtain an extract; 3) Membrane separation and nanofiltration: The extract was initially filtered through a 0.2 micron microporous membrane to remove large particle impurities; then the filtrate was further separated using a nanofiltration membrane with a molecular weight cutoff of 500 Daltons to remove small molecular impurities and residues in the solvent; 4) High performance countercurrent chromatography purification: The nanofiltered extract was used as a sample and injected into a high performance countercurrent chromatography system, using a n-hexane-ethanol-water system for gradient elution; By monitoring the contents of matrine and oxymatrine in the eluate, the eluate fraction rich in target components is collected; 5) Vacuum low temperature concentration and drying: The collected eluate segments are vacuum-low-temperature concentrated under the conditions of a vacuum degree of not less than 0.08 MPa and a temperature not exceeding 40°C until a concentrated solution is obtained; the concentrated solution is frozen at a temperature of -40°C to -50°C using vacuum freeze-drying technology, and then sublimated and dried under the condition of a vacuum degree of not less than 0.01 MPa to obtain a high-purity Sophora flavescens extract.

3. A pharmaceutical composition for reversing gemcitabine resistance in pancreatic cancer according to claim 1, characterized in that: The Poria cocos extract is prepared by the following steps: 1) Raw material pretreatment and activation: Dried Poria cocos rhizomes were selected as raw materials, and preliminary degreasing treatment was carried out through supercritical carbon dioxide fluid extraction technology to remove oil and volatile impurities in the raw materials and improve the subsequent extraction efficiency; The defatted Poria cocos raw material was subjected to microwave-assisted enzymatic hydrolysis, using a specific ratio of cellulase and pectinase at 40°C to 50°C for 30 minutes to destroy the cell wall structure, and the precipitate was obtained by centrifugation; 2) Dynamic pulsed ultrasound-microwave synergistic extraction: The pretreated Poria raw material is mixed with a 60%-80% (v / v) ethanol-water solution at a material-liquid ratio of 1:10-1:15 g / ml; Dynamic pulsed ultrasound-microwave synergistic extraction technology was applied, in which ultrasound was in intermittent pulse mode, with a frequency of 20kHz to 40kHz, a pulse interval of 1 second, a microwave power of 300W to 600W, and extraction at 45℃ to 55℃. The total extraction time was divided into 3 cycles, each cycle was 10 minutes, and the interval in between was 5 minutes. 3) Multi-stage membrane separation and nanofiltration: The extract first passes through a 0.45-micron microfiltration membrane to remove large particle impurities; Then, multi-stage membrane separation technology was used to gradually separate components of different molecular weights through ultrafiltration membranes with molecular weight cutoffs of 10,000 Daltons, 5,000 Daltons and 1,000 Daltons; Finally, a nanofiltration membrane with a molecular weight cutoff of less than 500 Daltons was used for further purification to remove small molecule impurities and residual solvents to obtain a high-purity Poria cocos extract; 4) High performance countercurrent chromatography-mass spectrometry purification: The nanofiltered extract was used as a sample and injected into a high performance countercurrent chromatography system, and gradient elution was performed using a n-hexane-acetonitrile system; At the same time, liquid chromatography-mass spectrometry was used to monitor the components in the eluent in real time, and the target active ingredients were identified and collected based on the mass spectrometry data: the eluent segment where polysaccharides, flavonoids and triterpenoids were located; 5) Vacuum low temperature concentration and spray drying: The collected target eluate segment is vacuum-concentrated at low temperature under the conditions of a vacuum degree of not less than 0.09 MPa and a temperature not exceeding 40° C. to obtain a concentrated solution; The concentrated solution is spray-dried using spray drying technology at an inlet temperature of 120° C. to 150° C. and an outlet temperature of 70° C. to 80° C. to obtain a powdered Poria extract.

4. A pharmaceutical preparation, characterized in that A pharmaceutical composition comprising the pharmaceutical composition according to any one of claims 1 to 3.

5. The pharmaceutical preparation according to claim 4, characterized in that The preparation is selected from the group consisting of tablets, capsules, granules, creams and injections.

6. An oral tablet for reversing gemcitabine resistance in pancreatic cancer, characterized in that: Prepared by the following steps: a) Raw material preparation and accurate weighing: Accurately weigh the following active ingredients by weight: 40 parts of Sophora flavescens extract, 20-40 parts of Poria cocos extract, 10-20 parts of emodin, 10-15 parts of glucosamine, 5-10 parts of gambogic acid, 5-10 parts of tranilast, and 3-6 parts of aspirin; Meanwhile, pharmaceutical excipients are prepared, including but not limited to fillers, binders, disintegrants, lubricants and glidants; b) Mixing and granulation: Using efficient mixing equipment, under a sterile and controlled environment, the active ingredient is fully mixed with the pharmaceutical excipients to form a uniform mixture; using wet granulation technology, an appropriate amount of wetting agent or binder is added to the mixture to form wet granules; the wet granules are dried in a fluidized bed or oven to remove excess water to obtain dry granules; c) Granulation and screening: Use granulation equipment to granulate the dried granules to ensure uniform particle size; use screening equipment to remove oversized or undersized particles and any possible impurities; d) Tableting: The sieved particles are fed into a tablet press and tableted according to the preset tablet specifications and weight; During tableting, the pressure, speed and tablet thickness need to be controlled to ensure uniform tablet quality and moderate hardness; f) Quality inspection and packaging: The compressed tablets are subjected to quality tests, including but not limited to weight variation, hardness, friability, dissolution, etc.

7. The oral tablet for reversing gemcitabine resistance in pancreatic cancer according to claim 6, characterized in that: The active ingredient content in each tablet is selected from 25 mg, 50 mg or 100 mg.

8. Use of the pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a drug for reversing gemcitabine resistance in pancreatic cancer, characterized in that: The drug resistance mechanism is formed by PKM2 mediated by histone lactylation upregulating the expression of SREBP1 through the AKT / mTOR pathway.

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