CN117100780A - New use of traditional Chinese medicine composition - Google Patents

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a novel application of a traditional Chinese medicine composition. The invention uses the traditional Chinese medicine composition of frankincense, myrrh, bezoar and musk as medicines, researches on the treatment of PC (pancreatic cancer) by utilizing in vivo and in vitro proliferation and apoptosis experiments, and uses CYP3A4 as a key target point of the traditional Chinese medicine composition for treating PC to play an anticancer role, and the traditional Chinese medicine composition can be used as an inhibitor of CYP3A4 for reducing the drug resistance of anticancer chemotherapeutics. The technical scheme of the invention has good application prospect in clinic.

Description

New use of traditional Chinese medicine composition

Technical Field

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a novel application of a traditional Chinese medicine composition.

Background

In recent years, the treatment of tumors by traditional Chinese medicine and traditional Chinese medicine compounds has become a hotspot in medical research at home and abroad. The traditional Chinese medicine has the advantages of multiple targets, multiple channels, stable structure, high safety and the like, and has small side effect. Traditional Chinese medicine extracts such as emodin, matrine, triptolide and the like have been found to be used for treating PC, and can improve clinical symptoms of patients and alleviate adverse reactions of radiotherapy and chemotherapy. Some traditional Chinese medicine compounds, such as pancreas-clearing and stasis-resolving formulas, compound kuh-seng injection and the like, also have good treatment effects.

The cytochrome P450 family (CYP) is a superfamily of proteins containing heme as a cofactor involved in many important reactions of drug synthesis, steroid and carcinogen metabolism. Although the liver is the main site of CYP-dependent drug metabolism, there are also different levels of CYP expression in extrahepatic tissues, especially the small intestine, and also the kidneys, lungs and brain, as can be seen, CYP expression can occur in a variety of tumour tissues. CYP plays a vital role in the metabolism of various exogenous substances, and it also affects the occurrence of tumors by closely correlating the ability to activate or inactivate carcinogens with chemical carcinogenesis. Treatment of cancer depends on the activity of the cytochrome P450 enzyme family, which is basically performed by the CYP3A4 and CYP3A5 enzymes. In addition, CYP3A4 is one of the most important enzymes in a CYP450 drug metabolism system, the expression level is the largest in various subtypes of CYP metabolic enzymes, and accounts for 30% -40% of all metabolic enzymes, and as a result of research, more than 50% of drugs are reaction substrates of the CYP3A4, and the enzymes have unsubstituted functions. Over-expression of this enzyme results in loss of efficacy, resulting in resistance to chemotherapeutic agents, a major problem in cancer patients. From a clinical point of view, although inhibition of CYP3A may be problematic, it may unexpectedly increase exposure to the substrate drug, resulting in toxicity. Inhibition may also be beneficial because considerable amounts of drugs are rapidly degraded by CYP3A, such that they cannot reach or maintain therapeutic plasma levels. Many drugs exert their pharmacological effects based on the mechanism of CYP3A4 inhibitors. Wherein irreversible CYP3A4 inhibitor drugs and related mechanisms are very important for rational use of therapeutic drugs. Mechanism-based CYP3A4 inhibition can reduce the first pass clearance of drugs in the liver and intestinal tract, thereby greatly altering pharmacokinetic behavior. To date, established inhibitors of CYP3A4 with clinically important mechanisms of action mainly include:

macrolide antibiotics (e.g., clarithromycin, erythromycin, etc.);

anti-HIV drugs (e.g., ritonavir, delavirdine);

antidepressants (e.g., fluoxetine, fluvoxamine);

calcium channel blockers (e.g., verapamil and diltiazem, etc.);

steroids and their modulators (e.g., gestodene gestone and mifepristone).

Because of the possible side effects of drugs and the different patient tolerance to drugs, there is still a need to develop a greater variety of CYP3A4 inhibitors.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a new application of a frankincense and myrrh traditional Chinese medicine composition, and aims to prepare a CYP3A4 inhibitor by using the traditional Chinese medicine composition of the frankincense and the myrrh and further prepare a cancer treatment medicine.

The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight:

55.7-278.5 parts of frankincense,

55.7-278.5 parts of myrrh,

1.52 to 7.6 portions of bezoar,

Musk 1.52-7.6 portions.

Preferably, the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight:

55.7 parts of frankincense,

55.7 parts of myrrh,

1.52 parts of bezoar,

Musk 1.52 parts.

Preferably, the olibanum is vinegar olibanum and the myrrh is vinegar myrrh.

Preferably, the traditional Chinese medicine composition is a preparation prepared by adding pharmaceutically acceptable auxiliary materials or auxiliary components into the active ingredients of the olibanum alcohol extract, the myrrh alcohol extract, the bezoar powder and the musk powder.

The invention also provides application of the traditional Chinese medicine composition or the extract thereof in preparing a CYP3A4 inhibitor.

Preferably, the CYP3A4 inhibitor is used to reduce resistance to anticancer chemotherapeutic agents.

The invention also provides application of the traditional Chinese medicine composition or the extract thereof in preparing medicines for treating cancers.

Preferably, the cancer therapeutic is for treating pancreatic cancer.

The invention also provides the application of the traditional Chinese medicine composition or the extract thereof in combination with a chemotherapeutic drug in preparing a cancer therapeutic drug.

Preferably, the cancer therapeutic is for treating pancreatic cancer.

Preferably, the chemotherapeutic agent is selected from at least one of gemcitabine, cisplatin, epirubicin hydrochloride, and te Mo Aozhong.

The invention also provides a combined medicament for treating cancers, which is prepared by taking the traditional Chinese medicine composition or the extract thereof and a chemotherapeutic medicament as active ingredients and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

Preferably, the combination is for use in the treatment of pancreatic cancer.

Preferably, the chemotherapeutic agent is selected from at least one of gemcitabine, cisplatin, epirubicin hydrochloride, and te Mo Aozhong.

In the invention, the musk is artificial musk.

The invention uses the traditional Chinese medicine composition of frankincense, myrrh, bezoar and musk as medicines, researches on the treatment of PC (pancreatic cancer) by utilizing in vivo and in vitro proliferation and apoptosis experiments, and uses CYP3A4 as a key target point of the traditional Chinese medicine composition for treating PC to play an anticancer role, and the traditional Chinese medicine composition can be used as an inhibitor of CYP3A4 for reducing the drug resistance of anticancer chemotherapeutics.

The invention provides a new clinical choice for CYP3A4 inhibitor and anticancer drugs, and has good application prospect.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is a graph of compound anti-PC cell SW1990 inhibition and IC 50;

FIG. 2 is a graph of clonogenic assay staining wherein A: a negative control group; b: compound 74.5 μg/ml group; c: compound 92.7 μg/ml group; b: compound 116.36 μg/ml group; e: compound 145.45 μg/ml group; f: compound 181.82 μg/ml group;

FIG. 3 shows the results of a cell morphology test in which, at 0h of administration: a: compound 0 mug/ml of control group; b: compound 74.5 μg/ml group; c: compound 92.7 μg/ml group; d: compound 116.36 μg/ml group; e: compound 145.45 μg/ml group; f: compound 181.82 μg/ml group; dosing for 24 h: h: compound 0 mug/ml of control group; i: compound 74.5 μg/ml group; j: compound 92.7 μg/ml group; k: compound 116.36 μg/ml group; l: compound 145.45 μg/ml group; m: compound 181.82 μg/ml group;

FIG. 4 is a Hoechst 33258 apoptosis staining experiment in which A: a negative control group; b: compound 74.5 μg/ml group; c: compound 92.7 μg/ml group; d: compound 116.36 μg/ml group; e: compound 145.45 μg/ml group; f: compound 181.82 μg/ml group; g, after drug addition, the number of apoptotic cells changes the bar graph, the abscissa represents the compound concentration, the ordinate represents the apoptotic cell rate (individual); * P <0.0001;

fig. 5 is a flow cytometry experimental result, wherein a: a negative control group; b: compound 74.5 μg/ml group; c: compound 92.7 μg/ml group; d: compound 116.36 μg/ml group; e: compound 145.45 μg/ml group; f: compound 181.82 μg/ml group; after the drug is added, the apoptosis rate bar graph is used, the abscissa represents the concentration of the compound, and the ordinate represents the apoptosis cell rate (%); * P <0.01, P <0.0001;

FIG. 6 is the effect of compound on body mass of SW1990 cell engrafted tumor Balb/c mice (x+ -SD, n=6);

FIG. 7 is the effect of compound on tumor volume of SW1990 cell engrafting tumor Balb/c mice (x+ -SD, n=6);

FIG. 8 is the effect of compound on SW1990PC mice growth tumor;

FIG. 9 is a microscopic image of PCNA, KI67 positive cell numbers, wherein KI67: FIG. A is a model set; panel B is the positive group (0.0065 g/ml); panel C shows that the compound group dose is 0.47g/kg; panel D shows that the compound group dose is 0.93g/kg; panel E shows that the compound group dose is 1.87g/kg; PCNA: FIG. F is a model set; panel G is the positive group (0.0065G/ml); panel H shows that the compound dose is 0.47g/kg; FIG. I shows that the compound dosage is 0.93g/kg; panel J shows that the compound group dose is 1.87g/kg;

fig. 10 is the results of TUNEL staining experiments to detect apoptosis in nude mouse tumor tissue, wherein positive expression: the apoptosis cell nucleus is light yellow or brown yellow, the negative expression (normal cell nucleus) is blue or light blue, and the background is white; ruler: 10 μm; FIG. A is a model set; panel B is the positive group (0.0065 g/ml); panel C shows that the compound group dose is 0.47g/kg; panel D shows that the compound group dose is 0.93g/kg; panel E shows that the compound group dose is 1.87g/kg.

Fig. 11 shows experimental results of verifying target CYP3A4 (n=3, x±sd) using WB method.

FIG. 12 shows the results of experiments using RT-PCR to verify the target CYP3A4 (n=3, x.+ -. SD).

Detailed Description

In the following examples and experimental examples, reagents and materials not specifically described are commercially available.

EXAMPLE 1 CYP3A4 enzyme inhibitory action and anti-cancer Activity of A Chinese medicinal composition

1. Chinese medicinal composition (hereinafter abbreviated as compound)

The traditional Chinese medicine composition used in the embodiment is as follows: 55.7g of vinegar olibanum, 55.7g of vinegar myrrh, 1.52g of bezoar fine powder and 1.52g of artificial musk fine powder.

The preparation method comprises the following steps:

extracting Olibanum 55.7g with 6 times of 85% ethanol twice for 1.5 hr, filtering the extractive solutions, mixing filtrates, recovering ethanol, concentrating to obtain fluid extract, adding 24g silicon dioxide, mixing, drying, pulverizing, and sieving with 80 mesh sieve twice. Adding 1.52g of calculus bovis fine powder (100 meshes), 1.52g of artificial musk fine powder and a proper amount of microcrystalline cellulose into the dry paste fine powder, uniformly mixing, preparing a soft material by using 50% ethanol, granulating by using a 14-mesh sieve, using anhydrous calcium chloride as a drying agent, drying at normal temperature under reduced pressure, and finishing granules by using a 14-mesh sieve. Adding 3.6g of croscarmellose sodium and 1.2g of magnesium stearate, uniformly mixing to obtain 106.82g of particles, and tabletting.

2. Experimental method

(1) Experimental materials

Cell lines: human pancreatic cancer SW1990 cells (Shanghai Fuheng organism)

Experimental animals: SPF-grade healthy BALB/C nude mice at 6 weeks of age were fed with body weight (20.+ -.2) g, 30 total, male, SPF-grade clean laboratory.

Experimental reagent:

(2) Experimental procedure

Extracting the compound:

taking the traditional Chinese medicine composition tablet prepared in the first part, grinding, precisely weighing 3g of medicinal powder, placing into a 50ml centrifuge tube, adding 12ml of DMSO for dissolution, and carrying out suction filtration to obtain 250mg/ml mother liquor. In the test, the mother solution is diluted by culture solution (L-15 culture medium containing 10% inactivated fetal bovine serum) to obtain the liquid medicine with the concentration required by the test.

Cell culture

Human pancreatic carcinoma SW1990 cells (supplied by Shanghai Fuheng biosciences Co., ltd.) were cultured in a sealed manner in L-15 complete medium containing 1% antibiotics (100U/ml penicillin and 100U/ml streptomycin) and 10% fetal bovine serum, 5% CO ₂ Culturing in an incubator at 37 ℃.

2.1cck-8 experiment

Human PC cells were inoculated at 104. Mu.L/ml concentration in 96-well plates, 100. Mu.L/well, 1d later, L-15 culture medium was added, and a blank group, DMSO solvent control group, compound five concentration gradients (74.5. Mu.g/ml, 92.7. Mu.g/ml, 116.36. Mu.g/ml, 145.45. Mu.g/ml, 181.82. Mu.g/ml) were set, 6 multiplex wells each, and cultured for 24h, 48h, 72h, respectively. 10ul CCK-8 reagent per well was added to the medium and incubated for 1.5h at 37 ℃. Absorbance at a wavelength of 450nm was measured using a microplate reader to reflect the activity of the cells. The following formula was used to determine the cell inhibition:

a non-linear regression plot was drawn between% inhibition of cells and Log10 concentration, and prism software 9.0 was used to determine semi-inhibitory concentration values. Each test was performed in 6 replicates and the results were the mean ± SD of the independent experiments. The median inhibitory concentration (IC 50) of the compound on SW1990PC cell proliferation inhibition was calculated.

2.2 clone formation assay

(1) SW1990 cells in logarithmic growth phase were digested with pancreatin, resuspended in cell suspension using L-15 complete medium and counted;

(2) Cell inoculation: inoculating 2000 cells in each hole of a 6-hole culture plate, sealing, placing the culture plate at 37 ℃, and incubating the culture plate in a 5% CO2 incubator for 24 hours;

(3) Adding 6 compound extracting solutions with concentration gradients: 0 μg/ml, 74.5 μg/ml, 92.7 μg/ml, 116.36 μg/ml, 145.45 μg/ml, 181.82 μg/ml, culturing continuously for 10 days, changing the medicinal liquid every 3 days, and observing cell state;

(4) After observing cell clone agglomeration under an ocular lens, discarding old solution, adding 1ml of precooled methanol into each hole for fixing for 5min, discarding the methanol, and washing for 1 time by PBS;

(5) 1ml of crystal violet dye solution is added into each hole in a dark place for fixation for 30min, the solution is washed for a plurality of times by PBS, and the solution is dried and photographed.

2.3 Effect on cell morphology

Logarithmic growth human PC cell line SW1990 cells were counted and inoculated into six-well plates at cell densities of 2X105 cells/ml for 24 hours, respectively, the culture medium was discarded, and six concentration gradient compound liquid medicines of 0. Mu.g/ml, 74.5. Mu.g/ml, 92.7. Mu.g/ml, 116.36. Mu.g/ml, 145.45. Mu.g/ml, 181.82. Mu.g/ml were added, and cell morphology was observed under a 10-fold microscope at each well of 2ml, respectively, for 0h, 24 hours and photographed.

2.4hoechst 33258 dyeing experiments

Morphological changes in apoptosis were observed using Hoechst 33258 detection kit. Logarithmically grown SW1990 cells were seeded in 6-well plates (n=3) at a density of 5×10 cells/well and treated with compound at different concentrations (0 μmol/L, 74.5 μg/ml, 92.7 μg/ml, 116.36 μg/ml, 145.45 μg/ml, 181.82 μg/ml) for 24h. Cells were washed twice with PBS and then fixed with 1ml methanol per well for 10 minutes at 4 degrees celsius. After fixation, the plates were washed once with PBS and incubated with Hoechst 33258 dye for 10min at room temperature, and washed 2-3 times with PBS. The results were observed under a T5A/H fluorescence microscope (Carl Zeiss, germany) and photographed. And counted using Image J V1.8.0.

2.5 flow cytometry

(1) SW1990PC cell line cells were inoculated in 6-well plates, 1X 106 cells per well after counting, 6 groups of 3 compound wells per group, and culture medium (0. Mu.g/ml, 74.5. Mu.g/ml, 92.7. Mu.g/ml, 116.36. Mu.g/ml, 145.45. Mu.g/ml, 181.82. Mu.g/ml) was replaced with the compound liquid medicine at 6 concentrations after sealed culture in an incubator at 37℃for 24 hours, and the culture was continued for 24 hours.

(2) Collecting supernatant of the 6-hole plate respectively, using pancreatin without EDTA to digest cells, centrifuging, discarding supernatant, pre-cooling PBS and washing for 2 times;

(3) Re-suspending and counting, diluting the cell density to 1X 106/ml, taking 105 cells per tube, centrifuging for 5min at 1000r/min, discarding the supernatant, and adding 195 μl Binding Buffer;

(4) After light weight and heavy suspension, 5 μl PI and 5 μl Annexin V-FITC reagent are added, mixed uniformly, reacted at room temperature in the dark for 15min, and then placed in ice bath;

(5) Apoptosis was detected by flow cytometry (ACEA NovoCyte, USA) within 1 h.

2.6 establishment and administration of a model of a graft tumor

Tumor cell lines were taken in a sterile environment, diluted with physiological saline, and subcutaneously injected on the back of nude mice near the right hind limb with 0.1ml of a SW1990 cell suspension (about 2X 106 tumor cells).

The diameter of the nude mice planted tumor is measured after 1 week of inoculation, and the model is judged to be successful by taking the tumor volume of not less than 62.5mm3, and the total of 30 nude mice are modeled successfully. After successful molding, the nude mice are divided into 5 groups according to the tumor volume, namely a model group, a gemcitabine group (the intraperitoneal injection amount is calculated according to 10ml/kg and is obtained for 1 time per week), and a compound dosage group (the compound corresponding filling amount is 0.47g/kg, 0.93g/kg and 1.87g/kg, and the compound dosage group is prepared by sodium carboxymethyl cellulose, namely, 3 groups of compound powder which is ground by a mortar are respectively taken, 0.47g, 0.93g and 1.87g are dissolved in 10ml sodium carboxymethyl cellulose and are fully dissolved by ultrasound), and 6 groups of compound powder are prepared. 6 model nude mice are selected, and an equal volume of physiological saline is injected at the same position to perfuse sodium carboxymethyl cellulose as a model group. The compound group is administrated by lavage once a day, and is continuously administrated for 12 times, and meanwhile, the volume of the physiological saline injected into the abdominal cavity is consistent with that of the positive medicine; the gemcitabine group nude mice were given the same amount of physiological saline as the compound group by intraperitoneal injection of gemcitabine for 12 days as described above and intragastric administration.

During the experiment, the mental state, activity, water intake, food intake, urination and defecation, and the presence or absence of bite wound and death of the mice were observed daily. If mice die, the number of deaths, and possible causes and dates should be recorded. Tumor diameters a, b were weighed daily and recorded. The next day of drug withdrawal, mice were sacrificed by cervical vertebra removal, tumor mass was peeled off, and the pictures were taken, and the tumor weight and tumor volume were weighed. Fixing tumor tissues by paraformaldehyde for each group of three samples (cut into halves), and storing for later use; the rest of the sample was filled into a freezing tube and placed in a liquid nitrogen tank overnight. The next day was transferred to a-80 ℃ refrigerator.

Tumor inhibition rate of each dosing group: tumor inhibition rate (%) = (average tumor weight of control group-average tumor weight of treatment group)/average tumor weight of control group х%.

2.7 immunocytochemistry Studies of antiproliferative Activity

(1) Paraffin sections dewaxed to water: sequentially placing the slices into xylene I for 15min, and xylene II

15min, xylene III 15min, absolute ethanol I5 min; absolute ethyl alcohol II for 5min,85% alcohol for 5min and 75% alcohol for 5min, and distilled water washing;

(2) Antigen retrieval: immersing the slices in citrate buffer (ph=6.0), heating with high fire in a microwave oven for 10min, stopping the fire for 8min, and heating with medium high fire for 10min again; after cooling, PBS is washed for 3 times, each time for 5min;

(3) Blocking endogenous peroxidases: placing the slices into 3% hydrogen peroxide, and standing at room temperature for 10min; washing with PBS for 3 times, each time for 5min;

(4) Serum blocking: dripping goat serum sealing liquid at room temperature for 20min;

(5) Dripping primary antibody (primary antibody'), and standing at 4deg.C overnight;

(6) Washing with PBS for 3 times, each time for 5min; dripping secondary antibody at 37 ℃ for 30min; washing with PBS for 3 times, each time for 5min;

(7) DAB color development: preparing fresh DAB color development liquid, dripping the liquid onto tissues, developing at room temperature, controlling the color development time under a microscope, and washing the slices with distilled water to stop color development, wherein the positive color is brown yellow;

(8) Counterstaining the nuclei: hematoxylin counterstain for 3min, washing with running water, and washing with running water after the clear water returns to blue;

(9) And (3) removing the water sealing piece: sequentially placing the slices into 75%, 85%, 95%, absolute ethyl alcohol and xylene, respectively soaking for 10 minutes, and sealing the neutral resin.

2.8 apoptosis TUNEL assay

Dehydrating reagent: absolute ethanol was diluted with purified water to make 75%, 85% and 95% ethanol solutions.

Hydrochloric acid-ethanol differentiation solution: taking 416ml of absolute ethyl alcohol and 178ml of purified water, pouring the absolute ethyl alcohol and the purified water into a beaker, uniformly mixing, slowly adding 6ml of concentrated hydrochloric acid, and uniformly stirring to obtain the aqueous solution.

The fixed tissue is dehydrated by a full-automatic dehydrator (dehydration time period: 75% alcohol 3h,85% alcohol 1h,95% alcohol 1h20min,100% alcohol I25 min,100% alcohol II 20min,100% alcohol

III 20min,100% alcohol: xylene is mixed for 20min in one-to-one mode, xylene I is mixed for 15min, xylene II is mixed for 15min, paraffin I is mixed for 1h, paraffin II is mixed for 2h, paraffin III is mixed for 3 h), embedded and sliced, and the following operation is carried out:

(1) Slide anti-slip processing: soaking with APES, taking out slices, and placing in an oven at 60deg.C for 60min to adhere the slices tightly;

(2) Slice dewaxing to water, specific operation: xylene I5-10 min, xylene II 5-10min, absolute ethanol I5 min, absolute ethanol II 5min,95% ethanol 5min,85% ethanol 5min,75% ethanol 5min, UP water soaking for 5min;

(3) Treating the tissue with trypsin K (Proteinase K) working solution at 37 ℃ for 25min;

(4) Rinsing 3 times with PBS;

(5) Preparation of TUNEL reaction mixture: mixing 50 μl of No. 1 solution and 450 μl of No. 2 solution;

(6) After the slide is dried, 50 μl TUNEL reaction mixture is added to the specimen, and the cover glass is added to react at 37 ℃ in a dark-wet box for 1h;

(7) Rinsing 3 times with PBS;

(8) After the slide is dried, adding 50 μl of No. 3 liquid (converter-POD) on the specimen, and reacting the cover glass or sealing film in a dark-wet box at 37deg.C for 30min;

(9) Rinsing 3 times with PBS;

(10) Dripping 50-100 mu l DAB color reagent, observing the reaction at 25 ℃ for 10min;

(11) Rinsing 3 times with PBS;

(12) Hematoxylin slight counterstain, and after a few seconds, washing with tap water;

(13) Alcohol dehydration, transparent xylene, and sealing with neutral resin.

(14) The above specimens were all processed according to the pathology test SOP procedure. Dehydrating, trimming, embedding, slicing, dyeing, sealing and the like, and finally performing microscopic examination.

2.9WB method for detecting CYP3A4 protein expression level

2.9.1 Total protein extraction from nude mouse tumor tissue samples

Taking out the sample, placing the sample into a 2mL grinding tube, adding 3mm steel balls and RIPA lysate (the sample is prepared by the mass ratio of the lysate to the 1:10) into each tube, and placing the mixture into a high-speed low-temperature tissue grinding instrument (the temperature is-20 ℃ C., grinding is carried out for 4 times, and each time is 60 seconds); taking out, placing in a refrigerator at 4deg.C for 30min for cracking, taking out, and placing in a centrifuge (at 4deg.C, 12000rpm for 10 min); after centrifugation, the supernatant was taken and the protein concentration was determined using BCA protein quantification kit.

2.9.2 determination of sample protein concentration (BCA method)

(1) Protein standard working solution configuration

1.2mL of the protein standard preparation is added to a tube of protein standard (30 mgBSA), and the protein standard solution of 25mg/mL is prepared after complete dissolution. Can be used immediately after preparation, or can be stored at-20deg.C for a long time.

A proper amount of 25mg/mL protein standard was taken and diluted to a final concentration of 0.5mg/mL. For example, 20. Mu.L of 25mg/mL protein standard can be prepared by adding 980. Mu.L of diluent to prepare 0.5mg/mL protein standard. In what solution the protein sample is in, the standard is preferably diluted with what solution. However, for simplicity, the standard may also be diluted with 0.9% NaCl or PBS. The diluted 0.5mg/mL protein standard can be stored for a long period of time at-20 ℃.

(2) Standard sample application

Taking an ELISA plate, and adding reagents according to the following table data:

TABLE 1

(3) BCA working solution preparation

Adding 1 volume BCA reagent B (50:1) into 50 volumes of BCA reagent A according to the number of samples to prepare a proper amount of BCA working solution, and fully and uniformly mixing; 200 mu L of BCA working solution was added to each well; after shaking and mixing, the mixture was left at 37℃for 30min.

(4) Drawing a standard curve

OD values at 562nm wavelength (shown in the following table) were measured by an enzyme-labeled instrument, and a standard curve was drawn with protein concentration (. Mu.g/. Mu.L) as an abscissa and OD values as an ordinate.

TABLE 2

2.9.2 sample assay

Diluting 2 mu L of a protein sample to be detected to 16 mu L by using a lysis solution, adding the diluted 2 mu L of the protein sample to be detected into a hole, adding the lysis solution to complement to 20 mu L, adding 200 mu L of BCA working solution, uniformly mixing, standing at 37 ℃ for 30 minutes, and then taking a 0-pore as a reference to determine the OD value at the wavelength of 562nm of the sample; the protein content (μg) of the sample can be found on a standard curve according to the OD value of the sample.

Protein concentration was calculated: the corresponding protein content (mug) searched on the standard curve is divided by the total volume of sample dilution (20 mug), and the actual concentration (mug/mug) of the sample to be detected is obtained by multiplying the sample dilution times.

2.9.3 protein denaturation

50 mu L of each experimental group is taken, 5 XLoding buffer is added according to the proportion of 4:1, and the mixture is uniformly mixed and then stored at the temperature of-80 ℃ for 15min by a thermal cycler at the temperature of 95 ℃.

2.9.4 sample application and electrophoresis

After the laminating adhesive is solidified, the two hands respectively pinch the two sides of the comb vertically upwards to slightly pull out the comb. Washing the laminated glue with water to clean broken glue in the holes, putting the broken glue into an electrophoresis tank, adding enough electrophoresis buffer solution, and then preparing for loading; loading: 30 μg (6 μg/μl), sample was slowly added to the loading well with a 10 μl pipette; electrophoresis: and (3) switching on a power supply, stabilizing the voltage for 100V for 15min, adjusting the voltage to 180V after the bromophenol blue dye reaches the separation gel, continuing electrophoresis, and stopping electrophoresis when the bromophenol blue dye reaches the bottom of the gel.

2.9.5 transfer film

Cutting 1 PVDF membrane and 6 filter papers according to the size of the separating glue, putting the membrane and the cut separating glue with a target strip into a membrane transferring liquid for balancing for 10min, and preparing two sponges at the same time; soaking 6 pieces of filter paper by using a film transferring liquid, stacking the sponge, the filter paper (3 pieces), the film, the gel, the filter paper (3 pieces) and the sponge in sequence, and removing bubbles between each two layers by using a glass rod; transferring: the gel surface is connected with the negative electrode, the PVDF film is connected with the positive electrode, the power supply is turned on, and the film is turned for 1-2 hours at 200 mA.

2.9.6 seal

PVDF membrane was placed in 5% skimmed milk diluted with TBST Buffer, placed in incubation box and gently shaken on a shaker for 2h.

2.9.7 incubation of antibodies

Incubation resistance: placing PVDF membrane into primary antibody (primary antibody concentration: CYP3A4 1:1000;GAPDH 1:50000), and lightly shaking on a shaking table, and incubating overnight at 4 ℃; washing PVDF membrane with TBST for 3 times and 5min each time;

secondary antibody incubation: placing PVDF membrane into secondary antibody (dilution concentration: 1:5000), and lightly shaking on a shaking table, and incubating at room temperature for 2-3h; the PVDF membrane was washed 3 times with TBST for 10min each.

2.9.8 development and fixation

Spreading PVDF film onto exposure plate, mixing A, B reagents of ECL luminous liquid in equal volume, dripping onto film, reacting for 1min, and placing the exposure plate with film into darkroom of chemiluminescent gel imager, and adjusting exposure time according to signal intensity.

Image analysis: the bands are subjected to exposure scanning by using the control software V2.0 of the natural energy GIS chassis, and the result is expressed as the relative expression quantity of the target protein.

2.10qRT-PCR method for detecting mRNA expression quantity of CYP3A4

2.10.1 Total RNA extraction from samples

(1) Before use, absolute ethyl alcohol is added into deproteinized liquid PL and rinsing liquid w, and the volume is added with reference to the label on the bottle.

(2) Fresh tissue was taken, 500ul of lysates LB was added per 10-20mg, and triturated with a high-speed cryogenic tissue mill.

(3) The homogenized slurry was transferred to a 1.5mL centrifuge tube and centrifuged at 12,000rpm for 5min.

(4) The supernatant was transferred to a DNA removal column A2, centrifuged at 12,000rpm for 2min, and the filtrate was retained.

(5) To the above-mentioned retained filtrate (volume should be about 500 uL) was added 1.6 volumes of deproteinized solution PL, immediately gently swirled and mixed.

(6) All the above mixture was put into RNA adsorption column A2, centrifuged at 12,000rpm for 1min, and the filtrate was discarded.

(7) 500ul of the binding solution BD was added, centrifuged at 12,000rpm for 1min, and the filtrate was discarded.

(8) The RNA adsorption column A2 was placed back into the collection tube, 700ul of rinse w was added, centrifuged at 12,000rpm for 1min, and the filtrate was discarded.

(9) Step 8 is repeated.

(10) The RNA adsorption column A2 was returned to the collection tube, and the empty column was centrifuged at 12,000rpm at room temperature for 2min to remove the residual rinse w.

(11) The RNA adsorption column A2 was placed in a new 1.5mL centrifuge tube, 50-100ul RNase-free H2O was added to the center of the membrane, and the mixture was left at room temperature for 2min. Then centrifuged at 12,000rpm for 1min. And collecting filtrate, namely the RNA solution, and preserving at the temperature of minus 80 ℃ to be detected.

2.10.2 removal reaction of genomic DNA

TABLE 3 genomic DNA removal reaction System

Note that: sequentially adding the reagents, and at a temperature of 42 ℃ for 2min

2.10.3 reverse transcription reaction

TABLE 4 reverse transcription reaction system

Note that: sequentially adding the reagents, and placing the mixture on a PCR instrument for reaction

2.10.4 real-time fluorescent quantitative polymerase chain reaction (real-time PCR)

(1) Primer design

The complete sequence of the gene was searched from the National Center for Biotechnology Information (NCBI) database and each gene-specific Primer was screened using Primer Premier Primer design software. All primers were designed and synthesized by Shanghai Bioengineering services Inc. and purified by ULTRAPAGE (see Table below for details).

TABLE 5 primers and nucleotide sequences for detection

(2) Real-time fluorescent quantitative PCR reaction

TABLE 6 PCR reaction System

TABLE 7 PCR reaction System

Note that: 45 cycles of

2.11 statistical analysis

Data were analyzed for one-way ANOVA using GraphPad Prism v6.0 software (GraphPad Software, inc., san Diego, CA, USA) and SPSS23.0 statistical analysis software and data were expressed as mean ± standard deviation (±sd). P <0.05 is considered statistically significant.

3. Experimental results

3.1 Compound experiments on PC cells SW1990 cck-8

As can be seen from table 8 below, DMSO group and 0 dosing group had p-values >0.05, with no statistical significance (n=6), thus it was seen that DMSO as solvent did not affect SW1990PC cell growth. The compound concentration is between 74.5 and 181.82 mug/ml, has obvious cell inhibition rate, and the cell inhibition rate is increased along with the increase of the concentration, so that the compound has concentration dependence. There was a statistical difference when the compound dosing concentration reached 92.7 μg/ml 48h after dosing (n=6, p < 0.0001). After 24h, 48h, 72h dosing, the cell inhibition rate increased with time, with time dependence, with statistical differences (n=6, p < 0.0001). As can be seen from FIG. 1B, C, the IC50 values after 24h, 48h and 72h of dosing were 128.4. Mu.g, 6. Mu.g/ml and 85.54. Mu.g/ml, respectively, and the IC50 values were lower as the dosing time was longer. The culture time was selected to be 24 hours, and the culture concentrations were 74.5. Mu.g/ml, 92.7. Mu.g/ml, 116.36. Mu.g/ml, 145.45. Mu.g/ml, 181.82. Mu.g/ml.

Table 8 Compound anti-PC cell SW1990 inhibition ratio

Note that: each group had P <0.0001 compared to the blank

3.2 Compound cloning test for PCSW1990

As shown in fig. 2, in the colony formation assay, the viable cells stained with crystal violet decreased with increasing compound concentration at compound concentrations between 74.5-181.82 μg/ml, and had a concentration dependence, thus demonstrating that the compound had an inhibitory effect on PC cells SW1990, with statistical significance (n=3).

3.3 Effect of the Compound on the morphology of PC cell SW1990 cells

As shown in FIG. 3, after 24 hours of compound action compared with the 0h cell morphology, SW1990 cells decreased with increasing administration concentration, the cell growth rate was significantly reduced, the cell fragmentation increased, and some fragments floated on the surface of the culture solution; as the time of administration increases, the cell morphology changes significantly, gradually changing from a cell population that is all integrated into one piece to a cell mass of one piece.

3.4hoechst 33258 dyeing experiments

As shown in FIG. 4, to study the effect of the compound on SW1990PC apoptosis, the compound was treated with different concentrations (0. Mu.g/ml, 74.5. Mu.g/ml, 92.7. Mu.g/ml, 116.36. Mu.g/ml, 145.45. Mu.g/ml, 181.82. Mu.g/ml) and stained with Hoechst 33258 for 24h. And observed by fluorescence microscopy. The result shows that the normal control group has less staining quantity of apoptotic cells and low fluorescence intensity. The cells after the compound treatment were microscopically seen as dense plaque brilliant blue apoptotic bodies, and the number of apoptosis was increased with increasing concentration, concentration-dependent, and statistically different when the concentration of administration was greater than 116.36 μg/ml (fig. 4). Thus, the compound has the capability of promoting PC cell apoptosis.

3.5 flow cytometry apoptosis experiments

As shown in fig. 5, to reveal the effects of compound pro-SW 1990 human PC apoptosis, detection was performed using flow cytometry. The results showed that after 24h of stimulation of apoptosis by the addition of the compound (74.5. Mu.g/ml, 92.7. Mu.g/ml, 116.36. Mu.g/ml, 145.45. Mu.g/ml, 181.82. Mu.g/ml), the total cell rate of apoptosis increased from 11.43% to 13.71%, 18.54%, 24.99%, 41.96% and 53.34%, respectively. As can be seen, the ratio of SW1990PC cells of (early apoptosis) annexin V+ -PI-and (late apoptosis) annexin V+ -PI+ increased with increasing compound concentration, and was concentration-dependent. And when the concentration was greater than 116.36 μg/ml, there was a statistical difference (P < 0.01) started. These results indicate that the compound has the ability to promote apoptosis of PC cells.

3.6 influence of the Compound on the weight and tumor volume of nude mice

As can be seen from Table 9 and FIG. 6, the Balb/c mice in each group had good status, had normal drinking water and activities, had decreased body weight, and were administered with obvious amounts, but showed no obvious differences (P > 0.05) from the model group at the same time points. Slow movement, reduced sensitivity, unchanged hair and normal urination and defecation. As can be seen from Table 10 and FIG. 7, the positive group (GEM-0.065 g/kg) and the compound (0.93 g/kg, 1.87 g/kg) showed significantly reduced tumor volumes compared with the model group after the first administration for 4 days, and had statistical significance (P <0.05 or P <0.01 or P < 0.001), and the compound-0.47 dose group showed reduced tumor volumes after the administration for 6 days, and had statistical difference (P < 0.01).

TABLE 9 influence of the Compound on the condition and body mass of SW1990 cell transplantation tumor Balb/c mice

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Table 10 Effect of the Compound on the tumor volume of SW1990 cell transplantation tumor Balb/c mice

3.7 tumor inhibition Rate

As shown in Table 11 and FIG. 9, the experimental results showed that the tumor weights of the compound 0.47g/kg dose group, the compound 0.93g/kg dose group, and the 1.87g/kg dose group were significantly reduced compared to the model group (positive group, compound 0.47g/kg dose group, compound 0.93g/kg dose group, and 0.187g/kg dose group vs model group: 0.127.+ -. 0.040, 0.113.+ -. 0.017, 0.080.+ -. 0.018, 0.053.+ -. 0.014, and tumor suppression rates of 32.75%, 40.00%, 57.51%, 71.85%, respectively). The compound has obviously reduced tumor volume and obviously reduced tumor quality along with the increase of concentration, and has extremely obvious difference (P < 0.01); the positive group had a significant difference (P < 0.05) in tumor volume, mass reduction compared to the model group.

Table 11 Compound anti-PC tumor inhibition rate

Note that: each group had P <0.05, < P <0.01, < P <0.0001; each of the compound dosing groups had a # P <0.01, # P <0.001, compared to the positive group

3.8 immunohistochemical Studies of Compound antiproliferative Activity

The results in table 12 and fig. 9 show that, compared with the model group, the numbers of ki67 positive cells of nude mice tumor tissues in the compound low-dose group, compound medium-dose group and compound high-dose group are obviously reduced, and the nude mice tumor tissues have concentration dependence and obvious statistical significance (P < 0.0001); the number of ki67 positive cells in the tumor tissue of the positive group nude mice is obviously changed, and the method has statistical significance (P < 0.001). Compared with the positive group, the number of ki67 positive cells in the tumor tissue of the nude mice in the compound medium-dose group and the compound high-dose group is obviously reduced, and the method has obvious statistical significance (p < 0.05); the number of ki67 positive cells of the compound low-dose group nude mice tumor tissue has no obvious change and has no statistical significance (P is more than 0.05). The results in table 13 and fig. 10 show that, compared with the model group, the number of PCNA positive cells in the tumor tissue of the nude mice in the low dose group, the medium dose group and the high dose group is obviously reduced, and the method has obvious statistical significance (P < 0.01); the number of PCNA positive cells of the tumor tissue of the positive group nude mice has no obvious change and has no statistical significance (P is more than 0.05).

TABLE 12 statistics of the number of positive cells positive for ki67

Note that: each group had P <0.001, P <0.0001; the compound dose groups were compared with the positive groups, with # p <0.05, # p <0.001

TABLE 13 statistical results of PCNA positive cell counts

Note that: each group had P <0.05, P <0.01 compared to the model group

3.9 Compound SW1990PC apoptosis TUNEL assay

The results in table 14 and fig. 10 show that: compared with the model group, the apoptosis rate in tumor tissues of the positive group, the compound low-dose group, the compound medium-dose group and the compound high-dose group is obviously increased, the significant difference (P < 0.0001) is generated, and the compound administration group has concentration dependence. Compared with the positive group, the apoptosis rate in the tumor tissues of the compound medium-dose group and the compound high-dose group is obviously increased, and has extremely obvious difference (P < 0.001), while the apoptosis rate in the tumor tissues of the compound low-dose group is reduced, and has obvious difference (P < 0.05).

Table 14 statistics of percent (%) apoptosis in tumor tissue of nude mice/>

Note that: each group had P <0.0001; each of the compound dosing groups had #p <0.05, # P <0.001, # P <0.0001, as compared to the positive group.

3.10WB

According to table 15 and fig. 11, the compound can down regulate the content of CYP3A4 protein, and compared with the compound administration group and the model group, the CYP3A4 protein expression in the tumor tissue of the compound nude mice is obviously reduced, and the compound nude mice has concentration dependence and statistical significance (p < 0.001). Therefore, CYP3A4 can be used as a key target point of the compound anti-PC.

TABLE 15 CYP3A4 expression in nude mouse tumor tissues of each experimental group

Note that: each group had P <0.001, P <0.0001, compared to the model group

3.11RT-PCR

The CT (Threshold cycle) value of each test sample of the PCR process was analyzed using Thermo Scientific PikoReal software (Thermo company). The laboratory calculates the relative mRNA expression level of X by 2- ΔΔct: Δct=ct gene of interest-CT internal reference; ΔΔct= - Δct experiment- Δct control; fold difference in XmRNA expression is expressed in terms of 2-DeltaCT. According to the results of table 16 and fig. 12, the compound can down-regulate the content of CYP3A4 mRNA, compared with the model group, the expression of CYP3A4 mRNA in nude mice tumor tissues of the compound in the middle-dose group and the high-dose group is reduced, and the compound has a significant difference (P < 0.05); the expression of CYP3A4 mRNA in the tumor tissue of the low-dose group nude mice has no obvious difference and has no statistical significance. Therefore, CYP3A4 can be used as a key target point of the compound anti-PC.

TABLE 16 expression Change of CYP3A4 mRNA in nude mouse tumor tissue

Note that: p <0.05 compared to group D

The experimental results of the above examples show that the traditional Chinese medicine composition of the frankincense, the myrrh, the bezoar and the musk has obvious curative effects on PC in vivo and in vitro. WB and PT-PCR experiments show that the traditional Chinese medicine composition can down regulate protein expression quantity and mRNA expression quantity of CYP3A4 in vivo. This demonstrates that the CYP3A4 target may be a potential key target for the treatment of PC with a traditional Chinese medicine composition. Therefore, the traditional Chinese medicine composition of the frankincense, the myrrh, the bezoar and the musk can inhibit the expression of CYP3A4, so that the effect of inhibiting the drug resistance of the chemotherapeutic medicine can be achieved.

Claims (13)

1. The traditional Chinese medicine composition is characterized by being prepared from the following raw materials in parts by weight:

55.7-278.5 parts of frankincense,

55.7-278.5 parts of myrrh,

1.52 to 7.6 portions of bezoar,

Musk 1.52-7.6 portions.

2. The traditional Chinese medicine composition according to claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight:

55.7 parts of frankincense,

55.7 parts of myrrh,

1.52 parts of bezoar,

Musk 1.52 parts.

3. The traditional Chinese medicine composition according to claim 1, wherein: the traditional Chinese medicine composition is a preparation prepared by adding pharmaceutically acceptable auxiliary materials or auxiliary components into the active ingredients of the olibanum alcohol extract, the myrrh alcohol extract, the bezoar powder and the musk powder.

4. Use of the traditional Chinese medicine composition or the extract thereof according to any one of claims 1-3 for preparing a CYP3A4 inhibitor.

5. Use according to claim 4, characterized in that: the CYP3A4 inhibitor is used for reducing the drug resistance of anticancer chemotherapeutics.

6. Use of the traditional Chinese medicine composition or the extract thereof according to any one of claims 1-3 in the preparation of a medicament for treating cancer.

7. Use according to claim 6, characterized in that: the cancer therapeutic agent is used for treating pancreatic cancer.

8. Use of a traditional Chinese medicine composition or an extract thereof according to any one of claims 1-3 in combination with a chemotherapeutic drug for the preparation of a cancer therapeutic drug.

9. Use according to claim 8, characterized in that: the cancer therapeutic agent is used for treating pancreatic cancer.

10. Use according to claim 8, characterized in that: the chemotherapeutic drug is selected from at least one of gemcitabine, cisplatin, epirubicin hydrochloride or temozolomide.

11. A combination medicament for treating cancer, which is prepared by adding pharmaceutically acceptable auxiliary materials or auxiliary components into the traditional Chinese medicine composition or the extract thereof as claimed in any one of claims 1-3 and a chemotherapeutic medicament as active ingredients.

12. A combination according to claim 11, wherein: the combination is used for treating pancreatic cancer.

13. A combination according to claim 11, wherein: the chemotherapeutic drug is selected from at least one of gemcitabine, cisplatin, epirubicin hydrochloride or temozolomide.