CN111999402A - Method for screening basis of antitumor effective substances of Kangai injection - Google Patents
Method for screening basis of antitumor effective substances of Kangai injection Download PDFInfo
- Publication number
- CN111999402A CN111999402A CN202010768640.2A CN202010768640A CN111999402A CN 111999402 A CN111999402 A CN 111999402A CN 202010768640 A CN202010768640 A CN 202010768640A CN 111999402 A CN111999402 A CN 111999402A
- Authority
- CN
- China
- Prior art keywords
- injection
- kang
- components
- ginsenoside
- screening
- Prior art date
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- Granted
Links
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- 239000007924 injection Substances 0.000 title claims abstract description 154
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- 230000004083 survival effect Effects 0.000 claims description 47
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 24
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Abstract
The invention relates to a method for screening an anti-tumor effective substance basis of a kang' ai injection in the technical field of medicines, which comprises the following steps: separating the KANGAI injection by high performance liquid chromatography, and measuring proliferation inhibition rate of the component of KANGAI injection on tumor cells; the conditions of the high performance liquid chromatography are as follows: mobile phase: phase A is 0.05 wt% formic acid-water, phase B is methanol; a linear elution gradient; flow rate: 11-14 mL/min; column temperature: 25-35 ℃; DAD detector wavelength: a full wavelength; sample introduction amount: 1-3 mL; starting with the first fraction collected at 3min, fractions were collected as one fraction every 3 min. The high performance liquid chromatography in the method can directly separate the kang ' ai injection, the separated components can be directly used for screening the basis of antitumor effective substances of the kang ' ai injection, and the proportion of effective components in the separated components is close to that of the kang ' ai injection.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a method for screening an anti-tumor effective substance basis of a kang' ai injection.
Background
The Kangai injection mainly comprises astragalus root, ginseng extract and kushenin, is a traditional Chinese medicine injection prepared by extracting traditional Chinese medicines of ginseng and astragalus root as main raw materials and mixing the extracted traditional Chinese medicines with kushenin, has the effects of tonifying qi and strengthening the body resistance and enhancing the organism immunity function, improves the life quality and survival rate of tumor patients, and is clinically used for treating primary liver cancer, lung cancer, rectal cancer, malignant lymphoma, gynecological malignant tumor, leucopenia and hypofunction caused by various reasons and chronic hepatitis B. The Kangai injection can be used as an adjuvant drug in combination with radiotherapy and chemotherapy, has direct treatment effect on various tumors, is one of the most widely used antitumor traditional Chinese medicine injections in clinic, shows good curative effect in clinically adjuvant treatment of tumors such as lung cancer, breast cancer and the like, and can improve the symptoms of patients, control the development of tumors, improve the quality of life and the like. And the research on the drug effect substance basis of the kang 'ai injection is helpful for the quality control and detection of the kang' ai injection.
The basic research method of drug-effect substances of Kangai injection is disclosed in Kangai injection (Ripeng, 2019.5.31), and the second chapter indicates that the separation of chemical components from injection is costly and difficult due to the low content of various components in Kangai injection. Therefore, starting from the prescription medicinal materials of the kang 'ai injection, the chapter refers to the preparation process of the kang' ai injection to respectively prepare the extracts of ginseng and astragalus, then separates the chemical components in the extracts by means of column chromatography, preparative liquid chromatography and the like, carries out structural identification through nuclear magnetism and standard substance contrast, then qualitatively analyzing with high performance liquid chromatography whether chemical components separated from the above extracts of human beings participating in radix astragali exist in the KANGAI injection, and finally preparing into various compositions with kushenin, ginsenoside compounds derived from Ginseng radix, flavonoids and saponin compounds derived from radix astragali, and the results of in vitro antitumor activity studies by adopting an MTT method show that the composition of rare ginsenoside derived from ginseng and the composition of flavone compound derived from astragalus are main antitumor effective substances of the kang' ai injection. In the above documents, on one hand, it is necessary to perform indirect detection of a compound possibly existing in a kang ' ai injection from a raw material of the kang ' ai injection, and then perform qualitative and quantitative detection of the kang ' ai injection by using a high performance liquid chromatography according to the detected compound, which requires many steps, and on the other hand, in the research on an antitumor drug, a composition artificially prepared by using monomer components has a large difference from the kang ' ai injection, and an antitumor drug base of the kang ' ai injection cannot be accurately screened and obtained.
Therefore, the invention provides a novel method for screening the basis of the antitumor effective substances of the kang' ai injection.
Disclosure of Invention
The invention aims to provide a novel method for screening an anti-tumor effective substance basis of a kang' ai injection.
A method for screening the basis of antitumor effective substances of Kangai injection comprises the following steps:
separating the kang 'ai injection by adopting high performance liquid chromatography to obtain components of the kang' ai injection; then, determining the proliferation inhibition rate of the components of the kang' ai injection on tumor cells by adopting an MTT method;
the conditions of the high performance liquid chromatography are as follows:
mobile phase: phase A is 0.05 wt% formic acid-water, phase B is methanol;
linear elution gradient: 0-35min, A: b is 85-95 wt%: 15-5 wt% → 54-64 wt%: 46-36 wt%; 35-55min, A: b is 54-64 wt%: 46-36 wt% → 0-5 wt%: 100-95 wt%; 55-65min, A: b is 0-5 wt%: 100-95 wt% → 0-5 wt%: 100-95 wt%;
flow rate: 11-14 mL/min;
column temperature: 25-35 ℃;
DAD detector wavelength: a full wavelength;
sample introduction amount: 1-3 mL;
starting with the first fraction collected at 3min, fractions were collected as one fraction every 3 min.
Preferably, the linear elution gradient is: 0-35min, A: b is 90 wt%: 10 wt% → 59 wt%: 41 wt%; 35-55min, A: b is 59 wt%: 41 wt% → 0 wt%: 100 wt%; 55-65min, A: b is 0 wt%: 100 wt% → 0 wt%: 100 wt%.
Preferably, the flow rate: 13 mL/min;
preferably, the column temperature: 30 ℃;
preferably, the DAD detector wavelength: a full wavelength;
preferably, the sample volume: 2 mL;
preferably, the kang' ai injection is concentrated according to the proportion of 50 (4-5), the concentrated solution is centrifuged, and the obtained supernatant is separated by high performance liquid chromatography. Preferably, the kang' ai injection is concentrated at a ratio of 50: 4.5.
Preferably, the method for screening the basis of the antitumor effective substances of the kang 'ai injection further comprises the step of determining the proliferation inhibition rate of the monomer components of the kang' ai injection on tumor cells by adopting an MTT method.
Further, the monomer component of the kang' ai injection comprises one or more of a kurarinone monomer component, a ginseng monomer component and an astragalus monomer component.
Further, the oxymatrine monomer component is selected from oxymatrine and oxysophocarpine;
the ginseng monomer component is selected from ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Re, ginsenoside Rg1, notoginsenoside R1, ginsenoside Rd, ginsenoside Rf and ginsenoside Ro;
the radix astragali monomer component is selected from calycosin, astragaloside IV, 9, 10-dimethoxy rosenane-3-O-b-D-glucopyranoside, formononetin, astragaloside 1, astragaloside 2, cycloastragenol and formononetin.
The MTT method is adopted to determine the components of the kang 'ai injection and the proliferation inhibition rate of the monomer components of the kang' ai injection on tumor cells, and comprises the following steps:
digesting tumor cells, inoculating the digested tumor cells in a culture medium, culturing for 24-48h, removing the original culture medium, diluting the components of the KANGAI injection, KANGAI injection or monomer components of the KANGAI injection with the culture medium to obtain a medicinal liquid at 37 deg.C and 5% CO2Incubating the cells for 24-48h, then removing the liquid medicine, adding MTT solution, incubating in the dark, removing the supernatant, adding DMSO, oscillating and mixing uniformly, measuring absorbance under the condition of 580nm wavelength by adopting an enzyme-labeling instrument, and calculating the cell survival rate of the components of the kang 'ai injection, the kang' ai injection or the monomer components of the kang 'ai injection with different concentrations after incubation, thereby calculating the proliferation inhibition rate of the kang' ai injection, the components of the kang 'ai injection or the monomer components of the kang' ai injection on the tumor cells;
the calculation formula of the cell survival rate is as follows: cell survival rate (%). absorbance of administration group/absorbance of negative control group × 100%;
the calculation formula of the proliferation inhibition rate is as follows: proliferation inhibition (%) is 1-cell survival (%).
Preferably, when the proliferation inhibition rate is more than 15%, the components of the kang ' ai injection or the monomer components of the kang ' ai injection are anti-tumor effective substances of the kang ' ai injection.
Preferably, the component 16, the component 17, the component 18 or the monomer components of oxysophorine, ginsenoside Rd, astragaloside 2, formononetin, 9, 10-dimethoxy pterocarpan-3-O-b-D-glucopyranoside, calycosin and cycloastragenol are main drug effect substances of the kang' ai injection for resisting lung cancer.
Preferably, the components 18, 19 and 20 or the monomer components cycloastragenol and ginsenoside Rd are the main drug effective substances of the kang' ai injection for resisting breast cancer.
The technical scheme of the invention has the following advantages:
1. the method for screening the basis of the antitumor effective substances of the kang' ai injection provided by the invention comprises the following steps: separating the kang 'ai injection by adopting high performance liquid chromatography to obtain components of the kang' ai injection; then measuring the proliferation inhibition rate of the components of the kang' ai injection on tumor cells; the conditions of the high performance liquid chromatography are as follows: mobile phase: phase A is 0.05 wt% formic acid-water, phase B is methanol; linear elution gradient: 0-35min, A: b is 85-95 wt%: 15-5 wt% → 54-64 wt%: 46-36 wt%; 35-55min, A: b is 54-64 wt%: 46-36 wt% → 0-5 wt%: 100-95 wt%; 55-65min, A: b is 0-5 wt%: 100-95 wt% → 0-5 wt%: 100-95 wt%; flow rate: 11-14 mL/min; column temperature: 25-35 ℃; DAD detector wavelength: a full wavelength; sample introduction amount: 1-3 mL; starting with the first fraction collected at 3min, fractions were collected as one fraction every 3 min; the high performance liquid chromatography in the method can directly separate the kang ' ai injection, the separated components can be directly used for screening the anti-tumor drug effect substance basis of the kang ' ai injection, the process is simple, the proportion of active ingredients in the separated components is close to that of the kang ' ai injection, and the problem that the difference between a composition manually prepared by utilizing monomer components and the kang ' ai injection is large, and the anti-tumor drug effect substance basis of the kang ' ai injection cannot be accurately screened and obtained is avoided.
2. The invention discloses a method for screening an anti-tumor effective substance basis of a kang 'ai injection, which is used for researching anti-tumor effective substances of the kang' ai injection from two aspects of components of the kang 'ai injection and monomer components of the kang' ai injection, aims to explain the anti-tumor effective substance basis of the kang 'ai injection and provides scientific basis for clinical curative effect of the kang' ai injection.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the effect of KANGAI injection and KANGAI injection components 2-6, 9-13, 15, 19, 20 on LLC cell survival;
FIG. 2 is a graph showing the effect of component 16 of a Kangai injection on LLC cell survival;
FIG. 3 is a graph showing the effect of component 17 of KANGAI injection on LLC cell survival;
FIG. 4 is a graph of the effect of component 18 of a Kangai injection on LLC cell survival;
FIG. 5 is a graph of the effect of KANGAI injection and KANGAI injection components on MCF-7 cell survival;
FIG. 6 shows the effect of monomer components oxymatrine, oxysophocarpine, ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, ginsenoside Ro, and notoginsenoside R1 in the kang' ai injection on LLC cell survival rate;
FIG. 7 shows the effect of monomer components including astragaloside 1, astragaloside 2, formononetin, astragaloside IV, 9, 10-dimethoxy-pteridine-3-O-b-D-glucopyranoside, calycosin, and cycloastragenol in the KANGAI injection on LLC cell survival rate;
FIG. 8 shows the effect of ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Ro, ginsenoside Rd, ginsenoside Rf, ginsenoside Rg1, ginsenoside Re, and notoginsenoside R1 on the survival rate of MCF-7 cells;
FIG. 9 shows the effect of formononetin, astragaloside 1, cycloastragenol, oxymatrine, and oxysophocarpine on the survival rate of MCF-7 cells in KANGAI injection.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The experimental materials and reagents of the invention are as follows:
(1) cell line
LLC cells: mouse Lewis lung carcinoma cells (LLC) were purchased from the cell bank of the culture Collection of the Chinese academy of sciences.
MCF-7 cells: human breast cancer cells were purchased from the cell bank of the culture Collection of the national academy of sciences.
(2) Experimental drugs and reagents
Kang ai injection (01150517): supplied by Changbai mountain pharmaceutical corporation;
oxymatrine monomer components: oxymatrine, oxysophocarpine; the ginseng comprises the following monomer components: ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Re, ginsenoside Rg1, and notoginsenoside R1; the astragalus membranaceus monomer component: calycosin, astragaloside, 9, 10-dimethoxy-pterocarpan-3-O-b-D-glucopyranoside, formononetin, astragaloside 1, astragaloside 2, cycloastragenol and formononetin are all purchased from Shanghai fusion grass medicine science and technology development Limited company. The ginseng comprises the following monomer components: ginsenoside Rd, ginsenoside Rf, and ginsenoside Ro, available from Doctorite Biotechnology Ltd;
methanol (CH)3OH): sigma, cat No.: 34860, respectively;
DMEM high-glucose medium: GIBCO, cat number: 12800017, respectively;
fetal Bovine Serum (FBS): GIBCO, cat number: 10099141;
100 × double antibody (Penicillin Streptomycin): GIBCO, cat number: 15140122, respectively;
digestive juice (0.25% Trypsin + EDTA): GIBCO, cat number: 25200072, respectively;
trypan Blue (0.4% Trypan Blue Stain): GIBCO, cat number: 15250061, respectively;
MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide salt): sigma, cat No.: m2128;
dimethylsulfoxide (DMSO): sigma, cat No.: 276855, respectively;
sodium chloride (NaCl): national drug group, product number: 10019318, respectively;
potassium chloride (KCl): biological, cargo number: PB 0440;
disodium hydrogen phosphate dodecahydrate (Na)2HPO4·12H2O): biological, cargo number: ST 1725;
anhydrous potassium dihydrogen phosphate (KH)2PO4): biological, cargo number: PB 0445;
sodium bicarbonate (NaHCO)3): national drug group, product number: 10018960, respectively;
formic acid (HCOOH): national drug group, product number: FA118P500
(3) Laboratory apparatus
CO2A cell culture box: model 3111, Thermo Scientific, usa;
the biological safety cabinet: 1300 series class ii, type a2, Thermo Scientific, usa;
inverted fluorescence microscopy: olympus IX53, Olympus, japan;
decompression rotary steaming instrument: model R-200, BUCHI rotavap, Germany;
freeze-drying the instrument: model Y14R-651981-YR, Thermo MicroMODULYO0230, USA;
a flat plate oscillator: eppendorf, germany;
a multifunctional microplate reader: model Tecan Infinite F200, Tecan, austria;
balance: METTLER TOLEDO AL104 and XS105, METTLER TOLEDO, switzerland;
Milli-Q ultrapure water System: millipore, usa;
preparing a liquid chromatograph: model SPD-M20A, SHIMADZU, Japan;
a centrifuge: model 5424R, Eppendorf, Germany.
The MTT method is also called as MTT colorimetric method, and is a method for detecting cell survival and growth. The MTT is fully called 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide salt, is also called thiazole blue, and is a yellow dye. The detection principle of the MTT method is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-violet crystalline Formazan (Formazan) and deposit the Formazan in the cells, but dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and an enzyme linked immunosorbent assay detector is used for measuring the light absorption value of the formazan at the wavelength of 570-580nm, so that the number of living cells can be indirectly reflected. Within a certain range of cell number, MTT crystals are formed in an amount proportional to the cell number.
Example 1
The embodiment provides a method for screening a basis of an anti-tumor effective substance of a kang' ai injection, which comprises the following steps:
(1)500mL kang' ai injection is suspended and concentrated to 45mL, centrifuged at 10000rpm for 10min, and then the supernatant is taken for component separation by adopting high performance liquid chromatography.
The conditions of the high performance liquid chromatography are as follows:
mobile phase: phase A is 0.05 wt% formic acid-water, phase B is methanol;
linear elution gradient: 0-35min, A: b is 90 wt%: 10 wt% → 59 wt%: 41 wt%; 35-55min, A: b is 59 wt%: 41 wt% → 0 wt%: 100 wt%; 55-65min, A: b is 0 wt%: 100 wt% → 0 wt%: 100 wt%; flow rate: 13 mL/min; column temperature: 30 ℃;
the wavelength range of the DAD detector is full wavelength;
sample introduction amount: 2 mL.
Fractions were collected from 3min, fractions were collected every 3min as one fraction, freeze-dried, and stored at 4 ℃. And (3) after freeze drying, obtaining 21 components (components 2-22). Because the amounts of the components 7, 8, 14, 21 and 22 after freeze drying are very small, the rest components are only used for screening the anti-tumor effective substances of the subsequent kang' ai injection.
The components of the KANGAI injection prepared by freeze drying are respectively dissolved in DMSO to prepare a mother solution of 100mg/mL, and the mother solution is stored in a refrigerator at the temperature of 80 ℃ below zero for subsequent experiments.
(2) And (3) screening the components of the kang' ai injection by using an MTT method for the antineoplastic active substances.
The kang' ai injection is diluted to 1/2, 1/4, 1/8, 1/16 and 1/32 concentrations by volume ratio with high-sugar DMEM medium for later use.
The components of the kang' ai injection are respectively diluted to 400 mug/mL, 200 mug/mL, 100 mug/mL and 50 mug/mL by using a high-sugar DMEM medium to be used as test solutions for LLC cell survival rate experiments. And (5) standby.
The components of the kang' ai injection are respectively diluted to 500 mu g/mL and 250 mu g/mL by using a high-sugar DMEM medium to be used as test solutions for MCF-7 cell survival rate experiments. And (5) standby.
LLC cell survival rate experiment: collecting LLC cells in logarithmic growth phase, digesting, counting, inoculating into 96-well plate at 4000/well, culturing in incubator for 24 hr, removing culture medium, diluting with culture medium, and adding 5% CO at 37 deg.C2And (3) incubating the cells in an incubator for 48h, then sucking and removing the liquid medicine, adding 100 mu L/hole of MTT solution, incubating for 4h in a dark place, sucking the supernatant, adding 100 mu L/hole of DMSO, shaking and uniformly mixing the mixture on a shaker at 37 ℃ for 10min to fully dissolve formazan, measuring absorbance under the condition of 580nm wavelength by using an enzyme labeling instrument, and calculating the cell survival rate of the drug groups with different concentrations.
MCF-7 cell viability assay: collecting MCF-7 cells in logarithmic growth phase, digesting and counting, inoculating into 96-well plate in the amount of 3000/well (group) and 5000/well (kang 'ai injection group), culturing in incubator for 24 hr (group) and 48 hr (kang' ai injection group), removing original culture medium, diluting with culture medium, and diluting at 37 deg.C with 5% CO2And (3) incubating the cells in an incubator for 24h, then sucking and removing the liquid medicine, adding 100 mu L/hole of MTT solution, incubating for 4h in a dark place, sucking the supernatant, adding 100 mu L/hole of DMSO, shaking and uniformly mixing on a shaker at 37 ℃ for 10min to fully dissolve formazan, measuring absorbance under the condition of 580nm wavelength by using an enzyme labeling instrument, and calculating the cell survival rate of the drug groups with different concentrations.
The cell viability was calculated as:
cell survival (%). absorbance of administration group/absorbance of negative control group × 100%. The negative control group is a culture medium containing equal volume of deionized water without adding liquid medicine, and the rest operations are the same.
LLC and MCF-7 cell viability experiments were performed in triplicate (3 replicates per concentration were set), and the dilutions or concentrations were plotted on the abscissa and cell viability on the ordinate. All data are expressed as mean ± standard deviation (mean ± SD) and processed using GraphPad Prism 6 software.
Example 2
The embodiment provides a method for screening a basis of an anti-tumor effective substance of a kang' ai injection, which comprises the following steps:
(1)500mL kang' ai injection is suspended and concentrated to 45mL, centrifuged at 10000rpm for 10min, and then the supernatant is taken for component separation by adopting high performance liquid chromatography.
The conditions of the high performance liquid chromatography are as follows:
mobile phase: phase A is 0.05 wt% formic acid-water, phase B is methanol;
linear elution gradient: 0-35min, A: b is 90 wt%: 10 wt% → 59 wt%: 41 wt%; 35-55min, A: b is 59 wt%: 41 wt% → 0 wt%: 100 wt%; 55-65min, A: b is 0 wt%: 100 wt% → 0 wt%: 100 wt%; flow rate: 13 mL/min; column temperature: 30 ℃;
the wavelength range of the DAD detector is full wavelength;
sample introduction amount: 2 mL.
Fractions were collected from 3min, fractions were collected every 3min as one fraction, lyophilized, and stored at 4 ℃. And (3) freeze-drying to obtain 21 components (components 2-22). Because the amount of the components 7, 8, 14, 21 and 22 after freeze-drying is very small, the rest components are only used for screening the anti-tumor effective substances of the subsequent kang' ai injection.
In this embodiment, the anti-tumor effective substances of the kang 'ai injection are screened from two layers of the components and the monomer components of the kang' ai injection. The monomer components adopted in the embodiment comprise kurarinone monomer components: oxymatrine, oxysophocarpine; the ginseng comprises the following monomer components: ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Re, ginsenoside Rg1, and notoginsenoside R1; the astragalus membranaceus monomer component: calycosin, astragaloside, 9, 10-dimethoxy-pterocarpan-3-O-b-D-glucopyranoside, formononetin, astragaloside 1, astragaloside 2, cycloastragenol and formononetin are all purchased from Shanghai fusion grass medicine science and technology development Limited company. The ginseng comprises the following monomer components: ginsenoside Rd, ginsenoside Rf, and ginsenoside Ro.
The components of the KANGAI injection prepared by freeze drying and the purchased monomer components are respectively dissolved in DMSO to prepare a mother solution of 100mg/mL, and the mother solution is stored in a refrigerator at-80 ℃ for subsequent experiments.
(2) And (3) screening the components of the kang' ai injection by using an MTT method for the antineoplastic active substances.
The kang' ai injection is diluted to 1/2, 1/4, 1/8, 1/16 and 1/32 concentrations by volume ratio with high-sugar DMEM medium for later use.
The components of the kang' ai injection are respectively diluted to 400 mug/mL, 200 mug/mL, 100 mug/mL and 50 mug/mL by using a high-sugar DMEM medium to be used as test solutions for LLC cell survival rate experiments. And (5) standby.
The components of the kang' ai injection are respectively diluted to 500 mu g/mL and 250 mu g/mL by using a high-sugar DMEM medium to be used as test solutions for MCF-7 cell survival rate experiments. And (5) standby.
The monomer component cycloastragenol is diluted to 50 mug/mL, 40 mug/mL, 35 mug/mL, 30 mug/mL, 20 mug/mL and 10 mug/mL by a high-sugar DMEM culture medium to be used as a test solution for LLC cell survival rate experiments. The rest monomer components are respectively diluted to 400 mug/mL, 200 mug/mL, 100 mug/mL and 50 mug/mL by using a high-sugar DMEM culture medium to be used as test solutions for LLC cell survival rate experiments. And (5) standby.
The monomer components of the kang' ai injection are diluted to 400 mug/mL, 200 mug/mL, 100 mug/mL and 50 mug/mL by using a high-sugar DMEM medium to be used as a test solution for MCF-7 cell survival rate experiments. And (5) standby.
LLC cell survival rate experiment: collecting LLC cells in logarithmic growth phase, digesting and counting, inoculating into 96-well plate at 4000/well, culturing in incubator for 24 hr, removing culture medium, diluting with culture medium, and adding 5% CO at 37 deg.C2Incubating cells in incubator for 48h, removing liquid medicine, adding MTT solution 100 μ L/hole, incubating for 4h in dark, removing supernatant, adding DMSO 100 μ L/hole, shaking and mixing at 37 deg.C for 10min to dissolve formazan, measuring absorbance with enzyme labeling instrument at 580nm wavelength, and calculating different concentrationsCell viability in the drug group.
MCF-7 cell viability assay: after MCF-7 cells in the logarithmic growth phase are digested and counted, the cells are inoculated into a 96-well plate in the number of 3000/well (group), 5000/well (conai injection group) and 5000/well (monomer group), culturing in incubator for 24h (group), 48h (kang ' ai injection group) and 48h (monomer component group), discarding the original culture medium, diluting the kang ' ai injection, kang ' ai injection or monomer component with culture medium, incubating the cells in incubator at 37 deg.C and 5% CO2 for 24h, then absorbing and removing the liquid medicine, adding 100 mu L/hole of MTT solution, incubating for 4h in a dark place, and (3) sucking a supernatant, adding DMSO (dimethylsulfoxide) at 100 mu L/hole, uniformly mixing on an oscillator at 37 ℃ for 10min by oscillation to fully dissolve formazan, measuring absorbance under the condition of 580nm wavelength by using an enzyme-labeling instrument, and calculating the cell survival rate of the drug groups with different concentrations.
The cell viability was calculated as:
cell survival (%). absorbance of administration group/absorbance of negative control group × 100%. The negative control group is culture medium without added medicinal liquid and containing equal volume of deionized water, and the rest operations are the same
LLC and MCF-7 cell viability experiments were performed in triplicate (3 replicates per concentration were set), and the dilutions or concentrations were plotted on the abscissa and cell viability on the ordinate. All data are expressed as mean ± standard deviation (mean ± SD) and processed using GraphPad Prism 6 software.
The effect of the components of the KANGAI injection and KANGAI injection on LLC cell viability is shown in FIGS. 1-4, and the effect on MCF-7 cell viability is shown in FIG. 5. The effect of the monomeric components on LLC cell viability is shown in FIGS. 6 and 7, and the effect of the monomeric components on MCF-7 cell viability is shown in FIGS. 8 and 9.
The kang ' ai injection can inhibit the growth of LLC cells, and the cell survival rate is about 0% when the kang ' ai injection acts on the cells for 48 hours under the dilution concentration of 1/2, which shows that the kang ' ai injection has strong effect of resisting LLC lung cancer. The component 17 has strong Lewis lung cancer resistance, and the cell survival rate is 50.66 percent after the cell is acted for 48 hours at the concentration of 400 mu g/mL; the components 16 and 18 have certain inhibition effect on the growth of LLC cells, and after the LLC cells act for 48 hours at the concentration of 400 mu g/mL, the cell survival rates are 76.54% and 71.84% respectively; after the other components act on cells for 48 hours at the concentration of 400 mu g/mL, the cell survival rate is over 85 percent. The results show that the components 16, 17 and 18 of the kang 'ai injection can be the main drug effective substances of the kang' ai injection for resisting the lung cancer.
The kang 'ai injection has a cell growth inhibition effect on MCF-7, and acts on cells for 24 hours under the dilution concentration of 1/2, the cell survival rate is 79.22%, and the kang' ai injection has a certain breast cancer resistance effect. The components 18, 19 and 20 of the kang' ai injection have certain cell growth inhibition effect on MCF-7, and after the component acts on cells at the concentration of 500 mu g/mL for 24 hours, the cell survival rates are 80.68%, 47.93% and 45.67% respectively; after the other components act on cells for 24 hours at the concentration of 500 mu g/mL, the cell survival rate is over 85 percent. The results show that the components 18, 19 and 20 of the kang 'ai injection can be the main drug effective substances of the kang' ai injection for resisting the breast cancer.
The cycloastragenol has strong inhibition effect on the growth of LLC cells, and the cell survival rate is about 0% after the cycloastragenol acts on the cells at the concentration of 50 mu g/mL for 48 hours; ginsenoside Rd, astragaloside 2, formononetin, 9, 10-dimethoxy rosenane-3-O-b-D-glucopyranoside and calycosin have strong inhibition effect on the growth of LLC, and after the cells are acted for 48 hours at the concentration of 400 mu g/mL, the cell survival rates are respectively 20.17%, 43.96%, 25.95%, 22.30%, 4.91% and 59.02%; the oxysophocarpine has certain effect of resisting LLC lung cancer, and the cell survival rate is 82.31% after the oxysophocarpine acts on cells at the concentration of 400 mu g/mL for 48 hours; after the other monomer components act on cells for 48 hours at the concentration of 400 mu g/mL, the cell survival rate is over 85 percent. The result shows that monomer components of sophocarpine oxide, ginsenoside Rd, astragaloside 2, formononetin, 9, 10-dimethoxy rosenane-3-O-b-D-glucopyranoside, calycosin and cycloastragenol can be main drug-effect substances of the KANGAI injection for resisting lung cancer.
The cycloastragenol has a strong cell growth inhibition effect on MCF-7, and the cell survival rate is 35.69% after the cell is acted for 24 hours at the concentration of 400 mu g/mL; ginsenoside Rd has certain cell growth inhibition effect on MCF-7, and the cell survival rate is 77.93% after the cell is acted at the concentration of 400 mu g/mL for 24 hours; after the rest monomer components act on cells for 24 hours at the concentration of 400 mu g/mL, the cell survival rate is over 85 percent. The result shows that the monomer components of the cycloastragenol and the ginsenoside Rd can be main drug-effect substances of the kang' ai injection for resisting the breast cancer.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A method for screening the basis of antitumor effective substances of Kangai injection is characterized by comprising the following steps:
separating the kang 'ai injection by adopting high performance liquid chromatography to obtain components of the kang' ai injection; then measuring the proliferation inhibition rate of the components of the kang' ai injection on tumor cells;
the conditions of the high performance liquid chromatography are as follows:
mobile phase: phase A is 0.05 wt% formic acid-water, phase B is methanol;
linear elution gradient: 0-35min, A: b is 85-95 wt%: 15-5 wt% → 54-64 wt%: 46-36 wt%;
35-55min, A: b is 54-64 wt%: 46-36 wt% → 0-5 wt%: 100-95 wt%; the time of the reaction lasts for 55-65min,
a: b is 0-5 wt%: 100-95 wt% → 0-5 wt%: 100-95 wt%;
flow rate: 11-14 mL/min;
column temperature: 25-35 ℃;
DAD detector wavelength: a full wavelength;
sample introduction amount: 1-3 mL;
starting with the first fraction collected at 3min, fractions were collected as one fraction every 3 min.
2. The method for screening basis of anti-tumor effective substances of kang' ai injection according to claim 1, wherein the linear elution gradient is: 0-35min, A: b is 90 wt%: 10 wt% → 59 wt%: 41 wt%; 35-55min, A: b is 59 wt%: 41 wt% → 0 wt%: 100 wt%; 55-65min, A: b is 0 wt%: 100 wt% → 0 wt%: 100 wt%.
3. The method for screening the basis of the anti-tumor effective substances of the kang 'ai injection as claimed in claim 1 or 2, wherein the kang' ai injection is concentrated according to the volume ratio of 50 (4-5), the concentrated solution is centrifuged, and the obtained supernatant is separated by high performance liquid chromatography.
4. The method for screening the basis of an antitumor agent for a kang ' ai injection according to claim 1 or 2, further comprising measuring the proliferation inhibition rate of the kang ' ai injection and monomer components of the kang ' ai injection on tumor cells.
5. The method for screening anti-tumor effective substance basis of kang 'ai injection as claimed in claim 4, wherein said monomer component of kang' ai injection comprises one or more of oxymatrine monomer component, ginseng monomer component and astragalus monomer component.
6. The method for screening the basis of an antitumor drug effective substance of Kangan injection as claimed in claim 5, wherein the oxymatrine monomer component is selected from oxymatrine and oxysophocarpine;
the ginseng monomer component is selected from ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Re, ginsenoside Rg1, notoginsenoside R1, ginsenoside Rd, ginsenoside Rf and ginsenoside Ro;
the radix astragali monomer component is selected from calycosin, astragaloside IV, 9, 10-dimethoxy rosenane-3-O-b-D-glucopyranoside, formononetin, astragaloside 1, astragaloside 2, cycloastragenol and formononetin.
7. The method for screening the basis of anti-tumor effective substances of kang 'ai injection according to any one of claims 1 to 6, wherein the proliferation inhibition rate of the kang' ai injection, the components of the kang 'ai injection and the monomer components of the kang' ai injection on tumor cells is determined by the MTT method, comprising:
digesting tumor cells, inoculating the digested tumor cells in a culture medium, culturing for 24-48h, removing the original culture medium, diluting the components of the KANGAI injection, KANGAI injection or monomer components of the KANGAI injection with the culture medium to obtain a medicinal liquid at 37 deg.C and 5% CO2Incubating the cells for 24-48h, then removing the liquid medicine, adding MTT solution, incubating in the dark, removing the supernatant, adding DMSO, oscillating and mixing uniformly, measuring absorbance under the condition of 580nm wavelength by adopting an enzyme-labeling instrument, and calculating the cell survival rate of the components of the kang 'ai injection, the kang' ai injection or the monomer components of the kang 'ai injection with different concentrations after incubation, thereby calculating the proliferation inhibition rate of the kang' ai injection, the components of the kang 'ai injection or the monomer components of the kang' ai injection on the tumor cells;
the calculation formula of the cell survival rate is as follows: cell survival rate (%). absorbance of administration group/absorbance of negative control group × 100%;
the calculation formula of the proliferation inhibition rate is as follows: proliferation inhibition (%) is 1-cell survival (%).
8. The method for screening the basis of anticancer active substances of kang 'ai injection as claimed in claim 7, wherein when said proliferation inhibition rate is > 15%, the components of kang' ai injection or the monomer components of kang 'ai injection are the anticancer active substances of kang' ai injection.
9. The method for screening the basis of anticancer active substances of kang 'ai injection as claimed in claim 1 or 2 or 5 or 6 or 8, wherein the component 16, the component 17, the component 18 or the monomer ingredients of sophocarpine oxide, ginsenoside Rd, astragaloside 2, formononetin, 9, 10-dimethoxy santaline-3-O-b-D-glucopyranoside, calycosin, and cycloastragenol are the main anticancer active substances of kang' ai injection.
10. The method for screening the basis of anti-tumor effective substances of KANGAI injection as claimed in claim 1 or 2 or 5 or 6 or 8, wherein component 18, component 19 and component 20 or monomer components of cycloastragenol and ginsenoside Rd are the main effective substances of KANGAI injection against breast cancer.
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