A combined drug of curcumin and tracheobic acid and its application in preparing anti-tumor drugs

Abstract

The present invention discloses a drug for combining curcumin and trabecular acid and its application in the preparation of an anti-tumor drug, and provides a treatment plan for improving the clinical efficacy of curcumin to ensure the therapeutic effect of curcumin on tumors and improve bioavailability. Curcumin and trabecular acid are combined to synergistically inhibit the proliferation of gastric cancer tumor cells both in vivo and in vitro, thereby achieving the effect of inhibiting tumors; curcumin and trabecular acid are combined to promote each other's absorption both in vivo and in vitro, and the promoting effect of TAB on CUR is greater than that of CUR on TAB; curcumin and trabecular acid are combined to promote each other's absorption by downregulating the expression of efflux transporters P-gp and BCRP, thereby improving bioavailability and enhancing the efficacy of curcumin and trabecular acid.

Description

Technical Field

[0001] The invention belongs to the technical field of curcumin application, and particularly relates to a drug combining curcumin and tracheobic acid, and the drug is applied in a drug for treating tumor cells. Background Art

[0002] Curcumin (CUR) is a diketone compound extracted from the rhizomes of some plants in the Zingiberaceae and Araceae families. 21 H 20 O6. Turmeric (Curcuma longa L.) contains approximately 3%-6% curcumin, a rare diketone pigment in the plant kingdom. It is widely used in spices, food colorants, cosmetic additives, and pharmaceutical preparations. Curcumin exhibits a wide range of pharmacological activities, including antioxidant, anti-inflammatory, anti-cancer, hypoglycemic, anti-atherosclerotic, and lipid metabolism disorder improvement. Scientists have also found that curcumin can help treat drug-resistant tuberculosis.

[0003] However, in actual applications, it is found that curcumin still has certain defects, such as low solubility, poor stability, low absorption rate, and is easily converted into complexes such as glucuronic acid or sulfonic acid in the intestine, with fast metabolism and short half-life. The existence of these problems has led to a low bioavailability of curcumin, which has seriously limited its application in this field. Therefore, improving the bioavailability of curcumin and improving its clinical efficacy have become important research directions. Current methods include new drug delivery systems, modified structures, combined adjuvants, etc. These methods each have their own characteristics, and combined adjuvants have better safety and stability than new drug delivery systems and modified structures. Chinese patent CN113907353A discloses a method for improving the water solubility and small intestinal digestion stability of curcumin. The method uses Protein Z from natural malt as the main raw material and applies it to improve the solubility and digestion stability of curcumin. Curcumin interacts with Protein Z, effectively protecting curcumin and thereby improving its bioavailability. Chinese patent CN101627969B discloses a curcumin self-emulsifying drug delivery system and its preparation. The method uses TPGS and Solutol HS15 as a mixed emulsifier to prepare curcumin into a curcumin self-emulsifying drug delivery system, greatly improving the absorption and oral bioavailability of curcumin. Other studies have shown that piperine improves bioavailability by reducing the activity of metabolic enzymes UGT and SULT in the liver and colon and inhibiting the inactivation of CUR; α-cyclodextrin may promote the intestinal absorption of CUR through paracellular and transcellular pathways, thereby improving bioavailability; emodin can also increase the bioavailability of CUR, and its effect may be related to P-glycoprotein (P-gp). P-gp inhibitors can prevent drugs from binding to P-gp by competing with drugs for binding sites on P-gp, thereby reducing drug excretion.

[0004] Curcumin can affect the pharmacokinetic process of P-gp substrates by regulating the activity or expression of P-gp-mediated transporters. In this process, CUR plays a dominant role in downregulating intestinal P-gp. Therefore, CUR itself is also a P-gp inhibitor, which can increase the absorption of combined drugs. Combined drugs should ideally have the same pharmacological effects to achieve synergistic effects. However, there are currently few combination drugs with the same pharmacological effects as curcumin, especially in the treatment of tumors. Summary of the Invention

[0005] Based on the above technical problems, the present invention provides a drug for the combined use of curcumin and tracheobic acid and its application in the preparation of anti-tumor drugs.

[0006] The present invention provides a combined drug of curcumin and tracheobacterial acid, wherein the mass concentration of curcumin is 3-30 μg·mL -1 The mass concentration of tracheobacterial acid is 10-80 μg·mL -1 .

[0007] Preferably, the mass concentration of curcumin is 12 μg·mL -1 The mass concentration of traemolic acid was 10, 20, 40, 60, and 80 μg·mL -1 Any one of .

[0008] Preferably, the mass concentration of curcumin is 24 μg·mL -1 The mass concentration of traemolic acid was 10, 20, 40, 60, and 80 μg·mL -1 Any one of .

[0009] The present invention also provides a combined medicament containing the above-mentioned curcumin and tracheobic acid, and the medicament further comprises a pharmaceutically acceptable excipient.

[0010] The present invention also provides a use of curcumin or tracheobic acid in preparing a medicine for treating gastric cancer cell SGC7901.

[0011] The present invention also provides use of a combined drug of curcumin and tracheobic acid in preparing a drug for treating gastric cancer cell SGC7901.

[0012] Preferably, the mass concentration of curcumin is 3-30 μg·mL -1 The mass concentration of tracheobacterial acid is 10-80 μg·mL -1 .

[0013] More preferably, the mass concentration of curcumin is 12 μg·mL -1 The mass concentration of traemolic acid was 10, 20, 40, 60, and 80 μg·mL -1 Any one of .

[0014] More preferably, the mass concentration of curcumin is 24 μg·mL -1 The mass concentration of traemolic acid was 10, 20, 40, 60, and 80 μg·mL -1 Any one of .

[0015] The beneficial effects of the present invention are as follows: the effects of CUR, TAB and their combination on the proliferation of gastric cancer cell SGC7901 were detected by MTT method, and the IC values ​​of CUR and TAB alone were calculated by SPSS software. 50 The combined use index was then calculated to determine whether the combination of CUR and TAB had a synergistic effect.

[0016] The present invention uses high performance liquid chromatography to detect the cellular uptake of CUR and TAB by SGC7901 cells, and finds that TAB is uptaken by 50-500 μg·mL -1 It has a good linear relationship with the peak area, and the standard curve equation is Y1=0.0168X+0.0916, R 2 =0.9967; CUR in the range of 10-250 μg·mL -1 It has a good linear relationship with the peak area, and the standard curve equation is Y2=0.0255X-0.0044, R 2 =0.9981. The cellular uptake experiment of CUR and TAB showed that TAB and CUR promoted each other's uptake in cells, and the promoting effect of TAB on CUR was greater than that of CUR on TAB.

[0017] The absorption of CUR and / or TAB in SD rats was determined by high performance liquid chromatography to determine whether the combination of CUR and TAB has a synergistic effect in promoting absorption and improving bioavailability.

[0018] The present invention focuses on the mechanism of action of CUR in improving bioavailability. By detecting the expression of efflux transporters P-gp and BCRP by Western Blot, it is found that CUR and TAB can promote absorption of each other by inhibiting the expression of efflux transporters, thereby improving bioavailability.

[0019] The present invention discovered that the combined use of CUR and TAB inhibits SGC7901 cell xenografts. Nude mice were cultured with SGC7901 cells to establish a xenograft tumor model. CUR, TAB, and the combination drug were administered continuously for 21 days. The volume and size of the xenograft tumors were observed daily. After the administration period, the mice were sacrificed, the tumors were dissected, weighed, and the drug concentrations within the tumors were measured. The results showed that both CUR and TAB, when used alone, inhibited the growth of gastric cancer cells in nude mice. Their combined use enhanced the inhibitory effect, significantly reducing the volume and mass of tumor cells. The combined use achieved an inhibition rate of 51.69%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of the combined use of TAB and CUR on the proliferation of gastric cancer cells in Example 1, in the figure, * indicates the significant difference compared with the blank control group, * indicates P < 0.05, the difference is significant, ** indicates P < 0.01, the difference is extremely significant; # indicates the significant difference compared with the single use group, # indicates P < 0.05, the difference is significant, ## indicates P < 0.01, the difference is extremely significant; ^ indicates the significant difference compared with the combination group, ^ indicates P < 0.05, the difference is significant, ^^ indicates P < 0.01, the difference is extremely significant.

[0021] Figure 2 These are the chromatographic results of blank cell lysate, blank cell lysate with reference substance, internal standard blank cell lysate, and drug-added cell lysate in the specificity experiment in Example 2. In the figure, A is blank cell lysate (203 nm), B is blank lysate with TAB reference substance and ursolic acid reference substance added (203 nm), C is drug-added cell lysate (203 nm), D is blank cell lysate (422 nm), E is blank lysate with CUR reference substance and emodin reference substance added (422 nm), and F is drug-added cell lysate (422 nm); in the figure, 1 is TAB, 2 is ursolic acid, 3 is CUR, and 4 is emodin.

[0022] Figure 3 This is an experiment on the promoting effect of the combination of CUR and TAB on drug absorption by gastric cancer cells in Example 2. In the figure, A is the promoting effect of CUR on TAB, and B is the promoting effect of TAB on CUR. In the figure, * indicates the significance of the difference compared with the single-dose control group, * indicates P < 0.05, the difference is significant, and ** indicates P < 0.01, the difference is extremely significant.

[0023] Figure 4The chromatograms are of blank plasma, blank plasma with reference substance and internal standard, and sample plasma of rats in Example 3. In the figure, A is blank plasma (203 nm), B is blank plasma with TAB reference substance and ursolic acid reference substance added (203 nm); C is plasma sample of dosed rats (203 nm); D is blank plasma (422 nm); E is blank plasma with CUR reference substance and emodin reference substance added (422 nm); F is plasma sample of dosed rats (422 nm); in the figure, 1 is TAB, 2 is ursolic acid, 3 is CUR, and 4 is emodin.

[0024] Figure 5 The mass concentration-time curves of CUR and TAB in rat plasma in Example 3 are shown in Figure A, which is the mass concentration-time curve of CUR and the mass concentration-time curve of TAB. In the figure, * indicates the significant difference from the single-use group, * indicates P < 0.05, the difference is significant, and ** indicates P < 0.01, the difference is extremely significant.

[0025] Figure 6 4 is a graph showing the results of the Western Blot experiment in Example 4, wherein A is a protein gel electrophoresis graph, and B is a bar graph showing the expression levels of each protein, where * indicates significant difference from the blank control group, * indicates P < 0.05, significant difference, and ** indicates P < 0.01, extremely significant difference.

[0026] Figure 7 This is the growth curve of the transplanted tumor after the combined use of CUR and TAB in Example 5. DETAILED DESCRIPTION

[0027] The instruments involved in the present invention are as follows:

[0028] DK-8A electric constant temperature water bath, purchased from Shanghai Jinghong Experimental Equipment Co., Ltd.;

[0029] Q / SGYM 1009 electronic balance, purchased from Ohaus Instruments Shanghai Co., Ltd.;

[0030] The EcoPu ultrapure water system was purchased from EcoPu International, Inc., USA;

[0031] LKQ-400DB CNC ultrasonic cleaner, purchased from Kunshan Ultrasonic Instrument Co., Ltd.;

[0032] LC-2030C 3D Plus high-performance liquid chromatograph was purchased from Shimadzu Corporation;

[0033] Vortex-Genie2 vortex oscillator, purchased from Scientific Industries, USA;

[0034] TD24-WS low-speed centrifuge, purchased from Changsha Pingfan Instrument Co., Ltd.;

[0035] Double-person single-sided clean bench, purchased from Suzhou Purification Equipment Co., Ltd.

[0036] The water-jacketed CO2 incubator was purchased from Shanghai Santeng Instrument Co., Ltd.

[0037] The electrophoresis apparatus was purchased from Beijing Liuyi Biological Instrument Company;

[0038] NanoQuant microplate reader was purchased from Tecan, Switzerland.

[0039] The drugs and reagents involved in the present invention are as follows:

[0040] TAB reference substance was homemade in our laboratory with a purity of >98%;

[0041] CUR reference substance was purchased from Shanghai Yien Chemical Technology Co., Ltd. with a purity of >98%, batch number: RH227528;

[0042] Rhein reference substance was purchased from Beijing Solarbio Technology Co., Ltd., purity >98%, batch number: 107D021;

[0043] Ursolic acid reference substance was purchased from Shanghai Yuanye Biotechnology Co., Ltd. with a purity of >98%, batch number: C15011Q126879;

[0044] Heparin sodium injection, 2 mL: 12,500 units, purchased from Wanbang Pharmaceutical, batch number: 52201102;

[0045] Fetal bovine serum was purchased from Biological Industries, Israel, batch number: 1907301;

[0046] 1640 dry powder culture medium, purchased from Gibco, USA, batch number 1937557;

[0047] Trypsin was purchased from Gibco, USA, batch number 2907B503;

[0048] MTT was purchased from Sigma, USA, batch number K0063;

[0049] SDS-PAGE gel preparation kit was purchased from Shanghai Biotech Biotechnology Co., Ltd., batch number 120920201217;

[0050] BCA protein quantification kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., batch number 090120201211;

[0051] Chloral hydrate and DMSO were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0052] Chromatographic grade acetonitrile and methanol were purchased from Tedia Company, USA;

[0053] Intravenous cannulas, made of PU material, were purchased from Beijing Siconos;

[0054] Flat head samplers, 22G, were purchased from Beijing Siconos.

[0055] The cell lines and animals involved in this invention are as follows:

[0056] SGC7901 gastric cancer cells were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences;

[0057] 7-week-old male SD rats, weighing 200±20 g; 6-week-old male BALB / c nude mice: 20±2 g. All were housed in the SPF animal laboratory of the Animal Center of China Three Gorges University. The experimental animal production license number is SYXK(E)2022-0012. The barrier system, drinking water, feeding, and lighting all comply with the "Quality Control Standards for Experimental Animals in Hubei Province".

[0058] The solutions prepared in this invention are as follows:

[0059] Ursolic acid internal standard solution: Weigh an appropriate amount of ursolic acid precisely and dissolve it in DMSO to obtain a ursolic acid internal standard stock solution of 5 mg·mL -1 . Take the internal standard stock solution and dilute the internal standard with an acetonitrile solution to a concentration of 0.5 mg·mL -1 . Store it in a -20°C refrigerator for later use. When using, dilute it with acetonitrile to a concentration of 40 μg·mL -1 to obtain the ursolic acid internal standard solution;

[0060] Emodin internal standard solution: Weigh an appropriate amount of emodin precisely and dissolve it in DMSO to obtain an emodin internal standard stock solution of 2 mg·mL -1 . Take the internal standard stock solution and dilute the internal standard with an acetonitrile solution to a concentration of 0.2 mg·mL -1 . Store it in a -20°C refrigerator for later use. When using, dilute it with acetonitrile to a concentration of 16 μg·mL -1 to obtain the emodin internal standard solution;

[0061] TAB reference substance solution: Weigh the TAB reference substance precisely, add it to DMSO, dissolve and mix well to obtain a concentration of 10 mg·mL -1 . Then dilute it with acetonitrile to obtain TAB reference substance solutions with concentrations of 1.25, 2.5, 3.75, 6.25, 7.5, and 10 mg·mL -1 respectively. Store it at -20°C for later use;

[0062] CUR reference substance solution: Weigh the CUR reference substance precisely, add it to DMSO, dissolve to obtain a concentration of 10 mg·mL-1 The stock solution was then diluted with acetonitrile to obtain concentrations of 0.5, 1.25, 2.5, 3.75, 5, and 6.25 mg mL -1 The CUR reference solution was stored at -20℃ for future use.

[0063] Example 1 Effect of combined use of CUR and TAB on the proliferation of gastric cancer cell line SGC7901

[0064] (1) Cell culture

[0065] Gastric cancer cells SGC7901 were cultured in 1640 medium containing 10% FBS and 1% double antibody, seeded in T-75 culture flasks, and cultured in an incubator at 37°C and 5% CO2. Medium was changed and passaged according to the cell growth status.

[0066] (2) MTT assay to detect cell viability when CUR or TAB was used alone

[0067] SGC7901 cells in the logarithmic growth phase were collected and cultured at a rate of 1×10 5 Cells were seeded in a 96-well plate at a concentration of 100 μL per well. The wells were sealed with PBS and incubated overnight in a 5% CO2, 37°C saturated humidity incubator. After the cells adhered to the wall, drugs were added. TAB and CUR stock solutions were prepared with DMSO at a concentration of 10 mg·mL. -1 After dilution with culture medium, TAB was added to a 96-well plate to make the final concentration of TAB 10, 20, 40, 60, and 80 μg mL -1 The final concentrations of CUR were 3, 6, 12, 24, and 30 μg mL -1 The blank control group was added with the same amount of 1640 complete medium, and three replicates were set up for each group. After culturing for 24 h, 20 μL MTT (5 mg mL -1 ), incubate in the dark for 4 h, discard the supernatant, add 150 μL DMSO to each well, shake at low speed for 10 min, measure the optical density (OD) value at a wavelength of 490 nm using a microplate reader, and calculate the growth inhibition rate according to formula (1). Repeat the experiment 3 times, and calculate the half-maximal inhibitory concentration (IC) of the cells to the drug using SPSS software. 50 ), the results are shown in Table 1:

[0068] Cell growth inhibition rate = (1-OD 实验组 / OD 对照组 )×100%(1);

[0069] Table 1 Effects of TAB and CUR alone on the proliferation of SGC7901 cells

[0070]

[0071] As shown in Table 1, the IC of CUR alone 50 is 37.23 μg·mL -1 , and the IC of TAB alone 50 is 119.36 μg·mL -1 .

[0072] (3) Detect the cell viability when CUR and TAB are used in combination by MTT method

[0073] Select the drug concentrations with single - use inhibition rates within 10% - 30% determined in (2) for combination. The combination concentration of CUR is 12, 24 μg·mL -1 , and the combination concentration of TAB is 20, 40 μg·mL -1 . Detect the cell viability by MTT method, and the method steps are the same as above. The results show that: compared with the single - use results, the cell viability significantly decreases after TAB and CUR are used in combination ( Figure 1 ), indicating that the combination of TAB and CUR can significantly improve the inhibitory effect on tumor cell proliferation. Then use Compusyn Software to calculate the combination index (CI). When 0 < CI < 1, it means that the combination of the two drugs has a synergistic effect, and the closer the CI value is to 0, the better the synergistic effect. The results show that when CUR is 12, 24 μg·mL -1 and TAB is 20, 40 μg·mL <00000​​​​​​​​​​​​​​​​​​​​​​​​​​​​SGC7901 cells were cultured according to the method described in Example 1. After the cells adhered to the culture flask, they were divided into TAB, CUR and two-drug combination groups, and 20 and 40 μg mL -1 TAB, 12, 24 μg·mL -1 After culturing CUR and the combination drug for 6 h, the upper culture medium was discarded and washed twice with 4 mL of PBS to remove floating cells and residual culture medium. The cells were collected by trypsin digestion and then washed twice with 2 mL of PBS. The cells were counted. After collecting the cells, 50 μL of cell lysis buffer was added, the supernatant was centrifuged and extracted three times with 200 μL of ethyl acetate. The extracts were combined, evaporated, and then 200 μL of acetonitrile solution containing internal standard was added for re-dissolution. The CUR group was treated with rhubarb internal standard solution (final concentration of 16 μg mL -1 ), TAB alone group plus ursolic acid internal standard solution (final concentration of 40 μg mL -1 ), CUR combined with TAB group plus rhubarb (final concentration of 16 μg mL -1 ) and ursolic acid (final concentration 40 μg mL -1 ) internal standard solution, centrifuge and collect the supernatant, and analyze the concentration of the two drugs in the cells by liquid chromatography, using the same chromatographic conditions as (1). Set up three parallel samples for each concentration, and calculate the amount of drug contained in 10,000 cells.

[0082] (3) Specificity experiment

[0083] The blank group cells were treated according to the method and steps of (2), and blank cell lysate was taken and TAB reference solution (final concentration of 150 μg mL -1 ) and ursolic acid internal standard solution (final concentration 40 μg mL -1 ) blank cell lysate, add CUR reference solution (final concentration of 50 μg mL -1 ) and emodin reference substance (final concentration of 16 μg·mL -1 ) and the cell lysate of the drug-treated group in Example 2 (2) were used as samples. After extraction, the samples were injected and analyzed according to the chromatographic conditions in (1). The results are shown in FIG. Figure 2 As shown in the figure, the endogenous substances in the blank cell lysate and the impurities in the mobile phase did not affect the determination of TAB and CUR, and TAB, CUR and the internal standard were completely separated, indicating that the chromatographic conditions can be used to determine the uptake of CUR and TAB by SGC7901 cells.

[0084] (4) Linear relationship

[0085] The cells were treated according to the method and steps of (2), and blank cell lysate was collected. Then, 48 μL of blank cell lysate was taken in 6 portions, and 2 μL of each series of TAB reference solution was added to obtain final TAB concentrations of 50, 100, 150, 250, 300, and 400 μg mL -1 The standard curve working solution was prepared, and then the sample was injected and analyzed according to the chromatographic conditions in (1). The chromatographic results were then statistically analyzed, with the ratio of the TAB peak area to the internal standard peak area as the Y axis, and the TAB concentration (μg mL -1 ) is the X-axis, and the least weighted square method is used for linear regression to obtain the standard curve equation: Y1=0.0168X+0.0916, R 2 =0.9967, TAB is 50-400 μg·mL -1 There is a good linear relationship between the peak area and the concentration of TAB in cells, indicating that this method can be used to quantitatively determine the concentration of TAB in cells within the limit of quantification.

[0086] The CUR stock solution was treated according to the above method to obtain final concentrations of 20, 50, 100, 150, 200, and 250 μg mL -1 The standard curve working solution was analyzed by liquid phase to obtain the standard curve equation Y2=0.0255X-0.0044, R 2 =0.9981, CUR is 20-250 μg·mL -1 There is a good linear relationship between the peak area and the concentration of CUR in cells, indicating that this method can be used to quantitatively determine the concentration of CUR in cells within the limit of quantification.

[0087] (6) Precision test

[0088] According to the method and steps in (2), the blank group SGC7901 cell lysate was collected, and CUR or TAB reference solution was added to obtain CUR quality control samples (75, 120, 200 μg mL -1 ) and TAB quality control samples (50, 150, 300 μg mL -1 ), each concentration was repeated three times, the quality control sample cell lysate was extracted three times with 200 μL ethyl acetate, the extracts were combined, and after evaporation, the CUR quality control sample was added with emodin internal standard solution (final concentration of 16 μg mL -1 ), ursolic acid internal standard solution (final concentration of 40 μg mL) was added to the TAB quality control sample. -1 ) was reconstituted and analyzed according to the chromatographic conditions in (1). TAB and CUR were measured six times per day at each concentration to calculate intra-day precision; each concentration was measured once per day for three consecutive days to calculate inter-day precision and accuracy. According to the "Guidelines for Quantitative Analysis of Biological Samples", an accuracy of no more than 15% meets the analytical requirements.

[0089] Table 3 Intra-day and inter-day precision and accuracy of CUR and TAB in cell lysate (n=6)

[0090]

[0091]

[0092] The determination results are shown in Table 3. It can be seen that the intra-day and inter-day precision of TAB and CUR are good, indicating that this method and instrument can be used to measure the concentrations of TAB and CUR in cells.

[0093] (7) Accuracy experiment

[0094] Take 48 μL of blank cell lysate and add 2 μL of CUR control solution (final concentration of 75, 120, and 200 μg mL -1 ), 2 μL TAB reference solution (final concentrations of 50, 150, and 300 μg mL -1 ), the sample was injected and measured according to the chromatographic conditions of (1), and the ratio of the obtained TAB peak area to the internal standard peak area was substituted into the standard curve equation to calculate the concentration. The ratio of the measured value to the actual value was used as the accuracy, as shown in Table 3. The results met the analysis requirements of biological samples.

[0095] (8) Effect of combined use of CUR and TAB on cellular uptake

[0096] Using the 40 and 80 μg·mL determined in Example 1 -1 TAB solution was mixed with 12 and 24 μg mL -1 The cell uptake was determined by combining TAB and CUR solutions with DMSO to prepare TAB and CUR stock solutions at a concentration of 10 mg mL -1 When adding drugs, dilute the two drugs with culture medium according to the required concentration and add them to the culture bottle. The results are as follows Figure 3 As shown in the figure, different concentrations of combined use have different promoting effects on drug absorption by gastric cancer cells, among which TAB (40 μg·mL -1 ) for CUR (12 μg·mL -1 ) had a promotion rate of 1.56, TAB (80 μg·mL -1 ) for CUR (24 μg·mL -1 ) had a promotion rate of 2.98; CUR (12 μg·mL -1 ) for TAB (40 μg·mL -1 ) had a promotion rate of 1.18, CUR (24 μg·mL -1 ) for TAB (80 μg·mL -1) was 1.63, indicating that TAB and CUR had a mutually promoting absorption effect on cells, and the promoting effect of TAB on CUR was greater than that of CUR on TAB.

[0097] Example 3 In vivo absorption experiment in rats

[0098] (1) Medication and sampling

[0099] After SD rats were anesthetized by intraperitoneal injection of 7% chloral hydrate (0.6 mL / 100 g), they were fixed in a dorsal position. The skin was cut longitudinally about 1 cm from the midpoint of the right clavicle 0.5 cm above the neck, the jugular vein was bluntly separated, and the distal end was ligated with silk thread. Then a small incision was made on the blood vessel, and the intravenous cannula was inserted about 2.5 cm along the incision toward the heart. It was fixed to the blood vessel with sterile surgical thread, and the uninserted part was led out from the back along the subcutaneous tissue, ligated and fixed, and the muscle layer and skin at the opening were sutured. After surgery, the rats were placed individually in metabolic cages for physiological recovery for at least 12 hours, and fasted but not watered for 12 hours before administration.

[0100] SD rats were divided into blank group, CUR group (300 mg·kg -1 ), TAB group (40 mg·kg -1 ) and CUR-TAB combined group, with 6 rats in each group; fasting for 12 h before the experiment, free access to water, oral administration, TAB, CUR were dissolved in 0.5% CMC-Na solution, prepared to the target concentration for use; CUR-TAB group, CUR and TAB were mixed and dissolved in 0.5% CMC-Na solution and administered simultaneously, and all rats were fed 2 h after administration; 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10 h after administration, about 200 μL of whole blood was collected each time through jugular vein cannula into heparinized EP tube, and immediately rotated at 4000 r·min -1 After centrifugation for 10 minutes, plasma samples were obtained and stored in a -20°C refrigerator for testing.

[0101] (2) Plasma sample processing

[0102] Take 100 μL of plasma sample obtained in (1) and add 300 μL of ethyl acetate, vortex for 5 min, mix well, and rotate at 12000 r·min. -1 Centrifuge for 10 min, take the upper solution, extract three times and combine the extracts, dry at 40°C, add 50 μL of acetonitrile solution containing internal standard to reconstitute, and add emodin internal standard solution (final concentration of 8 μg mL) to the plasma samples of CUR group. -1 ), plasma samples of TAB group were added with ursolic acid internal standard solution (final concentration of 20 μg mL -1), plasma samples of the CUR-TAB group were added with emodin (final concentration of 8 μg mL -1 ) and ursolic acid (final concentration of 20 μg mL -1 ) internal standard solution, the plasma samples of the CUR-TAB group were analyzed for the contents of CUR and TAB drugs, and chromatographic analysis was performed. The chromatographic conditions were the same as those in Example 2 (1).

[0103] (3) Specificity experiment

[0104] Blank plasma was taken and TAB reference solution (final concentration was 100 μg mL) was added. -1 ) and ursolic acid internal standard solution (final concentration of 20 μg mL -1 ) plasma, and CUR reference solution (final concentration of 20 μg mL -1 ) and emodin internal standard solution (final concentration 8 μg mL -1 ) plasma and rat plasma samples after administration were pre-treated according to the method steps in (2) and analyzed according to the chromatographic conditions in (1) of Example 2. The results are shown in Figure 2. Figure 4 As shown in the figure, endogenous substances in blank plasma and impurities in the mobile phase did not affect the determination of TAB and CUR. TAB, CUR and the internal standard were completely separated and the peak shapes were symmetrical, indicating that the chromatographic conditions could accurately determine TAB and CUR.

[0105] (4) Linear relationship

[0106] Take 6 portions of 96 μL of blank plasma and add 4 μL of TAB series concentration reference solution to obtain final concentrations of 10, 15, 25, 75, 100, and 150 μg mL -1 The standard curve working solution was prepared according to the method steps in (2) and the sample was analyzed according to the chromatographic conditions in (1) of Example 2. The ratio of the TAB peak area to the internal standard peak area was plotted on the Y axis, and the TAB concentration (μg·mL -1 ) as the X-axis, and the least weighted square method was used for linear regression to obtain the standard curve equation: Y3 = 0.054X ​​+ 0.4249, R2 = 0.9992, TAB in the range of 10-150 μg mL -1 The good linear relationship between the peak area and the concentration of TAB in rat plasma was observed, indicating that the method could be used to quantitatively determine the concentration of TAB in rat plasma within the limit of quantification.

[0107] The CUR reference solution was treated according to the above method to obtain 1, 10, 15, 20, 25, and 50 μg mL -1 The standard curve working solution was analyzed by liquid phase to obtain the standard curve equation Y4=0.0893X-0.1281, R 2 =0.9991, CUR is 1-50 μg·mL-1 There was a good linear relationship between the peak area and the concentration of CUR in rat plasma, indicating that the method could be used to quantitatively determine the concentration of CUR in rat plasma within the limit of quantification.

[0108] (5) Precision test

[0109] The rat blank plasma was treated according to the method and steps in (2) of Example 2, and the CUR reference solution was added to obtain final concentrations of 5, 15, and 30 μg·mL -1 The quality control samples were added with TAB reference solution to obtain final concentrations of 15, 100, and 150 μg mL -1 The quality control samples were prepared in triplicate at each concentration and chromatographic injection analysis was performed according to the conditions in (1) of Example 2. Each concentration of TAB and CUR was measured 6 times a day to calculate the intra-day precision; each concentration was measured once a day for three consecutive days to calculate the inter-day precision and accuracy. According to the "Guidelines for Quantitative Analysis of Biological Samples", an accuracy of no more than 15% meets the analysis requirements. The measurement results are shown in Table 4, which shows that the intra-day and inter-day precision of TAB and CUR are good, indicating that this method and instrument can be used to determine the content of TAB and CUR in rat plasma.

[0110] (6) Accuracy test

[0111] Blank plasma was taken and CUR reference solution was added to obtain final concentrations of 5, 15, and 30 μg mL -1 For quality control samples, TAB reference solution was added to obtain final concentrations of 15, 100, and 150 μg mL -1 The quality control samples were processed according to (2) of Example 3 and injected according to the chromatographic conditions in (1) of Example 2. The ratio of the obtained TAB peak area to the internal standard peak area was substituted into the standard curve equation to calculate the concentration. The ratio of the measured value to the actual value was used as the accuracy as shown in Table 4. The results met the analysis requirements of biological samples.

[0112] Table 4 Intra-day and inter-day precision and accuracy of CUR and TAB in rat plasma (n=6)

[0113]

[0114]

[0115] (7) Absorption in rats

[0116] The HPLC conditions in Example 1 were used to analyze the three groups of rats in (1) (CUR group (300 mg·kg -1 ), TAB group (40 mg·kg -1 ) and CUR-TAB combined group) plasma samples were tested and analyzed.

[0117] The CUR drug concentrations in the CUR group and CUR-TAB combination group were as follows: Figure 5 As shown in Figure A, the drug concentration in rat plasma was low when CUR was used alone, and the blood drug concentration increased significantly when used in combination; the TAB drug concentration in the TAB group and the CUR-TAB combination group was as shown in Figure A. Figure 5 As shown in Figure B, the TAB drug concentration in the combination group was higher than that in the TAB single-use group.

[0118] right Figure 5 The data from the were fitted and analyzed, resulting in metabolic parameters shown in Table 5. Pharmacokinetic results indicate that the peak concentration (Cmax) of TAB in the combination group was 2.88 times that of the TAB monotherapy group, and the Cmax of CUR in the combination group was 2.27 times that of CUR monotherapy. In summary, TAB and CUR enhance each other's absorption. TAB exhibits bimodal absorption at 0.75 and 2.5 hours, and the Cmax of both groups increases compared to TAB monotherapy.

[0119] Table 5 Pharmacokinetic parameters of CUR and TAB in rat plasma (n=6)

[0120]

[0121] Compared with the single-use group: *P<0.05, significant difference; **P<0.01, extremely significant difference.

[0122] Example 4 Western Blot Experiment

[0123] SGC7901 cells were divided into blank control group and positive control group (verapamil 50 μg·mL -1 or Ko143 50 μg mL -1 ), TAB group (40 μg·mL -1 ), CUR group (12 μg·mL -1 ), TAB+CUR group (40 μg·mL -1 +12 μg·mL -1 ) Five groups were administered drugs. The drugs were dissolved in DMSO to form a 10 mg / mL stock solution, diluted with culture medium to the required concentration and added to culture flasks. In the TAB+CUR group, the two drugs were mixed and administered simultaneously. After culturing in a 37°C 5% CO2 constant temperature incubator for 12 hours, intracellular proteins were extracted, and the protein samples were quantified using a BCA kit. Western Blot experiments were performed to observe the regulatory effects of TAB and / or CUR on the expression of P-gp and BCRP proteins.

[0124] The results are as follows Figure 6The results showed that compared with the blank group, TAB or CUR alone could reduce the expression of P-gp and BCRP proteins, but the effect was not significant. Compared with the positive control, the expression of the two proteins was higher. However, the combined treatment of TAB and CUR significantly reduced the expression of P-gp and BCRP proteins in SGC cells. After the combination of the two drugs, the expression of the two proteins was lower than that of the single-drug group and the positive control group, and the difference was extremely significant. These results indicate that TAB and CUR promote absorption by inhibiting the expression of efflux transporters P-gp and BCRP.

[0125] Example 5 Effects of CUR and TAB on SGC7901 transplanted tumors

[0126] (1) Animal modeling and drug administration

[0127] Twenty-four healthy male BALB / c nude mice, weighing 18-20 g and aged 5 weeks, were selected and fed for one week at a density of 4*10 7 100 μL of SGC7901 cell suspension was taken and inoculated into the armpit of nude mice. When the tumor volume reached 60-80 mm 3 After taking pictures, the nude mice were randomly divided into 4 groups: model group, TAB experimental group (40 mg·kg -1 ), CUR experimental group (40 mg·kg -1 ), combined experimental group; daily gavage administration, every other day the length and width of the transplanted tumors were measured and the nude mice were weighed; after 21 days of administration, the nude mice were sacrificed, the transplanted tumors were removed, the tumor volume was measured, and the weight was weighed; 5 nude mouse transplanted tumors were removed from each group, and tumor tissue of approximately 0.8 cm in diameter was cut and stored at -80°C for later use. Calculation was performed using the following formula:

[0128] (1) TV (tumor volume) = 1 / 2*a*b 2 , a tumor length; b tumor width;

[0129] (2) RTV (relative tumor volume) = V t / V0, V0 is the tumor volume of each group before drug administration, V t The tumor volume of each group was measured each time;

[0130] (3) Tumor inhibition rate (%) = [tumor weight of model group (C) - tumor weight of experimental group (T)] / tumor weight of model group (C) * 100%.

[0131] The tumor volume, weight and tumor inhibition rate of each group are shown in Table 6. The tumor growth curve is shown in Figure 7 shown.

[0132] Table 6 Tumor volume, weight and tumor inhibition rate of each group

[0133]

[0134] Compared with the model group: *P<0.1, significant difference; **P<0.05, extremely significant difference.

[0135] Table 6 and Figure 7 The results showed that both CUR and TAB could inhibit tumor growth. The tumor inhibition rate of CUR reached 28.09%, and that of TAB reached 21.35%. When TAB and CUR were used together, the tumor inhibition rate reached 51.69%, indicating that TAB and CUR had a synergistic effect when used together.

[0136] (2) Determination of drug concentration in tumor

[0137] Determination of drug concentration in tumors: 100 mg of collected tumor tissue was weighed and placed in EP tubes. 1 mL of ethyl acetate was added and ground in a grinder in an ice bath until the tumor was pulverized. The sample was centrifuged at 12,000 rpm for 20 min. The supernatant was removed and evaporated to dryness at 40°C. 100 μL of chromatography-grade acetonitrile was added for reconstitution. The concentrations of TAB and CUR in the tumors were determined according to the chromatographic conditions described in Example 2 (1). The results are shown in Table 7.

[0138] Table 7 Drug concentration in tumor

[0139]

[0140] Compared with the single-use group: *P<0.1, significant difference; **P<0.05, extremely significant difference.

[0141] The data in Table 7 show that the TAB concentration in the tumor of the combination group was higher than that of the TAB alone group, and the CUR concentration in the tumor of the combination group was higher than that of the CUR alone group, indicating that CUR and TAB mutually promoted the entry of drugs into the tumor.

Claims

1. A combined drug of curcumin and tracheobacterial acid, characterized in that: The mass concentration of curcumin is 3-30 μg·mL -1 The mass concentration of tracheobacterial acid is 10-80 μg·mL -1 .

2. The combined drug of curcumin and tracheobacterial acid according to claim 1, wherein: The mass concentration of curcumin was 12 μg·mL -1 The mass concentration of traemolic acid was 10, 20, 40, 60, and 80 μg·mL -1 Any one of .

3. The combined drug of curcumin and tracheobacterial acid according to claim 1, wherein: The mass concentration of curcumin was 24 μg·mL -1 The mass concentration of traemolic acid was 10, 20, 40, 60, and 80 μg·mL -1 Any one of .

4. A combined medicament comprising curcumin and tracheobacterial acid according to any one of claims 1 to 3, characterized in that: The medicament further includes a pharmaceutically acceptable excipient.

5. Use of a combination of curcumin and tracheobic acid in the preparation of a medicament for treating gastric cancer, characterized in that: The mass concentration of curcumin is 3-30 μg·mL -1 The mass concentration of tracheobacterial acid is 10-80 μg·mL -1 .

6. The use according to claim 5, characterized in that: The mass concentration of curcumin is 12 μg·mL -1 The mass concentration of traemolic acid was 10, 20, 40, 60, and 80 μg·mL -1 Any one of .

7. The use according to claim 5, characterized in that: The mass concentration of curcumin is 24 μg·mL -1 The mass concentration of traemolic acid was 10, 20, 40, 60, and 80 μg·mL -1 Any one of .

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