Evaluation method of marsdenia tenacissima tablets

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to an evaluation method of marsdenia tenacissima tablets. The method comprises the following steps: screening a quality marker for treating the liver cancer or / and the breast cancer in the marsdenia tenacissima tablets by combining a hemogenic component of the marsdenia tenacissima tablets with targeted network pharmacology; the quality markers comprise neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, marsdenia tenacissima glycoside I and marsdenia tenacissima glycoside H; detecting the influence of the quality marker on the proliferation of HepG2 and MCF-7 cells, the influence of the quality marker on the MDA content of the HepG2 and MCF-7 cells, the influence of the quality marker on the SOD enzyme activity of the HepG2 and MCF-7 cells and the influence of the quality marker on the GSH content of the HepG2 and MCF-7 cells to obtain an evaluation system; and determining the content of the quality marker in the to-be-evaluated marsdenia tenacissima tablet, and substituting the content into the evaluation system for evaluation. According to the invention, the quality marker of the marsdenia tenacissima tablet is combined with the therapeutic effect index, so that the effectiveness of the marsdenia tenacissima tablet in the aspect of treating liver cancer or / and breast cancer is evaluated, and the quality evaluation standard of the marsdenia tenacissima tablet is perfected.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and particularly relates to an evaluation method for Marsdenia tenacissima tablets. Background Art

[0002] Marsdenia tenacissima, also known as Marsdenia formosana, Marsdenia tenacissima Roxb., and Marsdenia sinensis, is the dried stem of the plant of the genus Marsdenia in the family Asclepiadaceae. Modern research shows that Marsdenia tenacissima has various pharmacological effects, such as anti-tumor, anti-inflammatory, antioxidant, etc., providing a theoretical basis for its application in cancer treatment. Many of its preparations also play a great adjuvant therapeutic role in the treatment of malignant tumors such as breast cancer, lung cancer, and liver cancer.

[0003] Currently, the preparation types of Marsdenia tenacissima extracts include injection, oral liquid, capsule, tablet, etc., all of which have been applied clinically. Among them, the current quality standard for Marsdenia tenacissima tablets has a single component control index and cannot comprehensively reflect the overall quality level of the drug and ensure its efficacy. In order to improve the quality standard of Marsdenia tenacissima tablets, based on the long history of using Marsdenia tenacissima as an ethnic medicine, its traditional anti-cancer efficacy, and its rich biological resources, a systematic study on the material basis and quality standard of Marsdenia tenacissima tablets is imminent.

[0004] The patent document with the publication number CN 104391065 A discloses a method for establishing and applying a fingerprint spectrum of Marsdenia tenacissima medicinal materials and preparations. For the to-be-detected Marsdenia tenacissima injection, phenolic acid compounds are detected by ultraviolet detector chromatography, and steroidal saponin compounds are detected by evaporative light scattering detector chromatography, and then fingerprint spectra are established respectively.

[0005] In this prior art, only the fingerprint spectrum of phenolic acid compounds of the to-be-detected Marsdenia tenacissima can be compared with the fingerprint spectrum of standard phenolic acid compounds to evaluate the phenolic acid compounds of the to-be-detected Marsdenia tenacissima alone; or only the fingerprint spectrum of steroidal saponin compounds of the to-be-detected Marsdenia tenacissima can be compared with the fingerprint spectrum of standard steroidal saponin compounds to evaluate the saponin compounds of the to-be-detected Marsdenia tenacissima alone; however, phenolic acid compounds and saponin compounds cannot be incorporated into a system to evaluate Marsdenia tenacissima as a whole. For example, there may be a situation where the phenolic acid compounds meet the standards but the saponin compounds do not meet the standards, so Marsdenia tenacissima still cannot be comprehensively evaluated. Summary of the Invention

[0006] The present invention aims to solve the above problems and provides an evaluation method for Marsdenia tenacissima tablets with comprehensive evaluation.

[0007] The technical solution for the present invention to solve the problem is to provide an evaluation method for Marsdenia tenacissima tablets, including the following steps: S1. Screening of quality markers: By combining the blood-forming components in Marsdenia tenacissima tablets with targeted network pharmacology, screen out the quality markers in Marsdenia tenacissima tablets for the treatment of liver cancer and / or breast cancer; The quality markers include neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, and tenacissoside H; S2. Establishment of an evaluation system: Detect the influence of the quality markers on the therapeutic efficacy to obtain an evaluation system; The influence on the therapeutic efficacy includes: Index A. Influence on the proliferation of HepG2 and MCF-7 cells, Index B. Influence on the MDA content of HepG2 and MCF-7 cells, Index C. Influence on the SOD enzyme activity of HepG2 and MCF-7 cells, Index D. Influence on the GSH content of HepG2 and MCF-7 cells; S3. Evaluation of the Cynanchum paniculatum tablets to be evaluated: Determine the content q of the quality markers in the Cynanchum paniculatum tablets to be evaluated 质量标准物 , and substitute it into the evaluation system for evaluation.

[0008] In step S1, Preferably, the present invention includes the following steps: S11. Analyze the blood components in the administered plasma by UPLC-Q / TOF-MS technology after administration; S12. Use the relevant databases of network pharmacology to collect the action targets corresponding to the blood components, construct a Cynanchum paniculatum tablet target library; construct an intersection target library of liver cancer and breast cancer; take the intersection of the Cynanchum paniculatum tablet target library and the intersection target library of liver cancer and breast cancer to obtain potential action targets; S13. Use the blood components and the potential action targets to construct a visual network diagram of "blood components-targets-diseases" of Cynanchum paniculatum tablets, and obtain the main active components in the blood components according to network analysis, which are the quality markers.

[0009] More preferably, it further includes the following steps: S14. Establish a PPI network for the potential action targets to obtain key action targets.

[0010] More preferably, it further includes the following steps: S15. Perform GO enrichment analysis and KEGG pathway enrichment on the potential action targets to obtain signal pathways with high enrichment significance.

[0011] In step S2, Preferably, the present invention detects the influence of the quality markers on the proliferation of HepG2 and MCF-7 cells through CCK-8 experiments; conducts cell experiments using an oxidative stress factor kit to verify the influence of the quality markers on the MDA content, SOD enzyme activity, and GSH content of HepG2 and MCF-7 cells.

[0012] In step S3, Preferably, the present invention includes the following steps: S31. Detect the contents of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in Marsdenia tenacissima tablets by HPLC-UV technology.

[0013] Among them, further preferably, the pretreatment step of Marsdenia tenacissima tablets is included before detection: Add the powder of Marsdenia tenacissima tablets into methanol solution, treat under ultrasonic conditions of 400 - 600W and 30 - 50kHz for 30 - 60min, let it cool, weigh it, and make up the lost weight with methanol solution; After mixing evenly, filter and centrifuge, and filter the supernatant through a 0.2 - 0.3μm microporous membrane.

[0014] Further preferably, it includes the step of preparing a mixed reference solution of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid, wherein the mass ratio of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid is 3:(3.8 - 4.2):(2.8 - 3.2).

[0015] Further preferably, the chromatographic conditions are as follows: Chromatographic column: Waters Symmetry-C18 (4.6 mm × 150, 5 μm), Flow rate: 0.8 - 1.2 mL / min, Column temperature: 30°C, Detection wavelength: 327 nm, Injection volume: 5 μL, Mobile phase A: Acetonitrile, Mobile phase B: Phosphoric acid or formic acid aqueous solution with pH of 1.0 - 3.0, Requirements for gradient elution are as follows: 0 - 10 min 5 - 8%A; 10 - 30 min 8 - 9%A; 30 - 35 min 9 - 13%A; 35 - 50 min 13 - 16%A; 50 - 65 min 16 - 20%A; 65 - 72min 20 - 25%A; 72 - 80 min 25 - 28%A; 80 - 88 min 28 - 40%A; 88 - 93 min 40 - 70%A; 93 - 95min 70 - 95%A; 95 - 98 min 95 - 100%A; 98 - 100 min 100%A.

[0016] Further preferably, the flow rate is 1mL / min.

[0017] Further preferably, the mobile phase B is 0.1% phosphoric acid aqueous solution.

[0018] S32. Detect the contents of marsdenioside I and marsdenioside H in Marsdenia tenacissima tablets by HPLC-ELSD technology.

[0019] Among them, further preferably, the pretreatment step of Marsdenia tenacissima tablets is included before detection: Add the powder of Marsdenia tenacissima tablets into methanol solution, treat under ultrasonic conditions of 400 - 600W and 30 - 50kHz for 30 - 60min, let it cool, weigh it, and make up the lost weight with methanol solution; After mixing evenly, filter and centrifuge, and filter the supernatant through a 0.2 - 0.3μm microporous membrane.

[0020] Further preferably, it includes the step of preparing a mixed control solution of tenacissoside I and tenacissoside H, wherein the mass ratio of tenacissoside I to tenacissoside H is (3-3.5):1.

[0021] Further preferably, the chromatographic conditions are as follows: chromatographic column: Agilent Extend-C18 (4.6 mm×150 mm, 5 μm), flow rate: 0.8-1.2 mL / min, column temperature: 30 °C, drift tube temperature: 110 °C, gas flow rate: 3.0 L / min, injection volume: 20 μL, mobile phase A: acetonitrile, mobile phase B: water, and the requirements for gradient elution are as follows: 21.5% A for 0-10 min; 21.5-35% A for 10-30 min; 35-50% A for 30-40 min; 50-60% A for 40-45 min; 60-80% A for 45-50 min; 80-21.5% A for 50-55 min; 21.5% A for 55-60 min.

[0022] Further preferably, the flow rate is 1 mL / min.

[0023] S33. Bring it into the evaluation system for evaluation.

[0024] In order to improve the evaluation accuracy and unify the evaluation results. As a preference of the present invention, according to the detection results of step S2, based on the influence effects of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, and tenacissoside H under a certain index, weights N are assigned to the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, and tenacissoside H under this index 指标-质量标志物 ; in step S3, for the content q of the quality marker 质量标准物 and the weight N 指标-质量标志物 the products are summed to obtain the evaluation score of the to-be-evaluated Tenacissus Stem Tablets. Specifically: the evaluation score of the to-be-evaluated Tenacissus Stem Tablets = (q 新绿原酸 ×N A-新绿原酸 + q 绿原酸 ×N A-绿原酸 + q 隐绿原酸 ×N A-隐绿原酸 + q 通关藤苷I ×N A-I + q 通关藤苷H ×N A-H ) + (q 新绿原酸 ×N B-新绿原酸 + q 绿原酸 ×N B-绿原酸 + q 隐绿原酸 ×N B-隐绿原酸 + q 通关藤苷I ×N B-I + q 通关藤苷H ×NB-H ) +(q 新绿原酸 ×N C-新绿原酸 + q 绿原酸 ×N C-绿原酸 + q 隐绿原酸 ×N C-隐绿原酸 + q 通关藤苷I ×N C-I + q 通关藤苷H ×N C-H ) +(q 新绿原酸 ×N D-新绿原酸 + q 绿原酸 ×N D-绿原酸 +q 隐绿原酸 ×N D-隐绿原酸 + q 通关藤苷I ×N D-I + q 通关藤苷H ×N D-H )。The evaluation of Marsdenia tenacissima tablets can be carried out by comparing the magnitudes of the evaluation scores.

[0025] Furthermore, for further optimization, weights M are assigned to the contents of the quality markers under each index according to the importance of the index. Based on this, the evaluation score of the Marsdenia tenacissima tablets to be evaluated = (q 新绿原酸 ×N A-新绿原酸 + q 绿原酸 ×N A-绿原酸 + q 隐绿原酸 ×N A-隐绿原酸 + q 通关藤苷I ×N A-I + q 通关藤苷H ×N A-H ) ×M A + (q 新绿原酸 ×N B-新绿原酸 + q 绿原酸 ×N B-绿原酸 + q 隐绿原酸 ×N B-隐绿原酸 + q 通关藤苷I ×N B-I + q 通关藤苷H ×N B-H ) ×M B +(q 新绿原酸 ×N C-新绿原酸 + q 绿原酸 ×N C-绿原酸 + q 隐绿原酸 ×N C-隐绿原酸 + q 通关藤苷I ×N C-I + q 通关藤苷H ×N C-H ) ×M C +(q 新绿原酸 ×N D-新绿原酸 + q 绿原酸 ×ND-绿原酸 + q 隐绿原酸 ×N D-隐绿原酸 + q 通关藤苷I ×N D-I + q 通关藤苷H ×N D-H ) ×M D 。

[0026] Among them, index A is to inhibit the viability of MCF-7 and HepG2 cells, which is the most direct and crucial therapeutic effect index, directly reflecting the inhibition of the proliferation or survival ability of cancer cells by the drug. In anti-cancer treatment, whether cancer cells can be effectively killed or the growth of cancer cells can be inhibited is the primary goal, so it has the highest importance. Index D is to increase the GSH content in MCF-7 and HepG2 cells. GSH (glutathione) is an important antioxidant and detoxifying molecule in cells. The increase in its content may assist in treatment by regulating the redox balance, enhancing drug metabolism, or affecting cancer cell drug resistance, so its importance is secondary. Index C is to increase the SOD enzyme activity in MCF-7 and HepG cells. Superoxide dismutase (SOD) reduces oxidative damage by scavenging superoxide radicals (O2⁻), but its scope of action is relatively narrow, mainly targeting specific types of free radicals, so its importance is lower than that of index D. Index B is to reduce the MDA content in HepG2 and MCF-7 cells. Malondialdehyde (MDA) is the end product of lipid peroxidation, reflecting the degree of oxidative damage. Its reduction is an indirect result of antioxidant intervention, rather than an active treatment mechanism, so its importance is the lowest.

[0027] Therefore, as a preference of the present invention, the importance: index A > index D > index C > index B.

[0028] In some embodiments, the weight M of index A A is 40%, the weight M of index D D is 30%, the weight M of index C C is 20%, and the weight M of index B B is 10%.

[0029] Among them, there are multiple embodiments for obtaining the weight N 指标-质量标志物 .

[0030] In some embodiments , rank the influencing effects of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, and tenacissoside H under a certain index, and assign the weight N to the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, and tenacissoside H under this index according to the order 指标-质量标志物 .

[0031] Specifically, for example, under index A, the influencing effects are: tenacissoside H > chlorogenic acid = cryptochlorogenic acid = neochlorogenic acid > tenacissoside I. Then, under index A, the weight value of tenacissoside H is x + 2y, the weight values of chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid are all x + y, and the weight value of tenacissoside I is x; where x + 2y + 3(x + y) + x = 100%. Thus, values can be assigned to x and y. For example, x = 15% and y = 5%.

[0032] As a preference of the present invention, in some other embodiments , and the weight N is assigned according to the influencing degrees of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, and tenacissoside H relative to the blank group 指标-质量标志物 .

[0033] Specifically, for example, the influencing degrees of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, and tenacissoside H on index A relative to the blank group are a, b, c, d, and e respectively. The weight values of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, and tenacissoside H under index A are a / (a + b + c + d + e), b / (a + b + c + d + e), c / (a + b + c + d + e), d / (a + b + c + d + e), and e / (a + b + c + d + e) respectively.

[0034] Among them, the influencing effects of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, and tenacissoside H under a certain index are related to the dosage.

[0035] In some embodiments , during the detection in step S2, the dosages of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, and tenacissoside H are the same.

[0036] As a preference of the present invention, in some other embodiments , to exclude the influence of content, during the detection in step S2, the dosage ratio of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, and tenacissoside H is the same as the content ratio of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tenacissoside I, and tenacissoside H in the tenacissoside tablets.

[0037] Advantages of the present invention: In the present invention, the quality markers of tenacissoside tablets are combined with the treatment efficacy indexes to evaluate the efficacy of tenacissoside tablets in the treatment of liver cancer and / or breast cancer, and the quality evaluation standard of tenacissoside tablets is improved. Specific embodiments

[0038] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described, but the present invention is not limited to these embodiments.

[0039] Example 1 A method for evaluating tenacissoside tablets includes the following steps: S1. Screening of quality markers : By combining the original components entering the blood of Marsdenia tenacissima tablets with targeted network pharmacology, quality markers for treating liver cancer and / or breast cancer in Marsdenia tenacissima tablets are screened out, and the specific steps are as follows: S11. After administration, use UPLC-Q / TOF-MS technology to analyze the components entering the blood in the plasma after administration: Experimental animals: Male SD rats, weighing 200±20 g.

[0040] Administration plan: Weigh an appropriate amount of Marsdenia tenacissima tablets, grind them finely, add an appropriate amount of ultrapure water, mix evenly with a vortex mixer, and then administer the suspension to the experimental rats by gavage (2.08 g / kg).

[0041] Sample collection: Continuously administer the suspension (2.08 g / kg) to the rats in the administration group by gavage for 7 days, and the blank group is given an equal amount of ultrapure water. All experimental rats are fasted for 12 h before gavage on the seventh day. After the fasting ends, the rats are decapitated to collect blood at 0.25, 0.5, 1, 2, and 4 h after gavage, placed in heparin tubes, and the collected samples are centrifuged at 3500 r / min at 4°C for 10 min. The supernatant is aspirated to obtain plasma samples, which are stored in a -80°C refrigerator for later use.

[0042] Sample pretreatment and detection: When analyzing the samples, take out the plasma samples and thaw them at 4°C. Add 4 mL of cold methanol to 1 mL of plasma, vortex for 5 min, shake for 20 min, centrifuge at 8000 r / min at 4°C for 10 min, take the supernatant and dry it with nitrogen, redissolve it with 100 µL of methanol, and centrifuge again at 13000 r / min at 4°C for 10 min. Take the supernatant for UPLC-Q-TOF-MS analysis.

[0043] Among them, the HPLC chromatographic conditions are as follows: chromatographic column: Agilent Poroshell-C18 (4.6 mm×150 mm, 2.7 μm), column temperature: 30°C, flow rate: 0.5 mL / min, injection volume: 5 μL; mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid water, and the gradient elution program is: 0 - 4 min, 5 - 20% A; 4 - 15 min, 20 - 25% A; 15 - 25 min, 25 - 35% A; 25 - 45 min, 35 - 50% A; 45 - 50 min, 50 - 75% A; 50 - 55 min, 75 - 95% A; 55 - 57 min, 95 - 100% A; 57 - 62 min, 100% A; 62 - 67 min, 100 - 5% A; 67 - 72 min, 5% A.

[0044] Mass spectrometry conditions: ESI electrospray ionization source was used, in MSE full scan mode, data were collected in positive and negative ion modes, the scan time was 0.3 s, and the scan range was 50 - 1200 Da. Argon was used as the collision gas, the low collision energy was 6 V, and the high collision energies were 20 - 30 V, 30 - 50 V, and 50 - 60 V. Sodium formate and leucine enkephalin were used to calibrate the mass spectrometer and for real-time calibration. The real-time reference ions generated by leucine enkephalin were [M+H]+ (m / z 556.2771) in the positive ion mode and [M - H]- (m / z 554.2615) in the negative ion mode.

[0045] The total ion chromatogram of rat plasma samples treated with methanol was obtained through analysis, and a total of 22 blood components were identified.

[0046] S12. Using relevant databases of network pharmacology, collect the action targets corresponding to the blood components, and construct the target library of Tongguanteng tablets; construct the intersection target library of liver cancer and breast cancer; take the intersection of the Tongguanteng tablet target library and the liver cancer and breast cancer intersection target library to obtain potential action targets: For the 22 identified blood prototype components, search for the corresponding target proteins of each component through the SwissTargetPrediction and Uniprot databases. After removing duplicate values, a total of 316 compound-related targets were obtained, which is the target library of Tongguanteng tablets.

[0047] In the GeneCards database, input "liver cancer" or "breast cancer" as keywords, and retrieve the disease-related targets of liver cancer and breast cancer. Among them, the algorithm of calculating the median was used to remove genes with relatively low relevance values, obtaining 2467 disease targets for liver cancer and 7783 for breast cancer. These target results were combined, and duplicate gene information was removed. An intersection target library of liver cancer and breast cancer containing 2842 targets was obtained.

[0048] Through the online tool interface of Venny, import the 316 Tongguanteng tablet blood component-related targets and the 2842 disease-related targets into venny 2.1.0, take the intersection of the two, and generate a Venn diagram. 146 potential action targets of Tongguanteng tablets for treating liver cancer and breast cancer were obtained.

[0049] S13. Using the blood components and potential action targets, construct a visualization network diagram of "blood component - target - disease" for Tongguanteng tablets, and based on network analysis, obtain the main active components in the blood components. The main active components are the composition: The blood - entering components and potential action targets in Marsdenia tenacissima tablets were respectively imported into the Cytoscape software to construct the "blood - entering component - target - disease" network of Marsdenia tenacissima tablets. According to the principles of network analysis, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, marsdenioside I and marsdenioside H among the blood - entering components are connected to more targets and are the quality markers for treating liver cancer or / and breast cancer in Marsdenia tenacissima tablets.

[0050] S14. Establish a PPI network for potential action targets to obtain key action targets: The 146 potential action targets were imported into the String database for PPI network construction. After hiding the free proteins, the network analysis graph was obtained and visualized through the Cytoscape software to obtain the key action targets of Marsdenia tenacissima tablets in treating liver cancer and breast cancer: HSP90AA1, PTPN11, PIK3CB, PIK3CD, KDR, MAPK3, ESR1, ERB2, JAK2, RXRA, MET, IL6, CDK1, STAT3, EGFR, PIK3CA, MAPK1.

[0051] Meanwhile, based on the main active components obtained in step S3, it was further confirmed that the key action targets connected to them include STAT3, PIK3CA, KDR, HSR90AA1, MET, PIK3CD and PIK3CB.

[0052] S15. Conduct GO enrichment analysis and KEGG pathway enrichment on potential action targets to obtain signal pathways with high enrichment significance: The potential action targets were imported into the Metascape database website to obtain the results of GO enrichment analysis and KEGG pathway enrichment analysis, and a triple - in - one bar graph of BP, CC and MF and a KEGG enrichment analysis bubble graph were drawn to analyze the pathways with high enrichment significance respectively. It was found that the five components of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, marsdenioside I and marsdenioside H in Marsdenia tenacissima tablets can play a therapeutic role through key pathways such as the PI3K - Akt signaling pathway, GnRH signaling pathway, IL - 17 signaling pathway, and tumor signaling pathway.

[0053] S2. Establishment of evaluation system : Detect the influence of quality markers on the therapeutic effect to obtain an evaluation system, which specifically includes the following steps: Among them, since neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid are isomers and their effects on cells are basically the same, the detection results of chlorogenic acid can represent those of neochlorogenic acid and cryptochlorogenic acid.

[0054] S21. Detect the influence of quality markers on the proliferation of HepG2 and MCF - 7 cells through CCK - 8 experiments: HepG2 cells and MCF-7 cells were placed in an incubator at 37 °C and 5% CO2 for incubation. HepG2 and MCF-7 cells in the logarithmic growth phase were respectively seeded into 96-well plates at a density of 1×10 4 cells / mL, and a total of 10 plates were seeded. After culturing for 24 hours, the supernatant was discarded, and drugs were administered to HepG2 and MCF-7 cells respectively, and then cultured in the incubator for 24 h.

[0055] The drug solution concentrations that need to be prepared separately before drug administration are as follows: the tylophorine I group (600 μg / mL, 100 μg / mL), the tylophorine H group (600 μg / mL, 100 μg / mL), and the chlorogenic acid group (400 μg / mL, 100 μg / mL).

[0056] After culturing for 24 hours, a mixed solution containing CCK-8 reagent and complete culture medium in a ratio of 1:10 was added to each well, and incubated in the dark. After incubation, the optical density (OD) value of the well was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0057] S22. The effects of quality markers on the MDA content, SOD enzyme activity, and GSH content of HepG2 and MCF-7 cells were detected by a cell experiment oxidative stress factor kit, and the specific steps are as follows: The cells were randomly divided into a blank control group and drug administration groups. The drug administration groups were the tylophorine I group (600 μg / mL, 100 μg / mL), the tylophorine H group (600 μg / mL, 100 μg / mL), and the chlorogenic acid group (400 μg / mL, 100 μg / mL).

[0058] After culturing for 24 h, the differences in MDA content, SOD enzyme activity, and GSH content among different groups were compared.

[0059] The detection results are shown in Table 1 below. Table 1. According to the detection results, weights N were assigned to the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tylophorine I, and tylophorine H under the index 指标-质量标志物 .

[0060] In this example, under the condition of the same drug dosage (100 μg / mL), the method of assigning weights to the total influence degree on HepG2 and MCF-7 was adopted.

[0061] Under Index A, compared with the blank example, the reduction in cell viability by 100 μg / mL of chlorogenic acid is about 31.59%; neochlorogenic acid and cryptochlorogenic acid are regarded as the same as chlorogenic acid. Compared with the blank example, the reduction in cell viability by 100 μg / mL of tenacissoside H is about 20.50%. Compared with the blank example, the reduction in cell viability by 100 μg / mL of tenacissoside H is about 35.11%. Therefore, the weight N of chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid A-绿原酸 / 新绿原酸 / 隐绿原酸 is 21%, and the weight N of tenacissoside I A-I is 14%, and the weight N of tenacissoside H A-H is 23%.

[0062] Under Index B, compared with the blank example, the reduction in the MDA content of cells by 100 μg / mL of chlorogenic acid is about 18.44%; neochlorogenic acid and cryptochlorogenic acid are regarded as the same as chlorogenic acid. Compared with the blank example, the reduction in the MDA content of cells by 100 μg / mL of tenacissoside I is about 17.55%. Compared with the blank example, the reduction in the MDA content of cells by 100 μg / mL of tenacissoside H is about 22.56%. Therefore, the weight N of chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid B-绿原酸 / 新绿原酸 / 隐绿原酸 is 19%, and the weight N of tenacissoside I B-I is 18%, and the weight N of tenacissoside H B-H is 24%.

[0063] Under Index C, compared with the blank example, the increase in the SOD enzyme activity of cells by 100 μg / mL of chlorogenic acid is about 34.79%; neochlorogenic acid and cryptochlorogenic acid are regarded as the same as chlorogenic acid. Compared with the blank example, the increase in the SOD enzyme activity of cells by 100 μg / mL of tenacissoside I is about 12.06%. Compared with the blank example, the increase in the SOD enzyme activity of cells by 100 μg / mL of tenacissoside H is about 17.14%. Therefore, the weight N of chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid C-绿原酸 / 新绿原酸 / 隐绿原酸 is 26%, and the weight N of tenacissoside I C-I is 9%, and the weight N of tenacissoside H C-H is 13%.

[0064] Under Index D, compared with the blank example, the increase in the GSH content of cells by 100 μg / mL of chlorogenic acid is about 59.25%; neochlorogenic acid and cryptochlorogenic acid are regarded as the same as chlorogenic acid. Compared with the blank example, the increase in the GSH content of cells by 100 μg / mL of tenacissoside I is about 43.40%. Compared with the blank example, the increase in the GSH content of cells by 100 μg / mL of tenacissoside H is about 11.70%. Therefore, the weight N of chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid D-绿原酸 / 新绿原酸 / 隐绿原酸 is 25%, and the weight N of tenacissoside I D-Iis 19%, and the weight N of tenacissoside H D-H is 5%.

[0065] In addition, the weight M of index A A is 40%, the weight M of index D D is 30%, the weight M of index C C is 20%, and the weight M of index B B is 10%.

[0066] S3. Evaluation of the Cynanchum paniculatum tablets to be evaluated : Determine the content q of the quality markers in the tenacissoside tablets to be evaluated 质量标准物 , which specifically includes the following steps: S31. Detect the contents of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in the tenacissoside tablets by HPLC-UV technology.

[0067] Prepare the test solution of tenacissoside tablets with 50% methanol: Take 10 tenacissoside tablets, grind them finely, mix well, weigh about 1.0 g, accurately weigh and place it in a 50 mL conical flask, add 20 mL of 50% methanol, and weigh. Ultrasonic treatment (power 500 W, frequency 40 kHz) for 45 min, let it cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, centrifuge for 6 min, take the supernatant solution, and filter it through a 0.22 μm microporous membrane to obtain the solution.

[0068] Prepare the mixed reference solution of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid with 50% methanol: Accurately weigh the reference substances and configure a mixed reference solution containing 0.1452 mg, 0.1976 mg, and 0.1484 mg of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid standards per 1 mL.

[0069] The chromatographic conditions are as follows: Chromatographic column: Waters Symmetry-C18 (4.6 mm × 150, 5 μm), flow rate: 1.0 mL / min, column temperature: 30 °C, detection wavelength: 327 nm, injection volume: 5 μL, mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid water, and the requirements for gradient elution are as follows: 0 - 10 min 5 - 8% A; 10 - 30 min 8 - 9% A; 30 - 35 min 9 - 13% A; 35 - 50 min 13 - 16% A; 50 - 65 min 16 - 20% A; 65 - 72 min 20 - 25% A; 72 - 80 min 25 - 28% A; 80 - 88 min 28 - 40% A; 88 - 93 min 40 - 70% A; 93 - 95 min 70 - 95% A; 95 - 98 min 95 - 100% A; 98 - 100 min 100% A.

[0070] Determination by HPLC: Separately aspirate the reference solution and the test solution, inject them into the liquid chromatograph, and determine according to the above chromatographic conditions. Measure the peak area value and calculate the content. That's all.

[0071] S32. Detect the contents of marsdenoside I and marsdenoside H in Marsdenia tenacissima Roxb. leaves tablets by HPLC-ELSD technology.

[0072] Prepare the test solution of Marsdenia tenacissima Roxb. leaves tablets with 50% methanol: Take 10 tablets of Marsdenia tenacissima Roxb. leaves tablets (batch number: 20221102), grind them finely, mix well, weigh about 1.0 g, accurately weigh and place it in a 50 mL conical flask, add 20 mL of 50% methanol, and weigh. Sonicate (power 500 W, frequency 40 kHz) for 45 min, let it cool, weigh again, make up the lost weight with 50% methanol, shake well, filter, centrifuge for 6 min, take the supernatant solution, and filter it through a 0.22 μm microporous membrane to obtain the solution.

[0073] Preparation of the mixed reference stock solution: Accurately weigh the reference substances and prepare a mixed reference solution containing 0.4768 mg of marsdenoside I and 0.1568 mg of marsdenoside H per 1 mL.

[0074] Chromatographic conditions: Chromatographic column: Agilent Extend-C18 (4.6 mm × 150 mm, 5 μm), flow rate: 1.0 mL / min, column temperature: 30 °C, drift tube temperature: 110 °C, gas flow rate: 3.0 L / min, injection volume: 20 μL, mobile phase A: acetonitrile, mobile phase B: water, and the requirements for gradient elution are as follows: (2) 0 - 10 min 21.5% A; 10 - 30 min 21.5 - 35% A; 30 - 40 min 35 - 50% A; 40 - 45 min 50 - 60% A; 45 - 50 min 60 - 80% A; 50 - 55 min 80 - 21.5% A; 55 - 60 min 21.5% A.

[0075] Separately aspirate the reference solution and the test solution, inject them into the liquid chromatograph, and determine according to the above chromatographic conditions. Measure the peak area value and calculate the content. That's all.

[0076] S33. The detection results of fifteen batches of Marsdenia tenacissima Roxb. leaves tablets are shown in Table 2 below.

[0077] Table 2. Based on the indicators obtained in step S2, the evaluation score of the Marsdenia tenacissima Roxb. leaves tablets to be evaluated = (q 新绿原酸 × 21% + q 绿原酸 × 21% + q 隐绿原酸 × 21% + q 通关藤苷I×14% + q 通关藤苷H ×23%) × 40% + (q 新绿原酸 ×19% + q 绿原酸 ×19% + q 隐绿原酸 ×19% + q 通关藤苷I ×18% + q 通关藤苷H ×24%) × 10% + (q 新绿原酸 ×26% + q 绿原酸 ×26% + q 隐绿原酸 ×26% + q 通关藤苷I ×9% + q 通关藤苷H ×13%) × 20% + (q 新绿原酸 ×25% + q 绿原酸 ×25% + q 隐绿原酸 ×25% + q 通关藤苷I ×19% + q 通关藤苷H ×5%) × 30% = q 新绿原酸 ×23.18% + q 绿原酸 ×23.18% + q 隐绿原酸 ×23.18% + q 通关藤苷I ×14.69% + q 通关藤苷H ×15.77%.

[0078] Therefore, the evaluation scores of fifteen batches of Marsdenia tenacissima tablets are shown in Table 3 below.

[0079] Table 3. According to the evaluation scores, the fifteen batches of Marsdenia tenacissima tablets can be ranked in terms of quality.

[0080] Example 2 This example is basically the same as Example 1, and the only difference is that in this example, under different dosing conditions, the influence degrees on the total of HepG2 and MCF-7 are weighted.

[0081] According to Table 2, it can be known from the average value that the content ratios of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, marsdenoside I, and marsdenoside H in Marsdenia tenacissima tablets are approximately 3:4:3:6:3. Therefore, in step S2, the influence degrees of 300 μg / mL neochlorogenic acid, 400 μg / mL chlorogenic acid, 300 μg / mL cryptochlorogenic acid, 600 μg / mL marsdenoside I, and 300 μg / mL marsdenoside H on indicators A, B, C, and D compared with the blank example are obtained. Among them, the influence degrees of 300 μg / mL neochlorogenic acid and 300 μg / mL cryptochlorogenic acid can be represented by the influence degree of 300 μg / mL chlorogenic acid.

[0082] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Those skilled in the art to which the present invention pertains may make various modifications or supplements to the described specific embodiments or use similar means for substitution, but will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A method for evaluating tongguan rattan slices, characterized in that: The following steps are involved: S1. Screening of quality markers: Through the blood-entering original components of Tongguanteng tablets combined with targeted network pharmacology, the quality markers in Tongguanteng tablets for the treatment of liver cancer and / or breast cancer were screened; The quality markers include neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tongguanteng glycoside I and tongguanteng glycoside H; S2. Establishment of an evaluation system: detecting the effect of the quality markers on the therapeutic efficacy and obtaining an evaluation system; The effects on the therapeutic efficacy include: Indicator A. Effect on the proliferation of HepG2 and MCF-7 cells, Indicator B. Effect on MDA content of HepG2 and MCF-7 cells, Indicator C. Effect on SOD enzyme activity of HepG2 and MCF-7 cells, Indicator D. Effect on GSH content of HepG2 and MCF-7 cells; S3. Evaluation of the Tongguanteng tablets to be evaluated: Determination of the content of quality markers in the Tongguanteng tablets to be evaluated 质量标准物 , and brought into the evaluation system for evaluation.

2. The method for evaluating the rattan slices according to claim 1, characterized in that: Step S3 includes the following steps: S31. The contents of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in Tongguanteng tablets were detected by HPLC-UV technology; S32. The contents of Tongguanteng glycoside I and Tongguanteng glycoside H in Tongguanteng tablets were detected by HPLC-ELSD technology.

3. The method for evaluating the rattan slices according to claim 2, characterized in that: In step S31, the detection wavelength is 327 nm, the chromatographic column is an acid-resistant column with octadecylsilane bonded silica gel as a filler, the column temperature is 30°C, and the mobile phase is gradient elution.

4. The method for evaluating the rattan slices according to claim 2, characterized in that: In step S32, the chromatographic column is an acid-resistant column with octadecylsilane bonded silica gel as a filler, the column temperature is 30°C, the pipetting temperature is 110°C, and the mobile phase is gradient elution.

5. The method for evaluating the rattan slices according to claim 2, characterized in that: Step S31 and step S32 both include a pretreatment step for the Tongguanteng slices: adding the Tongguanteng slice powder to a methanol solution, treating it under ultrasonic conditions of 400~600W and 30~50kH for 30~60min, weighing it after cooling to room temperature, and making up the lost weight with methanol solution; mixing evenly and filtering and centrifuging, removing the supernatant and filtering it through a 0.2~0.3μm microporous filter membrane.

6. The method for evaluating the vine slices according to claim 1, characterized in that: According to the detection results of step S2, based on the effects of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tongguanteng glycoside I and tongguanteng glycoside H on a certain indicator, a weight N is assigned to the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tongguanteng glycoside I and tongguanteng glycoside H under the indicator. 指标-质量标志物 ; In step S3, the content q of the quality marker is 质量标准物 With weight N 指标-质量标志物 The products are summed to obtain the evaluation score of the cleared rattan piece to be evaluated.

7. The method for evaluating the rattan slices according to claim 6, characterized in that: According to the importance of indicator A> indicator D> indicator C> indicator B, the content of the quality marker under each indicator is assigned a weight M. 指标 ; In step S3, the evaluation score of the cleared rattan slice to be evaluated = (q 新绿原酸 ×N A-新绿原酸 + q 绿原酸 ×N A-绿原酸 + q 隐绿原酸 ×N A-隐绿原酸 + q 通关藤苷I×NA-I + q 通关藤苷H×NA-H ) × M A + (q 新绿原酸 ×N B-新绿原酸 + q 绿原酸 ×N B-绿原酸 + q 隐绿原酸 ×N B-隐绿原酸 + q 通关藤苷I×NB-I + q 通关藤苷H×NB-H ) × M B +(q 新绿原酸 ×N C-新绿原酸 + q 绿原酸 ×N C-绿原酸 + q 隐绿原酸 ×N C-隐绿原酸 + q 通关藤苷I×NC-I + q 通关藤苷H×NC-H ) × M C +(q 新绿原酸 ×N D-新绿原酸 + q 绿原酸 ×N D-绿原酸 + q 隐绿原酸 ×N D-隐绿原酸 + q 通关藤苷I×ND-I + q 通关藤苷H×ND-H ) × M D .

8. The method for evaluating the vine slices according to claim 6 or 7, characterized in that: The weight N is assigned according to the influence of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tongguanteng glycoside I and tongguanteng glycoside H relative to the blank group. 指标-质量标志物 .

9. The method for evaluating the rattan slices according to claim 8, characterized in that: During the detection in step S2, the dosages of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tongguanteng glycoside I and tongguanteng glycoside H are the same.

10. The method for evaluating the rattan slices according to claim 8, characterized in that: During the detection in step S2, the dosage ratio of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tongguanteng glycoside I and tongguanteng glycoside H is the same as the content ratio of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, tongguanteng glycoside I and tongguanteng glycoside H in the tongguanteng tablets.

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