CN112336744B - New houttuynin sodium and its application in preparing antineoplastic medicine by combining with cisplatin - Google Patents
New houttuynin sodium and its application in preparing antineoplastic medicine by combining with cisplatin Download PDFInfo
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Abstract
The invention provides application of sodium new houttuyfonate and combination of sodium new houttuyfonate and cisplatin in treating malignant tumors such as colon cancer, breast cancer, lymphoma, melanoma, lung cancer and the like. The invention adopts MTT method and CCK-8 method to respectively measure the inhibition effect of sodium new houttuyfonate and the combined medication of sodium new houttuyfonate and cisplatin on various tumor cells (including SW620, A375, MCF-7, A549 and raji), and the test finds that: the sodium new houttuyfonate and the combined medication of the sodium new houttuyfonate and the cisplatin have obvious inhibition effect on various tumor cells, and the combined medication shows additive or synergistic effect, thereby providing valuable theory and data support for the clinical treatment of malignant tumor diseases.
Description
Technical Field
The invention belongs to the technical field of application of sodium new houttuyfonate, and particularly relates to application of sodium new houttuyfonate and combination of the sodium new houttuyfonate and cisplatin in preparation of antitumor drugs, such as application of the sodium new houttuyfonate in inhibition of malignant tumors of colon cancer, breast cancer, lymphoma, melanoma and the like, and application of the sodium new houttuyfonate and cisplatin in combination in inhibition of malignant tumors of colon cancer, lung cancer, breast cancer, melanoma and the like.
Background
Houttuynia cordata Thunb is fresh whole plant or dried aerial part of Houttuynia cordata Thunb of Saururaceae, and is recorded in miscellaneous records of famous physicians, and its descendants of herbal literature are recorded as essential drug for treating lung disorder. ([ 1] Liu Lian, liu Teng, pengbo, qiu Qiaochun. A compound pharmaceutical composition with anti-lymphoma effect and its use [ P ]. Guangdong province: CN107095870B, 2019-05-21.) mainly contain volatile oil, flavone, alkaloid, etc., and have a plurality of pharmacological activities such as anti-inflammatory, antibacterial, antiviral, anti-tumor, etc., and wide clinical application. Wherein, the houttuynin is one of the main components of the volatile oil of the houttuynia cordata, but is unstable and easy to degrade. The sodium bisulfite addition compound sodium new houttuyfonate is commonly used clinically at present. The sodium new houttuyfonate has more stable structure and similar pharmacological action to the volatile oil of herba Houttuyniae and houttuynin.
The New Sodium Houttuyfonate (SNH) has a chemical name of Sodium lauroyl-alpha-hydroxyethanesulfonate, has an obvious antibacterial effect, and has obvious inhibition effects on staphylococcus aureus, diplococcus pneumoniae, typhoid bacillus, escherichia coli, sporothrix bacteria and the like. Is mainly used as an anti-infective medicament and has the effects of resisting inflammation, relieving pain, clearing away heat and toxic materials, eliminating carbuncle, expelling pus and the like. At present, research on sodium new houttuyfonate is mostly focused on the aspects of antibiosis and anti-inflammation, while relatively few reports on the aspect of tumor resistance are reported.
Cisplatin (cissplatin, DDP) is a commonly used antitumor drug, belongs to the first platinum antitumor drugs with periodic non-specificity, and has the characteristics of wide antitumor spectrum and effectiveness on hypoxic cells. Is suitable for treating non-small cell lung cancer, testis cancer, ovarian cancer, cervical cancer, endometrial cancer, melanoma, sarcoma, head and neck tumor and various malignant lymphomas, and can be used as positive control drug. However, cisplatin generally has relatively high renal and pancreatic toxicity and neurotoxicity, and long-term use of cisplatin can increase the burden of organs, so that a new medicament needs to be searched to replace or reduce the toxic and side effects of cisplatin. At present, the method for improving the sensitivity of the drug action or reducing the toxic and side effects of the drug through the combined action between the traditional Chinese medicine and the chemotherapeutic drug clinically becomes a new method for treating the cancer and also becomes a development trend for treating the cancer in the future.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an application of sodium new houttuyfonate and a combination of the sodium new houttuyfonate and cisplatin in the aspect of preparing an anti-tumor medicament, such as the application of the sodium new houttuyfonate in the aspect of inhibiting malignant tumors of colon cancer, breast cancer, lymphoma, melanoma and the like and the application of the sodium new houttuyfonate and the combination of the cisplatin in the aspect of inhibiting the malignant tumors of the colon cancer, the lung cancer, the breast cancer, the melanoma and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
application of sodium new houttuyfonate in preparing antitumor drugs is provided.
The tumor is colon cancer, melanoma, breast cancer or lymphoma, namely the application of the sodium new houttuyfonate in inhibiting malignant tumors such as colon cancer, breast cancer, lymphoma and melanoma.
The invention also provides application of the combined compound of sodium new houttuyfonate and cisplatin in preparing antitumor drugs.
Specifically, in the application, the tumor is colon cancer, melanoma, breast cancer or lung cancer and the like, namely the application of the combined compound of sodium new houttuyfonate and cisplatin in the aspect of inhibiting malignant tumors such as colon cancer, lung cancer, breast cancer, melanoma and the like.
Compared with the prior art, the invention has the following beneficial effects:
the research of the application shows that the Sodium New Houttuyfonate (SNH) and the cisplatin (DDP) both have good anti-tumor effect, and the new opportunity is provided for treating malignant tumor by the combined administration of SNH and cisplatin. The combination of the excellent anti-tumor curative effect of SNH and the chemotherapeutic drug with good anti-tumor activity is used for synergistically treating malignant tumors such as colon cancer and the like, and the method is a remarkable and potential anti-tumor compound drug direction. The invention discusses the inhibiting effect of SNH on malignant tumors such as colon cancer, breast cancer, lymphoma and melanoma, and the inhibiting effect of the combination effect of SNH and cisplatin on malignant tumor cells such as colon cancer, lung cancer, breast cancer and melanoma, and provides valuable theoretical and data support for the clinical treatment of malignant tumor diseases.
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FIG. 1 shows, from left to right, CI values for 24h, 48h and 72h of inhibition of colon cancer cells (SW 620) by the combination of sodium new houttuyfonate and cisplatin, respectively;
FIG. 2 shows CI values corresponding to 24h, 48h and 72h for the inhibition of melanoma cells (A375) by the combination of sodium new houttuyfonate and cisplatin, respectively, from left to right;
FIG. 3 shows, from left to right, CI values corresponding to 24h and 48h for the combination of sodium new houttuyfonate and cisplatin in inhibiting breast cancer cells (MCF-7).
Detailed Description
The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.
The growth inhibition rate of colon cancer cells (SW 620) was measured by MTT method.
(1) Experimental procedure
(1.1) preparation of the drug
Sodium new houttuyfonate solution: 1.0mg of sodium new houttuyfonate standard substance was dissolved in 50. Mu.L of DMSO and diluted to the desired concentration using complete medium (i.e., DMEM medium containing 10% fetal bovine serum).
Cisplatin solution: 1.0mg of cisplatin standard substance was taken, and 100. Mu.L of physiological saline was added to dilute to the desired concentration.
Negative control group: complete medium (DMEM medium containing 10% fetal bovine serum).
(1.2) culturing SW620 colon cancer cells using complete medium. Taking cells in logarithmic phase, adding appropriate amount of pancreatin for digestion, centrifuging at 1000r/min for 5min, and collecting cells. Discarding old culture medium, adding new culture medium for resuspension, and adjusting cell density to 8.0 × 10 after counting 3 cells/mL, 100 μ L/well of cell suspension were seeded in 96-well plates, and the marginal wells were filled with autoclaved PBS solution. Culturing for 24h, and observing the cells under an optical microscopeShape and density, after the cells adhere to the wall, the old culture medium is discarded, and 100 mu L of the drug-containing culture medium is added. The final concentration of the sodium new houttuyfonate is respectively 20 mu g/mL, 40 mu g/mL, 60 mu g/mL, 80 mu g/mL and 100 mu g/mL; the final concentration of cisplatin was 2.5 μ g/mL, 5 μ g/mL, 7.5 μ g/mL, 10 μ g/mL, 15 μ g/mL, the final concentration of the combination of sodium new houttuyfonate and cisplatin was 20 × 2.5, 20 × 5, 30 × 2.5, 30 × 5, 40 × 2.5, 40 × 5, and a negative control group (i.e., a blank group to which an equal amount of the complete medium without drug was added) was provided, 3 wells were provided for each drug concentration, and the total volume of each well was 100 μ L. After 24h, 48h, 72h of drug induction, 20. Mu.L of MTT per well was added and the mixture was subjected to 37 ℃ and 5% CO 2 Culturing for 4 hours in an incubator with saturated humidity; then adding 150 mu L DMSO, shaking the shaking table for 10min, detecting the wavelength of 490nm by an enzyme-labeling instrument, recording the OD value, defaulting that the OD zero setting hole is zero, and calculating the result.
(2) Results of the experiment
The cell inhibition rate fa was calculated according to the formula (1).
fa =1- (OD dosing well-OD zeroing well)/(OD blank well-OD zeroing well) × 100% (1)
Processing data by using SPSS statics 21.0 software to obtain IC 50 The value is obtained.
The middle effect equation is shown in formula (2):
ln(fa/fu)＝mlnD-mlnD m (2)
wherein the survival rate fu =1-fa, D is the administration concentration, D m The concentration is the middle effect concentration, namely the administration concentration when the inhibition rate is 50%.
CI＝D1/D x1 +D2/D x2 (3)
According to the formula (2), the middle-effect concentration of single drug or combination of two drugs of SNH and DDP can be obtained, the concentration values corresponding to the two drugs at different inhibition rates when the SNH and DDP are combined are further calculated, and the Combination Index (CI) is calculated according to the formula (3). CI<1, exhibiting a synergistic effect; CI =1, as additive effect; CI>1, exhibiting antagonistic action. In the formula (3), D X1 And D X2 The administration concentrations of SNH and DDP at a certain inhibition rate are respectively shown, and D1 and D2 are respectively the administration concentrations of SNH and DDP when the two medicines are combined.
The CI values were calculated by the compucon software using the inhibition rates of two single drugs and two drugs in combination, according to the principles described above.
TABLE 1 inhibition ratio of SNH, DDP single drug to SW620
TABLE 2 IC of SNH, DDP vs SW620 50 Value of
As can be seen from tables 1 and 2: with the prolonging of the action time and the increasing of the concentration of the sodium new houttuyfonate, the inhibition rate is correspondingly increased, which shows that the sodium new houttuyfonate has obvious inhibition effect on SW620 cells and presents time and concentration dependence.
TABLE 3 inhibition of SW620 by SNH and DDP combinations
According to the inhibition rate of SNH and CIs-platinum DDP on SW620 cells, the CI value of SNH and CIs-platinum (hereinafter referred to as S X D) is calculated (shown in figure 1). The results in FIG. 1 show that CI is less than 1 in 24h, 48h and 72h of SW620 cells treated by administration after SNH and DDP are combined at different concentrations, which indicates that the combination of SNH and cisplatin has synergistic effect and good anti-tumor effect in 24h, 48h and 72h of administration. Namely, the sodium new houttuyfonate and the composition combined with cisplatin have the function of obviously inhibiting the proliferation of SW620 cells.
Application test 2 shows that the new sodium houttuyfonate and the combined application of the new sodium houttuyfonate and cisplatin have the effect of inhibiting melanoma.
The growth inhibition rate of melanoma cells (A375) was examined by MTT assay.
(1) The test steps are as follows: the difference was that the DMEM medium was replaced with RPMI1640 medium as described in the above experiment with colon cancer cells (1). Wherein the final concentration of sodium new houttuyfonate in combination with cisplatin is 20 × 5, 20 × 10, 30 × 5, 30 × 10, 40 × 5, 40 × 10.
(2) The experimental results are as follows: the calculation was performed in the same manner as described in the above item (2) for colon cancer cells.
TABLE 4 inhibition of A375 by SNH and DDP
TABLE 5 IC of SNH, DDP vs. A375 50 Value of
As can be seen from tables 4 and 5: with the prolonging of the action time and the increasing of the concentration of the sodium new houttuyfonate, the inhibition rate is correspondingly increased, and the increase trend of the inhibition rate after reaching a certain height is gentle, which indicates that the sodium new houttuyfonate has obvious inhibition effect on A375 cells and presents time and concentration dependence.
TABLE 6 inhibition of A375 by SNH and DDP combinations
Based on the inhibition rate of the SNH and DDP on A375 cells by the single drug and the combination of the two drugs, the CI value of the combination of SNH and cisplatin (hereinafter referred to as S X D) was calculated (see FIG. 2). The results in figure 2 show that CI is less than 1 when the A375 cells are subjected to administration treatment for 24h, 48h and 72h after the combination of SNH and DDP at different concentrations, which indicates that the combination of SNH and cisplatin has synergistic effect and good anticancer effect no matter the administration treatment is carried out for 24h, 48h or 72 h. Namely, the sodium new houttuyfonate and the composition combined with cisplatin have the function of obviously inhibiting the proliferation of A375 cells.
Application test 3 shows that the new sodium houttuyfonate and the combined use thereof with cisplatin have the effect of inhibiting breast cancer.
The growth inhibition rate of breast cancer cells (MCF-7) was measured by MTT method.
(1) The test contents are as follows: the same as the above-mentioned experimental contents of (1) in colon cancer cells.
(2) The experimental results are as follows: the calculation was performed in the same manner as described in item (2) above for colon cancer cells.
TABLE 7 inhibition of MCF-7 by SNH and DDP
TABLE 8 IC of SNH, DDP vs. MCF-7 50 Value of
As can be seen from tables 7 and 8: with the prolonging of the action time and the increasing of the concentration of the sodium new houttuyfonate, the inhibition rate is correspondingly increased, the increase trend of the inhibition rate after reaching a certain height is gentle, and the sodium new houttuyfonate has obvious inhibition effect on MCF-7 cells and shows time and concentration dependence.
TABLE 9 inhibition of MCF-7 by SNH and DDP combinations
The CI values for the combination of SNH and cisplatin were calculated based on the inhibition rates of the SNH and cisplatin alone and in combination on MCF-7 cells as described above (see FIG. 3). The results of FIG. 3 show that CI is almost less than 1 in 24h and 48h of MCF-7 cells treated by administration after SNH and cisplatin with different concentrations are combined, which indicates that SNH and cisplatin combined have synergistic or additive effects in 24h and 48h of administration treatment and have good anti-tumor effects. Namely, the sodium new houttuyfonate and the composition combined with the cisplatin have the function of inhibiting the MCF-7 cell proliferation.
Application test 4 shows the effect of sodium new houttuyfonate and its combined medicine in inhibiting lung cancer.
The growth inhibition rate of the lung cancer cells (A549) is detected by adopting an MTT method.
(1) The test contents are as follows: the difference was that the DMEM medium was replaced with RPMI1640 medium as described in the above experiment of (1) on colon cancer cells. Wherein the final concentration of sodium new houttuyfonate is 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 40 μ g/mL and 80 μ g/mL; the final concentration of cisplatin is 1.25 mug/mL, 2.5 mug/mL, 5 mug/mL, 7.5 mug/mL and 10 mug/mL; the final concentration of sodium new houttuyfonate in combination with cisplatin was 1.25 × 0.3125,2.5 × 0.625,5 × 1.25, 10 × 2.5, 20 × 5,1.5 × 0.25,3 × 0.5,6 × 1, 12 × 2, and the duration of action was 48h.
(2) The experimental results are as follows: the calculation was performed in the same manner as described in the above item (2) for colon cancer cells.
TABLE 10 inhibition of A549 by SNH and DDP single drugs
TABLE 11 IC of SNH, DDP vs. A549 50 Value of
As can be seen from tables 10, 11: the inhibition rate is correspondingly increased along with the prolonging of the action time or the increasing of the concentration of the sodium new houttuyfonate, which shows that the sodium new houttuyfonate has obvious inhibition effect on A549 cells and shows time and concentration dependence.
TABLE 12 inhibition of A549 by SNH and DDP combination
And calculating the CI value of the combination of the SNH and the cisplatin according to the inhibition rate of the single drug and the combination of the SNH and the cisplatin on the A549 cells. The results of the administration treatment of A549 cells for 48h after the combination of SNH and cisplatin with different concentrations show that the CI is less than or equal to 1, which shows that the combination of SNH and cisplatin shows synergistic or additive effect after the administration treatment for 48h and has good anti-tumor effect. Namely, the sodium new houttuyfonate and the composition combined with the cisplatin both have the effect of inhibiting the proliferation of A549 cells and have concentration dependence and time dependence.
Application test 5 shows the effect of sodium new houttuyfonate in inhibiting lymphoma.
The growth inhibition rate of lymphoma cells (raji) was examined by the CCK-8 method.
(1) The test steps are as follows:
(1.1) preparing a medicament: the same as described in (1.1) above for colon cancer cells.
(1.2) raji lymphoma cells were cultured using complete medium (1640 medium containing 10% fetal bovine serum). Taking cells in logarithmic phase, centrifuging at 1000r/min for 5min, and collecting cells. Discarding old culture medium, adding new culture medium for resuspension, counting, and adjusting cell density to 1.8 × 10 4 cells/mL, 100 μ L/well of cell suspension were seeded in 96-well plates, and the marginal wells were filled with autoclaved PBS solution. Continuously culturing for 24h, observing cell morphology and density under an optical microscope, removing the old culture medium after the cells adhere to the wall, adding 100 μ L of the drug-containing culture medium to make the final concentrations of sodium new houttuyfonate be 2.5 μ g/mL, 5 μ g/mL, 7.5 μ g/mL, 10 μ g/mL and 15 μ g/mL respectively; the final concentration of cisplatin was 2.5. Mu.g/mL, 5. Mu.g/mL, 7.5. Mu.g/mL, 10. Mu.g/mL, 15. Mu.g/mL. After 24h, 48h and 72h of drug induction, 10. Mu.l of CCK-8 (Cell Counting Kit-8) was added to each well, and the mixture was incubated at 37 ℃ for 5% CO 2 And culturing for 4h in an incubator with saturated humidity, detecting the wavelength of 450nm by using an enzyme-labeling instrument, recording an OD value, setting the OD zero setting hole to be zero by default, and calculating a result.
(2) The experimental results are as follows: the calculation method is the same as that described in the above item (2) of colon cancer cells.
TABLE 13 inhibition ratio of single SNH and DDP drugs on raji
TABLE 14 IC of SNH, DDP vs. raji 50 Value of
As can be seen from tables 13, 14: with the prolonging of the action time and the increasing of the concentration of the sodium new houttuyfonate, the inhibition rate is correspondingly increased at 24, 48 and 72h, and even exceeds the positive control cisplatin at 24 and 48h, which shows that the sodium new houttuyfonate has very obvious inhibition effect on lymphoma cells (raji) and presents time and concentration dependence.
To summarize: the above experimental results show that: the sodium new houttuyfonate has obvious inhibiting effect on malignant tumors such as colon cancer, melanoma, breast cancer, lymphoma and the like, and the combination of the sodium new houttuyfonate and the cisplatin shows additive or synergistic effect, so that the growth of tumor cells (such as colon cancer, melanoma, breast cancer, lung cancer and the like) can be more effectively inhibited, the using amount of the cisplatin is greatly reduced, the toxic and side effects of the cisplatin can be reduced, and the novel antitumor drug has great research value and development potential in the aspect of developing novel antitumor drugs.
Claims (1)
1. The application of the combined compound of sodium new houttuyfonate and cisplatin in the preparation of antitumor drugs is characterized in that the tumor is colon cancer, melanoma or lung cancer.
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| WO2002041828A2 (en) * | 2000-11-17 | 2002-05-30 | Dajun Yang | Houttuyninum compositions and methods for inhibiting the activity of erbb-2 based thereon |
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