Composition for resisting multidrug resistant tumors and preparation method thereof

Abstract

The invention relates to the technical field of drugs for resisting multidrug-resistant tumors, in particular to a composition for resisting multidrug-resistant tumors and a preparation method thereof.The composition comprises a venenum bufonis extract, a tripterygium wilfordii extract and a flos carthami extract, and the mass ratio of the venenum bufonis extract to the tripterygium wilfordii extract to the flos carthami extract is (0.8-1.2): (0.8-1.2): (0.8-1.2). The IC50 value of the composition to various drug-resistant tumor cells is reduced by 80% or above compared with that of a single component, and the remarkable synergistic effect is extremely rare in the prior art; westernblot and PCR (Polymerase Chain Reaction) analysis prove that the composition can regulate and control the expression of a plurality of drug resistance related genes at the same time to form an omnibearing drug resistance reversal network. A cytotoxicity experiment shows that the toxicity of the composition to normal cells is far lower than that of an existing anti-drug-resistant drug, the selectivity is derived from the ingenious mutual restriction effect among the three components, the nano preparation shows excellent targeting and slow release characteristics in in-vivo and in-vitro experiments, and the enrichment degree of the drug in tumor tissue is 3.6 times that of a common preparation.

Description

Technical Field

[0001] The invention relates to the technical field of drugs for resisting multidrug-resistant tumors, and in particular to a composition for resisting multidrug-resistant tumors and a preparation method thereof. Background Art

[0002] At present, tumor multidrug resistance has become the main obstacle to the effectiveness of chemotherapy. The generation of drug resistance mainly stems from the overexpression of drug efflux pumps on the tumor cell membrane, especially the abnormal activation of members of the ABC transporter family such as P-glycoprotein, MRP1 and BCRP. The existing technology mainly solves this problem in the following ways: first, develop P-glycoprotein inhibitors, such as verapamil drugs; second, design drug delivery systems that can bypass efflux pumps; third, use Chinese medicine compound to improve chemotherapy sensitivity. However, these methods all have obvious shortcomings. Inhibitors with a single mechanism of action often produce serious toxic side effects due to insufficient selectivity; simple delivery systems are difficult to cope with complex resistance mechanisms; although traditional Chinese medicine compound has the advantage of multiple targets, the active ingredients are unclear and the quality is difficult to control. Summary of the invention

[0003] In view of this, there is an urgent need to develop new formulations that can synergistically reverse drug resistance through multiple targets while ensuring controllable quality.

[0004] The object of the present invention is to provide a composition for resisting multidrug-resistant tumors, comprising toad venom extract, tripterygium wilfordii extract and safflower extract, wherein the mass ratio of the toad venom extract, tripterygium wilfordii extract and safflower extract is (0.8-1.2):(0.8-1.2):(0.8-1.2).

[0005] Preferably, the mass ratio of the toad venom extract, the tripterygium wilfordii extract and the safflower extract is 0.8:0.8:0.8.

[0006] The preparation method of the composition comprises the following steps:

[0007] (1) preparing the toad venom extract, comprising:

[0008] First, toad venom powder was mixed with dichloromethane at a weight-to-volume ratio of 1:20 and extracted three times at 20 ± 2 °C for 4 h each time;

[0009] Secondly, the extract is concentrated under reduced pressure until there is no solvent smell;

[0010] Then, the concentrate was dissolved in methanol and subjected to silica gel column chromatography using chloroform:methanol:acetic acid=95:8:5 as the mobile phase;

[0011] Finally, the collected components were purified by Sephadex LH-20 gel column, eluted with methanol, and the second part was collected and freeze-dried to obtain the toad venom extract.

[0012] Preferably, the preparation method further comprises:

[0013] (2) preparing the Tripterygium wilfordii extract, comprising:

[0014] First, the powder of Tripterygium wilfordii was mixed with 75% ethanol at a weight-to-volume ratio of 1:12 and refluxed for extraction at 78±2°C for 3 times, each time for 30 min;

[0015] Secondly, the extract is concentrated under reduced pressure to a relative density of 1.15-1.20;

[0016] Again, the concentrate was chromatographed on a HPD-100 macroporous resin column, eluting with water, 40% ethanol, 60% ethanol and 80% ethanol in sequence;

[0017] Finally, the 60% ethanol eluate was collected, concentrated and dried under reduced pressure to obtain the Tripterygium wilfordii extract.

[0018] Preferably, the preparation method further comprises:

[0019] (3) preparing the safflower extract, comprising:

[0020] First, safflower powder was mixed with 75% ethanol at a weight-to-volume ratio of 1:15;

[0021] Secondly, ultrasonic extraction was performed twice at 45±2℃, power 300W, and frequency 40kHz, each time for 30 minutes;

[0022] Then, the extract was chromatographed on a D101 macroporous resin column and eluted with a gradient of 50-70% ethanol;

[0023] Finally, the target eluate was collected, concentrated and dried under reduced pressure to obtain the safflower extract.

[0024] 6 Preferably, the particle sizes of the toad venom powder, tripterygium wilfordii powder and safflower powder are 100 mesh, 80 mesh and 100 mesh respectively.

[0025] Preferably, the stationary phase of the silica gel column chromatography is 200-300 mesh silica gel, and the flow rate is 2.0 mL / min; the flow rate of the HPD-100 macroporous resin column chromatography is 1.5 BV / h; the pH of the D101 macroporous resin column chromatography is 5.0-6.0, and the flow rate is 1.0 BV / h.

[0026] The preparation of the composition is an injection or a nano preparation; wherein the pH of the injection is 6.8-7.2, and the osmotic pressure is 270-330 mOsm / L; the particle size of the nano preparation is 80-200 nm, the polydispersity index PDI is ≤0.25, the Zeta potential is -20 to -30 mV, and the encapsulation rate is ≥85%.

[0027] Based on the in-depth study of three traditional Chinese medicines, namely, toad venom, tripterygium wilfordii and safflower, the present invention discovered for the first time that their specific active ingredients formed a unique synergistic network at the molecular level: toad toxic glycoside compounds change the cell membrane potential and increase drug transmembrane transport by specifically binding to Na+ / K+-ATPase; tripterygium wilfordii lactone compounds achieve epigenetic regulation of MDR1 gene transcription by regulating the MAPK signaling pathway; and hydroxysafflower yellow not only has an antioxidant effect, but also can stabilize the cell membrane structure, forming a complete action chain with the first two. What is even more surprising is that the optimal ratio of the three ingredients of 1:1:1 not only maximizes the activity, but also significantly reduces the toxicity of each ingredient through mutual restraint.

[0028] The present invention adopts an innovative extraction process and successfully solves the problems of low purity of active ingredients and large batch differences in traditional processes. By accurately controlling key parameters such as extraction temperature and pH value, the stable acquisition of high-purity active ingredients is ensured. In particular, in the extraction process of bufotoxin, the method of low-temperature extraction with dichloromethane combined with macroporous resin separation is creatively adopted, which not only ensures the integrity of the active ingredients, but also significantly improves the yield.

[0029] In terms of formulation technology, the nanoliposome system developed in the present invention exhibits unique advantages: first, by optimizing the lipid composition, efficient encapsulation of the three components is achieved; second, the nanosize enables it to be enriched at the tumor site through the EPR effect; more importantly, the liposome delivers the drug directly into the cell through endocytosis, cleverly bypassing the efflux pump system on the cell membrane.

[0030] The present invention has the following beneficial effects:

[0031] 1. Breakthrough anti-drug resistance activity: Experimental data show that the IC50 value of the combination against a variety of drug-resistant tumor cells is more than 80% lower than that of a single prescription. This significant synergistic effect is extremely rare in the prior art.

[0032] 2. Unique multiple synergistic mechanism: Western blot and PCR analysis confirmed that the combination can simultaneously regulate the expression of multiple drug resistance-related genes, forming a comprehensive drug resistance reversal network.

[0033] 3. Remarkable selectivity: Cytotoxicity experiments show that the combination is much less toxic to normal cells than existing anti-resistance drugs. This selectivity stems from the subtle mutual restraint between the three components.

[0034] 4. Excellent drug delivery effect: Nanoformulations have shown excellent targeting and sustained-release properties in both in vivo and in vitro experiments, and the drug enrichment in tumor tissue is 3.6 times that of ordinary preparations.

[0035] 5. Good industrialization prospects: The innovative extraction process and preparation technology not only ensure the stability of product quality, but also have a high cost-effectiveness ratio, laying the foundation for industrialization.

[0036] In summary, the present invention has achieved breakthroughs in component selection, ratio optimization, process improvement and formulation innovation, providing a new solution to overcome the problem of tumor drug resistance. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0038] Example 1

[0039] Composition for resisting multidrug-resistant tumors and preparation method thereof

[0040] The composition of this embodiment consists of toad venom extract, tripterygium wilfordii extract and safflower extract, and the mass ratio of the three extracts is 0.8:0.8:0.8.

[0041] The specific preparation method comprises the following steps:

[0042] (1) Preparation of Toad Venom Extract

[0043] First, take 100 mesh toad venom powder, add 20 times the volume of dichloromethane, and extract 3 times at 20°C, 4 hours each time. Secondly, the extract is concentrated under reduced pressure at 35°C and -0.08MPa until there is no solvent smell. Then, the concentrate is dissolved in methanol, and chromatographed using a 200 mesh silica gel column with chloroform: methanol: acetic acid = 95:8:5 as the mobile phase at a flow rate of 2.0mL / min. Finally, the collected components are purified using a Sephadex LH-20 gel column, eluted with methanol, and the second part is collected, and freeze-dried at -50°C for 48 hours to obtain the toad venom extract.

[0044] (2) Preparation of Tripterygium wilfordii extract

[0045] First, take 80 mesh Tripterygium wilfordii powder, add 12 times the volume of 75% ethanol, and reflux extract at 78°C for 3 times, each time for 30 minutes. Secondly, the extract is concentrated under reduced pressure at 45°C and -0.06MPa to a relative density of 1.15. Thirdly, the concentrate is chromatographed on an HPD-100 macroporous resin column at a flow rate of 1.5BV / h, and eluted with 3 column volumes of water, 2 column volumes of 40% ethanol, 3 column volumes of 60% ethanol, and 2 column volumes of 80% ethanol. Finally, the 60% ethanol eluate is collected, concentrated under reduced pressure, and dried to obtain the Tripterygium wilfordii extract.

[0046] (3) Preparation of safflower extract

[0047] First, take 100 mesh safflower powder and add 15 times the volume of 75% ethanol. Secondly, ultrasonic extraction is performed twice at 45°C, power 300W, frequency 40kHz, each time for 30 minutes. Then, the extract is chromatographed on a D101 macroporous resin column, pH 5.0, flow rate 1.0BV / h, and gradient eluted with 50% ethanol. Finally, the target eluate is collected, concentrated and dried under reduced pressure to obtain a safflower extract.

[0048] Preparation of formulations:

[0049] The three extracts prepared above were mixed in a mass ratio of 0.8:0.8:0.8, and the preparation process was adopted for preparation. Specifically, the mixture was dissolved in water for injection, the pH was adjusted to 6.8, the osmotic pressure was controlled to 270mOsm / L, and sterilized by filtration with a 0.22μm filter membrane. The prepared injection was stored in a sterile vial and sterilized at 121°C for 15 minutes. Quality testing showed that the prepared injection met the injection quality standards and had good stability. The preparation prepared in this embodiment showed significant anti-multidrug resistance activity in in vitro cell experiments, and the IC50 value of MCF-7 / ADR resistant cells was significantly reduced.

[0050] Example 2

[0051] Composition for resisting multidrug-resistant tumors and preparation method thereof

[0052] The composition of this embodiment is composed of toad venom extract, tripterygium wilfordii extract and safflower extract, and the mass ratio of the three extracts is 1:1:1. Preferably, the toad venom extract in this embodiment contains toad toxic glycosides (50%, purity 98.5%), cinobuccal venom (30%, purity 96%) and monacolic toxin (20%, purity 96%). Experiments show that this ratio can synergistically enhance the anti-resistance activity of each component. Tripterygium wilfordii extract contains triptolide A (55%, purity 96%), triptolide B (30%, purity 92%) and triptolide A (15%, purity 92%). Safflower extract contains hydroxysafflor yellow A (65%, purity 92%) and safflor yellow B (30%, purity 87%).

[0053] The specific preparation method comprises the following steps:

[0054] (1) Preparation of Toad Venom Extract

[0055] First, take 100 mesh toad venom powder, add 20 times the volume of dichloromethane, and extract 3 times at 22°C, 4 hours each time. Secondly, the extract was concentrated under reduced pressure at 35°C and -0.07MPa until there was no solvent smell. Then, the concentrate was dissolved in methanol, and chromatographed using a 250 mesh silica gel column with chloroform: methanol: acetic acid = 95:8:5 as the mobile phase at a flow rate of 2.0mL / min. Finally, the collected components were purified using a Sephadex LH-20 gel column, eluted with methanol, and the second part was collected and freeze-dried at -50°C for 48 hours to obtain the toad venom extract.

[0056] (2) Preparation of Tripterygium wilfordii extract

[0057] First, take 80 mesh Tripterygium wilfordii powder, add 12 times the volume of 75% ethanol, and reflux extract at 78°C for 3 times, each time for 30 minutes. Secondly, the extract is concentrated under reduced pressure at 45°C and -0.06MPa to a relative density of 1.18. Thirdly, the concentrate is chromatographed on an HPD-100 macroporous resin column at a flow rate of 1.5BV / h, and eluted with 3 column volumes of water, 2 column volumes of 40% ethanol, 3 column volumes of 60% ethanol, and 2 column volumes of 80% ethanol. Finally, the 60% ethanol eluate is collected, concentrated under reduced pressure, and dried to obtain the Tripterygium wilfordii extract.

[0058] (3) Preparation of safflower extract

[0059] First, take 100 mesh safflower powder and add 15 times the volume of 75% ethanol. Secondly, ultrasonic extraction is performed twice at 45°C, power 300W, frequency 40kHz, each time for 30 minutes. Then, the extract is chromatographed on a D101 macroporous resin column, pH 5.5, flow rate 1.0BV / h, and gradient eluted with 60% ethanol. Finally, the target eluate is collected, concentrated and dried under reduced pressure to obtain a safflower extract.

[0060] Preparation of formulations:

[0061] The nanoformulation process was adopted. The three extracts were mixed in a mass ratio of 1:1:1, and the nanoemulsion was prepared by high-pressure homogenization. The particle size was controlled to be 140nm, the PDI was 0.20, and the Zeta potential was -25mV. Finally, a nanoformulation with an encapsulation rate of 90% was obtained. The preparation showed excellent stability and bioavailability.

[0062] Example 3

[0063] Composition for resisting multidrug-resistant tumors and preparation method thereof

[0064] The composition of this embodiment is composed of toad venom extract, tripterygium wilfordii extract and safflower extract, and the mass ratio of the three extracts is 1.1:1.1:1.1. Preferably, in this embodiment, the composition of toad toxin compounds is optimized and screened, wherein the combination of toad toxic glycosides (52%, purity 99%), cinobufacturing toxins (32%, purity 97%) and monacolin (22%, purity 97%) can significantly improve the sensitivity of tumor cells to chemotherapeutic drugs. Tripterygium wilfordii extract contains triptolide A (57%, purity 97%), triptolide B (32%, purity 93%) and triptolide A (17%, purity 93%), and this ratio shows the best MDR1 gene inhibition effect in in vitro experiments. Safflower extract contains hydroxysafflower yellow A (67%, purity 93%) and safflower yellow B (32%, purity 88%), and this group ratio is outstanding in anti-oxidation and cell protection.

[0065] The specific preparation method comprises the following steps:

[0066] (1) Preparation of Toad Venom Extract

[0067] First, take 100 mesh toad venom powder, add 20 times the volume of dichloromethane, and extract 3 times at 22°C, 4 hours each time. Secondly, the extract was concentrated under reduced pressure at 35°C and -0.075MPa until there was no solvent smell. Then, the concentrate was dissolved in methanol, and chromatographed using a 275 mesh silica gel column with chloroform: methanol: acetic acid = 95:8:5 as the mobile phase at a flow rate of 2.0mL / min. Finally, the collected components were purified using a Sephadex LH-20 gel column, eluted with methanol, and the second part was collected and freeze-dried at -50°C for 48 hours to obtain the toad venom extract.

[0068] (2) Preparation of Tripterygium wilfordii extract

[0069] First, take 80 mesh Tripterygium wilfordii powder, add 12 times the volume of 75% ethanol, and reflux extract at 78°C for 3 times, each time for 30 minutes. Secondly, the extract is concentrated under reduced pressure at 45°C and -0.065MPa to a relative density of 1.19. Thirdly, the concentrate is chromatographed on an HPD-100 macroporous resin column at a flow rate of 1.5BV / h, and eluted with 3 column volumes of water, 2 column volumes of 40% ethanol, 3 column volumes of 60% ethanol, and 2 column volumes of 80% ethanol in sequence. Finally, the 60% ethanol eluate is collected, concentrated under reduced pressure, and dried to obtain the Tripterygium wilfordii extract.

[0070] (3) Preparation of safflower extract

[0071] First, take 100 mesh safflower powder and add 15 times the volume of 75% ethanol. Secondly, ultrasonic extraction is performed twice at 45°C, power 300W, frequency 40kHz, each time for 30 minutes. Then, the extract is chromatographed on a D101 macroporous resin column, pH 5.7, flow rate 1.0BV / h, and gradient eluted with 65% ethanol. Finally, the target eluate is collected, concentrated and dried under reduced pressure to obtain a safflower extract.

[0072] Preparation of formulations:

[0073] Combining the advantages of injection and nanoformulation, this embodiment adopts the preparation process of nanoinjection. The three extracts are mixed in a mass ratio of 1.1:1.1:1.1, and a nanosuspension is prepared by high-pressure homogenization. The particle size is controlled to be 160nm, the PDI is 0.22, the Zeta potential is -27mV, and the encapsulation efficiency is 92%. The pH is further adjusted to 7.0, the osmotic pressure is 300mOsm / L, and the nanoinjection is obtained after sterilization with a 0.22μm filter membrane. The preparation shows excellent anti-multidrug resistance effect in both in vitro and animal experiments.

[0074] Example 4

[0075] Composition for resisting multidrug-resistant tumors and preparation method thereof

[0076] The composition of this embodiment is composed of toad venom extract, tripterygium wilfordii extract and safflower extract, and the mass ratio of the three extracts is 1.2:1.2:1.2. Preferably, the toad venom extract in this embodiment contains toad toxic glycosides (55%, purity 99.5%), cinobuccal venom (35%, purity 98%) and monacolic toxin (25%, purity 98%). Studies have shown that this ratio is most outstanding in inhibiting the growth of drug-resistant tumor cells. Tripterygium wilfordii extract contains triptolide A (60%, purity 98%), triptolide B (35%, purity 95%) and triptolide A (20%, purity 95%). Safflower extract contains hydroxysafflor yellow A (70%, purity 95%) and safflor yellow B (35%, purity 90%).

[0077] The specific preparation method comprises the following steps:

[0078] (1) Preparation of Toad Venom Extract

[0079] First, take 100 mesh toad venom powder, add 20 times the volume of dichloromethane, and extract 3 times at 24°C, 4 hours each time. Secondly, the extract is concentrated under reduced pressure at 35°C and -0.08MPa until there is no solvent smell. Then, the concentrate is dissolved in methanol, and chromatographed using a 300 mesh silica gel column with chloroform: methanol: acetic acid = 95:8:5 as the mobile phase at a flow rate of 2.0mL / min. Finally, the collected components are purified using a Sephadex LH-20 gel column, eluted with methanol, and the second part is collected, and freeze-dried at -50°C for 48 hours to obtain the toad venom extract.

[0080] (2) Preparation of Tripterygium wilfordii extract

[0081] First, take 80 mesh Tripterygium wilfordii powder, add 12 times the volume of 75% ethanol, and reflux extract at 78°C for 3 times, each time for 30 minutes. Secondly, the extract is concentrated under reduced pressure at 45°C and -0.07MPa to a relative density of 1.20. Thirdly, the concentrate is chromatographed on an HPD-100 macroporous resin column at a flow rate of 1.5BV / h, and eluted with 3 column volumes of water, 2 column volumes of 40% ethanol, 3 column volumes of 60% ethanol, and 2 column volumes of 80% ethanol in sequence. Finally, the 60% ethanol eluate is collected, concentrated under reduced pressure, and dried to obtain the Tripterygium wilfordii extract.

[0082] (3) Preparation of safflower extract

[0083] First, take 100 mesh safflower powder and add 15 times the volume of 75% ethanol. Secondly, ultrasonic extraction is performed twice at 45°C, power 300W, frequency 40kHz, each time for 30 minutes. Then, the extract is chromatographed on a D101 macroporous resin column, pH 6.0, flow rate 1.0BV / h, and gradient eluted with 70% ethanol. Finally, the target eluate is collected, concentrated and dried under reduced pressure to obtain a safflower extract.

[0084] Preparation of formulations:

[0085] This embodiment adopts the nanoliposome preparation process. The three extracts are mixed in a mass ratio of 1.2:1.2:1.2, and liposomes are prepared by thin film dispersion method, with a controlled particle size of 200nm, a PDI of 0.25, a Zeta potential of -30mV, and an encapsulation efficiency of 95%. The preparation shows the best targeting and sustained release effect, and can significantly increase the enrichment of the drug at the tumor site. In the HepG2 / ADR drug-resistant cell line, the preparation shows the strongest drug resistance reversal effect.

[0086] Comparative Example 1 (single component control, corresponding to Example 1)

[0087] This comparative example is intended to verify the synergistic advantage of the three components of toad venom-tripterygium wilfordii-safflower. Only toad venom extract is used, and its composition and content are the same as those of the toad venom extract in Example 1, and the preparation method is also the same as the preparation method of the toad venom extract in Example 1. Finally, an injection is prepared, and its formulation parameters are the same as those in Example 1. The IC50 value of MCF-7 / ADR cells was determined by the MTT method. The results showed that the IC50 value when the toad venom extract was used alone was 25.6±2.3μg / mL, which was significantly higher than the IC50 value of the combination of the three components in Example 1 (8.2±0.7μg / mL), P<0.01, indicating that the three components do have a significant synergistic effect.

[0088] Comparative Example 2 (component ratio optimization comparison, corresponding to Example 2)

[0089] This comparative example uses a non-optimal ratio to verify the ratio optimization effect of the present invention. The toad venom extract, tripterygium wilfordii extract and safflower extract are mixed in a mass ratio of 2:1:1, and the composition, purity and preparation method of each extract are the same as those in Example 2. Prepared using the same nanoformulation process as Example 2. The expression level of the MDR1 gene was detected by flow cytometry, and the results showed that the MDR1 inhibition rate of this comparative example was 45.3±4.2%, while the inhibition rate of Example 2 reached 72.6±5.1%, and the difference was statistically significant (P<0.01). Further verification by Western blot found that the optimal ratio of 1:1:1 can significantly enhance the inhibitory effect of P-glycoprotein.

[0090] Comparative Example 3 (Purity Impact Verification, Corresponding to Example 3)

[0091] In order to verify the effect of the purity of active ingredients on the efficacy, low-purity extracts were used in this comparative example. Specifically, the content ratios of toad toxicity glycosides (purity 85%), cinobufon venom (purity 80%) and monacolin (purity 82%) are the same as those in Example 3, but the purification requirements are reduced. In the preparation method, the Sephadex LH-20 gel column purification step was omitted, and the remaining steps were the same as in Example 3. The drug accumulation experiment of the final nanoinjection in HepG2 / ADR cells showed that compared with Example 3, the accumulation of doxorubicin in the cells was reduced by about 45% (P<0.01), indicating that high-purity active ingredients are crucial for reversing drug resistance.

[0092] Comparative Example 4 (Process Optimization Verification, Corresponding to Example 3)

[0093] This comparative example is intended to verify the importance of optimizing the preparation process parameters. The same raw material ratio as in Example 3 was used, but the key process parameters were changed: 35°C was used for toad venom extraction (instead of 22°C), the relative density was controlled at 1.10 (instead of 1.19) for Tripterygium wilfordii extraction, and the pH value was not controlled for safflower extraction. Among them, the preparation method of toad venom extract is as follows: first, take 100 mesh toad venom powder, add 20 times the volume of dichloromethane, and extract 3 times at 35°C, each time for 4 hours. The subsequent steps are the same as in Example 3. In addition to the above changes, the preparation methods of Tripterygium wilfordii extract and safflower extract are the same as in Example 3. The content of each active ingredient was determined by high performance liquid chromatography, and it was found that high temperature extraction resulted in a decrease in the content of toad venom glycosides by about 25%, and failure to control the pH value significantly reduced the extraction selectivity of safflower yellow pigment. MTT experiment was carried out in A549 / DDP cells, and the results showed that the IC50 value of this comparative example was about 2.8 times higher than that of Example 3 (P<0.001), which fully confirmed the important influence of process parameter optimization on product quality.

[0094] Comparative Example 5 (delivery system innovation verification, corresponding to Example 4)

[0095] This comparative example is designed to verify the superiority of the nanoliposome delivery system. Three extracts were obtained by using the same component ratio and preparation process as Example 4, but the common injection preparation process was used, that is, the three extracts were mixed at a mass ratio of 1.2:1.2:1.2, directly dissolved in water for injection, adjusted to pH 7.0, and controlled to have an osmotic pressure of 300mOsm / L. The tissue distribution of the two preparations in tumor-bearing mice was compared by fluorescent tracing technology, and the results showed that compared with this comparative example, the nanoliposome preparation of Example 4 had a drug concentration 3.6 times higher in tumor tissue (P<0.001), and the maintenance time was extended by 2.5 times. At the same time, it was observed by confocal microscopy that nanoliposomes significantly increased the accumulation of drugs in drug-resistant cells, confirming the important role of advanced delivery systems in improving drug bioavailability.

[0096] Comparative Example 6 (Verification of multidrug resistance mechanism, corresponding to Example 2)

[0097] This comparative example verifies the multi-drug resistance reversal mechanism of the present invention by changing the type of active ingredients. While keeping the total ratio of 1:1:1 unchanged, some active ingredients are replaced: only toad venom extract retains tofu glycosides (50%), and other ingredients are replaced by toad venom saponins; only triptolide A (55%) is retained in tripterygium wilfordii extract, and is replaced by triptolide; only hydroxysafflor yellow A (65%) is retained in safflower extract. The preparation method is basically the same as that of Example 2, but the elution conditions are adjusted during chromatographic separation to adapt to the changes in ingredients. The drug resistance reversal effect was investigated by a multi-index evaluation system: (1) Flow cytometry showed that the P-glycoprotein inhibitory activity of this comparative example was 46% lower than that of Example 2; (2) Real-time fluorescence quantitative PCR results showed that the transcriptional inhibitory effects of this comparative example on MDR1, MRP1 and BCRP were reduced by 52%, 38% and 41%, respectively; (3) In the three drug-resistant cell lines of MCF-7 / ADR, HepG2 / ADR and A549 / DDP, the chemotherapeutic drug sensitization effect of this comparative example was significantly lower than that of Example 2 (P<0.01). These results fully confirmed the multi-target synergistic advantages of the original active ingredient combination of the present invention.

[0098] In order to further study the mechanism of action of the composition of the present invention, the following experiments were conducted:

[0099] Anti-multidrug resistance activity evaluation experiment

[0100] 1. Experimental Methods

[0101] In order to comprehensively evaluate the anti-multidrug resistance activity and mechanism of action of the composition of the present invention, the following experiments were designed:

[0102] 1. Cell culture: Human breast cancer resistant cells MCF-7 / ADR, human liver cancer resistant cells HepG2 / ADR and human lung cancer resistant cells A549 / DDP were selected as experimental subjects. RPMI-1640 medium containing 10% fetal bovine serum was used for culture at 37°C and 5% CO2. Among them, 1μg / mL doxorubicin was added to the culture medium of MCF-7 / ADR and HepG2 / ADR cells to maintain drug resistance, and 2μg / mL cisplatin was added to the culture medium of A549 / DDP cells to maintain drug resistance.

[0103] 2. Cell proliferation inhibition experiment: The MTT method was used to evaluate the inhibitory effect of each preparation on the proliferation of drug-resistant cells. The specific steps are: the cells were inoculated at a density of 5×103 / well in a 96-well plate, and different concentrations of test samples were added after culturing for 24 hours, and the culture was continued for 48 hours. MTT solution (5mg / mL) 20μL / well was added and the culture was continued for 4 hours. The supernatant was discarded, DMSO 150μL / well was added, and the plate was shaken for 10 minutes, and the absorbance value at 490nm was measured.

[0104] 3. Drug accumulation experiment: Flow cytometry was used to detect the accumulation of doxorubicin in cells. Cells were seeded in a 6-well plate at a density of 1×106 cells / well, and the test sample was added and co-cultured with doxorubicin (5 μg / mL) for 2 hours. Cells were collected and the fluorescence intensity was detected by flow cytometry.

[0105] 4. Molecular mechanism study: The expression level of P-glycoprotein was detected by Western blot, and the transcription levels of MDR1, MRP1 and BCRP genes were detected by real-time fluorescence quantitative PCR. At the same time, the membrane localization of P-glycoprotein was observed by immunofluorescence.

[0106] 2. Experimental Results

[0107] Table 1. IC50 values ​​of each preparation against different drug-resistant cell lines (μg / mL, n=6, Mean±SD)

[0108]

[0109]

[0110] Table 2. Inhibition rate of each preparation on drug resistance gene expression (%, n = 6, Mean ± SD)

[0111] Formulation type MDR1 MRP1 BCRP Example 1 65.3±5.8 58.4±5.2 55.7±4.9 Example 2 72.6±6.4* 65.8±5.8* 62.3±5.5* Example 3 78.4±6.9** 70.5±6.2** 68.9±6.1** Example 4 85.2±7.5** 76.3±6.7** 73.5±6.5** Comparative Example 1 32.4±2.9 28.5±2.5 26.8±2.4 Comparative Example 2 45.3±4.2 41.2±3.8 38.6±3.5 Comparative Example 3 40.6±3.8 36.9±3.4 34.7±3.2 Comparative Example 4 43.5±4.0 39.8±3.6 37.2±3.4 Comparative Example 5 52.8±4.7 47.5±4.3 44.9±4.1 Comparative Example 6 38.9±3.6 35.2±3.3 33.1±3.0 Note: Compared with comparative example 1, *P<0.05, **P<0.01

[0112] 3. Unexpected technical effects

[0113] 1. Multiple synergistic effects: The experimental results show that the IC50 value of Example 4 is 83.6% lower than that of Comparative Example 1. This significant synergistic effect is far beyond expectations. In-depth analysis found that the three components not only play their own roles, but also form a complex synergistic network: toad glycosides reduce cell membrane potential by inhibiting Na+ / K+-ATPase and increase cell permeability to drugs; triptolide downregulates MDR1 gene expression by regulating the MAPK signaling pathway; and hydroxysafflor yellow stabilizes cell membrane structure through antioxidant effects and prolongs the drug action time. The discovery of this multiple synergistic effect has important theoretical significance.

[0114] 2. Targeted enhancement of nanoformulations: The nanoliposome preparation used in Example 4 not only improves the bioavailability of the drug, but also achieves selective targeting of drug-resistant cells. Confocal microscopy observations revealed that nanoliposomes can bypass the efflux of P-glycoprotein through endocytosis and directly deliver drugs into cells. This discovery provides a new idea for overcoming multidrug resistance.

[0115] 3. Broad-spectrum anti-drug resistance activity: As can be seen from Tables 1 and 2, the present invention exhibits significant inhibitory effects on different types of drug-resistant cells, and can simultaneously inhibit three major drug resistance genes, MDR1, MRP1 and BCRP. This broad-spectrum anti-drug resistance activity is extremely rare in the prior art, suggesting that the present invention may have other unknown mechanisms of action.

[0116] 4. Safety advantage: In the cytotoxicity experiment, it was unexpectedly found that the toxicity of the present invention to normal cells was significantly lower than that of any component used alone. This selectivity may be related to the mutual restraint of the three components. This discovery provides a new idea for the development of safer anti-resistance drugs.

[0117] The above experimental results and analysis fully confirm the innovation and superiority of the present invention in the field of multidrug resistance, and its mechanism of action and clinical application value deserve further in-depth study.

[0118] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A composition for treating multidrug-resistant tumors, characterized in that: The invention comprises toad venom extract, tripterygium wilfordii extract and safflower extract, wherein the mass ratio of the toad venom extract, tripterygium wilfordii extract and safflower extract is (0.8-1.2):(0.8-1.2):(0.8-1.2).

2. The composition according to claim 1, characterized in that The mass ratio of the toad venom extract, the tripterygium wilfordii extract and the safflower extract is 0.8:0.8:0.

8.

3. The method for preparing the composition according to claim 1 or 2, characterized in that: The following steps are involved: (1) preparing the toad venom extract, comprising: First, toad venom powder was mixed with dichloromethane at a weight-to-volume ratio of 1:20 and extracted three times at 20 ± 2 °C for 4 h each time; Secondly, the extract is concentrated under reduced pressure until there is no solvent smell; Then, the concentrate was dissolved in methanol and subjected to silica gel column chromatography using chloroform:methanol:acetic acid=95:8:5 as the mobile phase; Finally, the collected components were purified by Sephadex LH-20 gel column, eluted with methanol, and the second part was collected and freeze-dried to obtain the toad venom extract.

4. The method according to claim 3, characterized in that The preparation method further comprises: (2) preparing the Tripterygium wilfordii extract, comprising: First, the powder of Tripterygium wilfordii was mixed with 75% ethanol at a weight-to-volume ratio of 1:12 and refluxed for extraction at 78±2°C for 3 times, each time for 30 min; Secondly, the extract is concentrated under reduced pressure to a relative density of 1.15-1.20; Again, the concentrate was chromatographed on a HPD-100 macroporous resin column, eluting with water, 40% ethanol, 60% ethanol and 80% ethanol in sequence; Finally, the 60% ethanol eluate was collected, concentrated and dried under reduced pressure to obtain the Tripterygium wilfordii extract.

5. The method according to claim 3, characterized in that: The preparation method further comprises: (3) preparing the safflower extract, comprising: First, safflower powder was mixed with 75% ethanol at a weight-to-volume ratio of 1:15; Secondly, ultrasonic extraction was performed twice at 45±2℃, power 300W, and frequency 40kHz, each time for 30 minutes; Then, the extract was chromatographed on a D101 macroporous resin column and eluted with a gradient of 50-70% ethanol; Finally, the target eluate was collected, concentrated and dried under reduced pressure to obtain the safflower extract.

6. The method according to any one of claims 3 to 5, characterized in that: The particle sizes of the toad venom powder, tripterygium wilfordii powder and safflower powder are 100 mesh, 80 mesh and 100 mesh respectively.

7. The method according to any one of claims 3 to 5, characterized in that: The stationary phase of the silica gel column chromatography is 200-300 mesh silica gel, and the flow rate is 2.0 mL / min; the flow rate of the HPD-100 macroporous resin column chromatography is 1.5 BV / h; the pH of the D101 macroporous resin column chromatography is 5.0-6.0, and the flow rate is 1.0 BV / h.

8. The preparation of the composition according to claim 1 or 2, characterized in that: It is an injection or a nano preparation; wherein the pH of the injection is 6.8-7.2, and the osmotic pressure is 270-330mOsm / L; the particle size of the nano preparation is 80-200nm, the polydispersity index PDI≤0.25, the Zeta potential is -20 to -30mV, and the encapsulation rate is ≥85%.

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