CN113322298A - Biological limit value determination method for Qingjin phlegm-resolving decoction - Google Patents
Biological limit value determination method for Qingjin phlegm-resolving decoction Download PDFInfo
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Abstract
The invention discloses a method for measuring biological limit of Qingjin Huatan Tang, belonging to the technical field of drug analysis and detection and comprising the following steps: step 1, preparing a clear gold phlegm-resolving decoction water extract freeze-dried powder solution and an LPS solution, and culturing RAW264.7 macrophages; step 2, monitoring the relation between the inoculation density of RAW264.7 macrophage and the incubation time; step 3, monitoring the influence of the clear gold phlegm-resolving decoction water extract freeze-dried powder solution on the activity of RAW264.7 cells; step 4, determining the bioactivity limit of the Qingjin Huatan decoction water extract freeze-dried powder based on the secretion function of RAW264.7 macrophages; step 5, determining the bioactivity limit of the Qingjin Huatan decoction water extract freeze-dried powder based on the phagocytic function of RAW264.7 macrophages; step 6, data processing; the invention has important significance for the quality control of the Qingjin phlegm-resolving decoction by establishing a biological activity determination method for correlating the efficacy and the pharmacological action of the Qingjin phlegm-resolving decoction.
Description
Technical Field
The invention belongs to the technical field of drug analysis and detection, and particularly relates to a biological limit value determination method of Qingjin Huatan decoction.
Background
The Qingjin Huatan Tang is originated from the medical science of Ming Dynasty leaf ages, and comprises 11 medicines of scutellaria baicalensis, gardenia, fritillaria, cortex mori, semen trichosanthis, exocarpium citri rubrum, platycodon grandiflorum, radix ophiopogonis, rhizoma anemarrhenae, poria cocos and liquorice, has remarkable effects of clearing heat, moistening lung, reducing phlegm and relieving cough, and is one of 100 ancient classic famous prescriptions. In order to fully ensure and exert the clinical curative effect, the establishment of a scientific and comprehensive quality control method has important significance. The commonly used traditional Chinese medicine quality control method comprises appearance shape identification, chemical qualitative identification, index component detection, chemical fingerprint spectrum and the like of medicinal materials. However, because the chemical components of the traditional Chinese medicine are complex, the quality of the traditional Chinese medicine is difficult to be comprehensively controlled and the clinical curative effect is reflected by applying a single quality control method. The biological activity measurement refers to that the proper reaction on organisms is searched by combining the functional indications or toxic and side effects of the traditional Chinese medicine and the preparation thereof, so that the quality of the traditional Chinese medicine is more comprehensively and effectively evaluated and controlled, the traditional Chinese medicine quality evaluation system is perfected, and the safety and effectiveness of the clinical use of the medicine are ensured. In recent years, many studies have reported that biological activity measurement is applied to the research of evaluating the biological effect of traditional Chinese medicines, and leech biological activity measurement is recorded in pharmacopoeia as one of quality control methods, and the application of biological activity measurement to the field of quality control of traditional Chinese medicines has become a trend.
Because the formula and the contained chemical components of the Qingjin phlegm-resolving decoction are complex, the research reports on the quality reference and the quality control of the Qingjin phlegm-resolving decoction are few through literature retrieval, so that the establishment of the biological activity determination method related to the efficacy and the pharmacological action of the Qingjin phlegm-resolving decoction has important significance on the quality control of the Qingjin phlegm-resolving decoction.
The invention combines the pharmacological actions of the anti-inflammatory and immunoregulation of the Qingjin Huatan decoction, establishes a biological limit value measuring method based on the phagocytic and secretory functions of RAW264.7 macrophages, and is used for controlling the quality of the Qingjin Huatan decoction.
Disclosure of Invention
The invention aims to provide a gold-clearing phlegm-reducing biological limit determination method.
The technical problems to be solved by the invention are as follows:
because the formula and the contained chemical components of the Qingjin phlegm-resolving decoction are complex, the research reports on the quality reference and the quality control of the Qingjin phlegm-resolving decoction are few through literature retrieval, so that the establishment of the biological activity determination method related to the efficacy and the pharmacological action of the Qingjin phlegm-resolving decoction has important significance on the quality control of the Qingjin phlegm-resolving decoction.
The purpose of the invention can be realized by the following technical scheme:
a method for measuring biological limit of Qingjin Huatan Tang specifically comprises the following steps:
step 1, preparing a clear gold phlegm-resolving decoction water extract freeze-dried powder solution and an LPS solution, and culturing RAW264.7 macrophages;
step 2, monitoring the relation between the inoculation density of RAW264.7 macrophage and the incubation time;
step 3, monitoring the influence of the clear gold phlegm-resolving decoction water extract freeze-dried powder solution on the activity of RAW264.7 cells;
step 4, determining the bioactivity limit of the Qingjin Huatan decoction water extract freeze-dried powder based on the secretion function of RAW264.7 macrophages;
step 5, determining the bioactivity limit of the Qingjin Huatan decoction water extract freeze-dried powder based on the phagocytic function of RAW264.7 macrophages;
and 6, data processing, namely carrying out statistical processing on experimental data by using IBM SPSS Statistics19.0 statistical software, wherein single-factor variance analysis is adopted for comparison among groups, LSD (least squares) test is adopted for comparison among groups, and P <0.05 has statistical significance.
Wherein, the step 4 specifically comprises the following steps:
step A1, testing the influence of the freeze-dried powder of the water extract of the Qingjin Huatan decoction on the content of IL-1 beta and IL-6 secreted by LPS-induced RAW264.7 macrophage
Logarithmic growthAdjusting cell suspension concentration to 3 × 10 with RAW264.7 macrophage5one/mL, inoculated in a 96-well plate at 100. mu.L/well, placed at 37 ℃ with 5% CO2After being cultured in an incubator for one night in a wall pasting way, the group is divided into a control group, a model group and a Qingjin phlegm-resolving decoction water extract freeze-dried powder administration group, wherein the model group and the Qingjin phlegm-resolving decoction water extract freeze-dried powder administration group are added into the model group and the Qingjin phlegm-resolving decoction administration group at the same time, and the final mass concentration is 1 mu g/mL-1The final mass concentrations of the LPS and each concentration gradient drug of (1) are respectively 62.5, 125, 250 and 500 mug. multidot.mL-16 multiple wells in each group, after administration, 5% CO at 37 deg.C2Culturing in an incubator for 24h, collecting supernatant, detecting the content of IL-1 beta and IL-6, and calculating the inhibition rate after administration (%) (model group content-administration group content)/model group content × 100%;
step A2, determining the limit dosage of the clear gold phlegm-resolving decoction water extract freeze-dried powder
Taking RAW264.7 macrophage in logarithmic growth phase, adjusting cell suspension concentration to 3 × 105one/mL, inoculated in a 96-well plate at 100. mu.L/well, placed at 37 ℃ with 5% CO2After being cultured in an incubator for one night in a wall-pasting way, the materials are divided into a control group, a model group and a Qingjin phlegm-reducing decoction water extract freeze-dried powder group, wherein the model group and each administration group of the Qingjin phlegm-reducing decoction are added with the final mass concentration of 1 mu g/mL-1The final mass concentration of the LPS and the drugs in each batch is 500 mug. multidot.mL-16 multiple wells in each group, after administration, 5% CO at 37 deg.C2Culturing in an incubator for 24h, collecting supernatant, detecting the content of IL-6, and calculating the inhibition rate (%) after administration (model group content-administration group content)/model group content × 100%;
step A3, performing repeatability and method suitability verification
Repeatability: collecting the lyophilized powder of QINGJINHUATAN decoction water extract to final mass concentration of 500 μ g/mL-1Then, the amount of the reaction solution was measured for 1. mu.g.mL-1The inhibition rate of LPS induced RAW264.7 macrophage secretion IL-6 is repeated for 3 times, and the experimental result is evaluated;
the method has the following applicability: taking the freeze-dried powder of the water extract of the Qingjin Huatan decoction of each batch to act on RAW264.7 macrophage, and measuring the ratio of the freeze-dried powder to the RAW264.7 macrophage to 1 microgram/mL-1LPS-induced RAW264.7 hugeThe inhibition rate of IL-6 secretion by phagocytes was evaluated.
Further, the preparation method of the clear golden sputum-reducing decoction water extract freeze-dried powder solution comprises the following specific steps:
precisely weighing each batch of QINGJINHUATAN decoction water extract lyophilized powder, adding DMEM complete culture medium for dissolving, filtering with 0.22 μm microporous membrane to obtain 1.0 g.L-1The basic mother liquor of the Qingjin Huatan decoction is diluted to the concentration of the corresponding working solution by using a DMEM complete culture medium, and is prepared when being used.
Further, the specific steps of preparing the LPS solution are as follows:
LPS powder and PBS buffer were mixed according to 1 mg: 1mL of the mixture is mixed evenly and then filtered by a 0.22 mu m microporous membrane to prepare 1 g.L-1The mother liquor is frozen at-20 ℃, and DMEM complete culture medium is diluted to the corresponding working solution concentration when the mother liquor is used.
Further, the procedure for culturing RAW264.7 macrophages is as follows: culturing RAW264.7 macrophage in DMEM complete medium containing 10% fetal calf serum, placing at 37 deg.C and 5% CO2And culturing in an incubator with saturated humidity.
Further, the specific method of step 2 is:
taking RAW264.7 macrophage in logarithmic growth phase, adjusting cell suspension concentration to 1 × 105、3×105、5×105one/mL of the cells were inoculated into a 96-well plate at 100. mu.L/well, and then 100. mu.L of complete medium of LDMEM was added thereto, and the mixture was incubated at 37 ℃ with 5% CO2Culturing in an incubator, adding 20 μ L CCK-8 after culturing for 12h, 24h, 36h, 48h and 60h respectively, and measuring the absorbance value under 450nm of an enzyme-labeling instrument after 2 h.
Further, the specific method of step 3 is:
taking RAW264.7 macrophage in logarithmic growth phase, adjusting cell suspension concentration to 3 × 105one/mL, inoculated in a 96-well plate at 100. mu.L/well, placed at 37 ℃ with 5% CO2Culturing in incubator for 3 hr, and dividing into control group and QINGJINHUATANG water extract lyophilized powder group with final mass concentration of 62.5, 125, 250, 500, 1000 μ g/mL-13 multiple wells per group, containing at 37 ℃ after administration5％CO2Culturing in incubator for 24h, adding 20 μ L CCK-8 reagent into each well, measuring absorbance at 450nm wavelength of microplate reader after 2h, calculating proliferation rate (%) (drug OD value-control group OD value)/control group OD value × 100%, and repeating the test for 3 times.
Further, the determination of the bioactivity limit of the Qingjin Huatan decoction aqueous extract freeze-dried powder based on the phagocytic function of RAW264.7 macrophages in the step 5 specifically comprises the following steps:
step B1, determination of limit dosage of the clear gold phlegm-resolving decoction water extract freeze-dried powder
Taking RAW264.7 macrophage in logarithmic growth phase, adjusting cell suspension concentration to 3 × 105one/mL, inoculated in a 96-well plate at 100. mu.L/well, placed at 37 ℃ with 5% CO2Culturing in incubator for 3 hr, and separating into control group, LPS group, and QINGJINHUATANG water extract lyophilized powder group with final mass concentration of 500 μ g/mL-1At least 3 multiple wells in each group, after administration, at 37 deg.C with 5% CO2Culturing for 24h in an incubator, discarding the supernatant, adding 100 muL of neutral red staining solution into each well, incubating for 1h, discarding neutral red, washing with PBS once, 200 muL/well each time, adding 100 muL/well of prepared cell lysate, shaking with a micro-shaking apparatus for 15min, standing for 2h, detecting the absorbance at 562nm, and calculating the phagocytosis index of each group, wherein the phagocytosis index (%) -is the OD value of the drug group/the OD value of the control group multiplied by 100%;
step B2, repeatability monitoring
Taking a group of clear gold phlegm-resolving decoction water extract freeze-dried powder solutions to make the final mass concentration be 500 mug/mL-1Determining the influence of the protein on the phagocytic index of RAW264.7 macrophages, repeating the experiment for 6 times, and evaluating the experiment result;
step B3, intermediate precision monitoring
500 mu g/mL is adopted by different experimenters in the same laboratory-1After the freeze-dried powder of the Qingjin Huatan decoction water extract acts on RAW264.7 macrophage, a neutral red experiment is adopted to detect the phagocytosis index of the freeze-dried powder, and the experimental result is evaluated;
step B4, method suitability monitoring
According to the method of the step B1, after the batch to be detected acts on RAW264.7 macrophages, the phagocytosis index of the batch is measured, and the experimental result is evaluated.
Further, the test result in the step 3 shows that the mass concentration of the clear golden sputum-reducing decoction water extract freeze-dried powder is 62.5-250 mug/mL-1The proliferation of RAW264.7 cells is promoted.
Further, the test result in the step 4 shows that the mass concentration of the clear golden sputum-reducing decoction water extract freeze-dried powder is more than or equal to 125 mu g/mL-1Obviously inhibiting LPS-induced RAW264.7 cells from secreting IL-6; the mass concentration of the freeze-dried powder of the water extract of the Qingjin Huatan decoction is more than or equal to 500 mu g/mL-1Inhibit IL-6 secretion of RAW264.7 cells induced by LPS, and inhibit rate>45% is a preferred mass.
Further, the test result in the step 5 shows that the mass concentration of the clear golden sputum-reducing decoction water extract freeze-dried powder is more than or equal to 500 mug.mL-1Enhance the phagocytic function of RAW264.7 cells.
Furthermore, the mass concentration of the freeze-dried powder of the water extract of the Qingjin Huatan decoction is more than or equal to 125 mu g/mL-1Inhibit LPS-induced IL-6 secretion from RAW264.7 cells, or more than or equal to 500 μ g/mL-1Enhance the phagocytic function of RAW264.7 cells, reach any one of the phagocytic functions and serve as a bioactive quality control index.
The invention has the beneficial effects that:
the invention combines the anti-inflammatory biological activity of the Qingjin Huatan decoction, establishes an LPS (LPS) -induced RAW264.7 macrophage inflammation model, and discovers 125, 250 and 500 mu g/mL by monitoring the influence of the Qingjin Huatan decoction on the content of IL-1 beta and IL-6 secreted by RAW264.7 macrophages-1The freeze-dried powder of the water extract of the Qingjin Huatan decoction has obvious inhibition effect on the content of IL-6 and shows dose-effect relationship, particularly when the final mass concentration is 500 mu g.mL-1In this case, 500. mu.g/mL was selected because the inhibitory effect was the strongest and the experimental results were stable-1As a limit dose, the dose is 1. mu.g.mL-1The inhibition rate of the content of IL-6 secreted by RAW264.7 macrophage induced by LPS is more than 45 percent, and the quality can be judged to be qualified;
in addition, according to the existing research, the clinic symptoms of a patient can be improved by the qing jin huan tang through the pharmacological action of immune regulation, the invention establishes a biological limit value measuring method of the qing jin huan tang aqueous extract freeze-dried powder based on the phagocytic function of macrophages aiming at the pharmacological activity of the qing jin huan tang immune regulation, and the result shows that when the mass concentration of the qing jin huan tang aqueous extract freeze-dried powder is 62.5-250 mug/mL, the biological limit value measuring method has obvious promotion effect on the proliferation of the macrophages, and when the mass concentration is 125 mug/mL, the biological limit value measuring method has the strongest promotion effect on the proliferation, when the concentration is 500ug/mL, the biological limit value measuring method has obvious toxicity and no obvious promotion effect on the proliferation to the macrophages, but has obvious promotion effect on the phagocytic function under the dosage, the phagocytic index is 111-121%, and the analysis on the influence of the qingjin huan tang aqueous extract freeze-dried powder on the phagocytic function of the macrophages shows, in the research of the invention, in order to increase the reliability of the method and ensure the repeatability of the experimental result, the mass concentration of 500 mug/mL is selected as the limit dosage, the phagocytic index is more than 111%, and the quality can be judged to be qualified;
in summary, the invention combines the pharmacological activities of anti-inflammation and immunoregulation of the Qingjin Huatantang, establishes a biological effect model of inflammation of RAW264.7 macrophages and neutral red phagocytosis, takes the inhibition capability of the Qingjin Huatantang aqueous extract freeze-dried powder on the secretion of inflammatory factors IL-6 of RAW264.7 macrophages and the phagocytosis capability on neutral red as evaluation indexes, and is used as a biological evaluation method of the Qingjin Huatantang aqueous extract freeze-dried powder.
Drawings
In order to more clearly illustrate the technical solutions of 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 only 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 is a line graph showing the results of the measurement of the relationship between the inoculation density of the macrophage monitoring RAW264.7 and the incubation time in the present invention.
FIG. 2 is a bar chart of the effect of the freeze-dried powder of the water extract of Qingjin Huatan Tang in each batch of the invention on the activity of RAW264.7 cells.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in the figure 1-2, the method for measuring the biological limit of the Qingjin Huatan Tang specifically comprises the following steps:
step 1, preparing a clear gold phlegm-resolving decoction water extract freeze-dried powder solution and an LPS solution, and culturing RAW264.7 macrophages;
the preparation process of the clear golden sputum-reducing decoction water extract freeze-dried powder solution is as follows: adding 19112606, 19112607, 19112608, 19112609, 19112610 batches of QINGJINHUATAN water extract lyophilized powder provided by Tianjin pharmaceutical research institute, Inc. into DMEM complete culture medium respectively for dissolving, filtering with 0.22 μm microporous membrane, and making into 1.0 g.L-1The golden clear phlegm-resolving decoction substance reference mother solution is diluted to the corresponding working solution concentration by a DMEM complete culture medium;
the LPS solution was prepared as follows:
25mg of LPS powder was completely dissolved in 25mg of PBS, and the solution was filtered through a 0.22 μm microporous membrane to prepare 1 g.L-1Freezing the mother liquor at-20 deg.C, and diluting with DMEM complete culture medium to corresponding working solution concentration;
the process of culturing phagocytes is as follows:
culturing RAW264.7 macrophage in DMEM complete medium containing 10% fetal calf serum, placing at 37 deg.C and 5% CO2And culturing in an incubator with saturated humidity;
step 2, monitoring the relation between the inoculation density of RAW264.7 macrophage and the incubation time;
taking RAW264.7 macrophage in logarithmic growth phase, adjusting cell suspension concentration to 1 × 105、3×105、5×105one/mL of the cells were inoculated into a 96-well plate at 100. mu.L/well, and then 100. mu.L of complete medium of LDMEM was added thereto, and the mixture was incubated at 37 ℃ with 5% CO2Culturing in an incubator, adding 20 μ L CCK-8 after culturing for 12h, 24h, 36h, 48h and 60h respectively, and measuring the absorbance value under 450nm of an enzyme-labeling instrument after 2h, wherein the test result is shown in figure 1;
when the cell density is 3X 10 in combination with microscopic observation5When the number of cells per mL is less than 48 hours, the cell shape is stable, the growth state is good, and the cell density is in a saturated state; when the cell density is 1X 105At one/mL, the cells continued to grow and the morphology was changed by 3X 105More than one/mL; when the cell density is 5X 105At each/mL, the cell morphology is easy to change, the overlarge density is not beneficial to monitoring the action time of the medicament, the factors of the experimental stability and the experimental time are comprehensively considered, and the inoculation density of RAW264.7 macrophage is selected to be 3 multiplied by 105one/mL, the experiment is completed within 36 h;
step 3, monitoring the influence of the clear gold phlegm-resolving decoction water extract freeze-dried powder solution on the activity of RAW264.7 cells;
taking RAW264.7 macrophage in logarithmic growth phase, adjusting cell suspension concentration to 3 × 105one/mL, inoculated in a 96-well plate at 100. mu.L/well, placed at 37 ℃ with 5% CO2After being cultured for 3h in an incubator in a sticking way, the culture medium is divided into a control group, batches of 19112607, 19112608 and 19112609 freeze-dried powder groups with the final mass concentration of 62.5, 125, 250, 500 and 1000 mu g/mL-13 multiple wells in each group, after administration, 5% CO at 37 ℃2Culturing in incubator for 24 hr, adding 20 μ L CCK-8 reagent into each well, measuring absorbance at 450nm wavelength of microplate reader after 2 hr, and calculating proliferation rate (%) (drug group OD value-control group OD value)/control group OD value × 100%; the test was repeated 3 times;
the test results are shown in FIG. 2, where A represents batch 19112607, B represents batch 19112608, and C represents batch 19112609;
as can be seen from FIG. 2, the quality is goodThe quantitative concentration is 62.5-500 mug/mL-1In the meantime, the freeze-dried powder of the water extract of the Qingjin Huatan decoction of each batch has no toxic effect on RAW264.7 macrophage, and the concentration is 62.5-250 mug.mL-1When the compound is used, the batch 19112607 and the batch 19112609 both have obvious proliferation promoting effect; when the mass concentration of the freeze-dried powder of the water extract of the Qingjin Huatan decoction is 500 mug/mL-1When compared with the control group, the three batches have no influence on the proliferation of RAW264.7 macrophages, and the dosage of 500 mu g/mL which has no obvious influence on the proliferation of the macrophages is selected in comprehensive consideration-1Monitoring the limit dosage as the administration concentration;
step 4, determining the bioactivity limit of the Qingjin Huatan decoction water extract freeze-dried powder based on the secretion function of RAW264.7 macrophages;
step A1, testing the influence of the freeze-dried powder of the water extract of the Qingjin Huatan decoction on the content of IL-1 beta and IL-6 secreted by LPS-induced RAW264.7 macrophage
Taking RAW264.7 macrophage in logarithmic growth phase, adjusting cell suspension concentration to 3 × 105one/mL, inoculated in a 96-well plate at 100. mu.L/well, placed at 37 ℃ with 5% CO2After being cultured in an incubator in a sticking way overnight, respectively comprising a control group, a model group and a freeze-dried powder administration group of the water extract of the Qingjin Huatan Tang with the batch of 19112607, wherein the model group and the Qingjin Huatan Tang are added into the administration groups with the final mass concentration of 1 mu g/mL-1The final mass concentrations of the LPS and each concentration gradient drug of (1) are respectively 62.5, 125, 250 and 500 mug. multidot.mL-16 multiple wells in each group, after administration, 5% CO at 37 deg.C2Culturing in an incubator for 24h, collecting supernatant, detecting the content of IL-1 beta and IL-6, and calculating the inhibition rate after administration (%) (model group content-administration group content)/model group content × 100%; the test results are shown in table 1;
TABLE 1 influence of QINGJINHUATANG on IL-1 beta and IL-6 content in RAW264.7 macrophage: (
n＝3)
Note: p <0.05, P < 0.01, compared to the blank control group; compared with LPS model group, # P <0.05, # P < 0.01
As is clear from the results of the experiments in Table 1, the final mass concentrations were 62.5, 125, 250 and 500. mu.g/mL-1The 19112607 Qingjin Huatan Tang water extract freeze-dried powder of 1 microgram mL-1The content of IL-1 beta and IL-6 secreted by RAW264.7 macrophage induced by LPS shows obvious inhibition effect, and shows obvious dose-effect relationship to the secretion of IL-6, therefore, IL-6 is selected as a quality control index, and biological limit value is searched;
step A2, determining the limit dosage of the clear gold phlegm-resolving decoction water extract freeze-dried powder
Taking RAW264.7 macrophage in logarithmic growth phase, adjusting cell suspension concentration to 3 × 105one/mL, inoculated in a 96-well plate at 100. mu.L/well, placed at 37 ℃ with 5% CO2After being cultured in an incubator in a sticking way overnight, the materials are divided into a control group, a model group and freeze-dried powder groups with the batches of 19112607, 19112608 and 19112609 QINGJINHUATANG water extract, wherein the model group and each administration group of QINGJINHUATANG are added with the final mass concentration of 1 mug/mL-1The final mass concentration of the LPS and the drugs in each batch is 500 mug. multidot.mL-16 multiple wells in each group, after administration, 5% CO at 37 deg.C2Culturing in an incubator for 24h, collecting the supernatant, detecting the content of IL-6, and calculating the inhibition rate (%) after administration (model group content-administration group content)/model group content × 100%, with the test results shown in table 2;
TABLE 2 influence of lyophilized powder of QINGJINHUATAN decoction on the content of IL-6 secreted by LPS-induced RAW264.7 macrophage (see below)
n＝3)
Note: p <0.05, P < 0.01, compared to the blank control group; compared with LPS model group, # P <0.05, # P < 0.01
As can be seen from Table 2, the final mass concentration of the freeze-dried powder of the Qingjin Huatan Tang aqueous extract is 500 mug/mL-1In case of three lots of 19112607, 19112608 and 19112609, the amount of each lot is 1. mu.g.mL-1The content of IL-6 secreted by RAW264.7 macrophage induced by LPS shows obvious inhibition, and the inhibition rates are 68.80%, 63.90% and 63.79% respectively;
step A3, performing repeatability and method suitability verification
Repeatability: respectively taking the final mass concentration of 500 mu g/mL-119112607, 19112608 and 19112609 three batches of clear-gold phlegm-reducing decoction water extract freeze-dried powder to make the final mass concentration be 500 mug/mL-1Then, the amount of the reaction solution was measured for 1. mu.g.mL-1The inhibition rate of LPS induced RAW264.7 macrophage secretion IL-6 is repeated for 3 times, the results are shown in Table 3,
TABLE 3 influence of lyophilized powder of QINGJINHUATAN decoction on the content of IL-6 secreted by LPS-induced RAW264.7 macrophage (see below)
n＝3)
| Group of | Final mass concentration (. mu.g.mL)-1) | Inhibition ratio (%) | RSD(％) |
| LPS | 1 | - | - |
| Batch 19112607 | 500 | 52.16±2.93 | 5.62 |
| Batch 19112608 | 500 | 49.97±1.82 | 3.65 |
| Batch 19112609 | 500 | 46.37±1.14 | 2.46 |
The result of multiple tests of 19112607 batches of preparations is integrated, 125 mug. multidot.mL-1The concentration can show obvious effect of inhibiting IL-6 secretion of RAW264.7 macrophage (P)<0.01); referring to the variation degree of 15% between ELISA test board and board, and integrating the dose limit monitoring and repeatability test results of 3 batches of preparations, the mass concentration of lyophilized powder of Qingjin Huatan Tang lyophilized powder decoction is 500 mug. multidot.mL-1The average inhibition rate of IL-6 secretion of RAW264.7 macrophage is 56.02%, the 90% confidence interval is 52.22-59.82%, the RSD value range of the test is referred, and 80% of the average inhibition rate is set as the quality control standard, namely 500 mug.mL-1The liquid medicine shows that the quality is better when the liquid medicine inhibits RAW264.7 macrophage from secreting IL-6 to 45 percent, and the results show that the three batches are 1 mu g/mL-1The content of IL-6 secreted by RAW264.7 macrophage induced by LPS has obvious inhibition effect, the inhibition rate is 45.32-55.09%, the RSD value is less than 10%, and the repeatability is good;
the method has the following applicability: taking 19112607, 19112610 two batches of QINGJINHUATANG water extract lyophilized powder to make final mass concentration be 500 μ g/mL-1，1μg·mL-1The inhibition rate of LPS for inducing RAW264.7 macrophage to secrete IL-6 is repeated for 3 times, the test results are shown in Table 4,
TABLE 4 influence of lyophilized powder of QINGJINHUATAN decoction on the content of IL-6 secreted by LPS-induced RAW264.7 macrophage (see below)
n＝3)
Note: p <0.05, P < 0.01 compared to blank; compared with the model group, # P <0.05, # P < 0.01
As can be seen from Table 4, batches 19112606 and 19112610 are for 1. mu.g.mL-1The inhibition rates of IL-6 secretion of RAW264.7 macrophage induced by LPS are 66.66% and 59.47%, and the judgment is qualified;
step 5, determining the bioactivity limit of the Qingjin Huatan decoction water extract freeze-dried powder based on the phagocytic function of RAW264.7 macrophages;
step B1, determination of limit dosage of the clear gold phlegm-resolving decoction water extract freeze-dried powder
Taking RAW264.7 macrophage in logarithmic growth phase, adjusting cell suspension concentration to 3 × 105one/mL, inoculated in a 96-well plate at 100. mu.L/well, placed at 37 ℃ with 5% CO2Culturing in incubator for 3 hr, and separating into control group, LPS group, 19112607, 19112608 and 19112609 lyophilized powder with final mass concentration of 500 μ g/mL-1At least 3 multiple wells in each group, after administration, at 37 deg.C with 5% CO2Culturing for 24h in an incubator, discarding the supernatant, adding 100 muL of neutral red staining solution into each well, incubating for 1h, discarding neutral red, washing with PBS once, 200 muL/well each time, adding 100 muL/well of prepared cell lysate, shaking with a micro-shaking apparatus for 15min, standing for 2h, detecting the absorbance at 562nm, and calculating the phagocytosis index of each group, wherein the phagocytosis index (%) -is the OD value of the drug group/the OD value of the control group multiplied by 100%; the results of the measurement are shown in Table 5,
TABLE 5 Effect of the lyophilized powder of the decoction for eliminating phlegm and LPS on the phagocytic function of macrophages (
n＝3)
| Group of | Mass concentration (. mu.g.mL)-1) | Phagocytosis index (%) |
| Control group | - | 100.00 |
| LPS | 0.25 | 124.03±1.81 |
| Batch 19112607 | 500 | 116.20±5.27 |
| Batch 19112608 | 500 | 117.63±4.41 |
| Batch 19112609 | 500 | 116.16±0.12 |
As is clear from Table 5, the final mass concentration was 500. mu.g/mL-119112607, 19112608 and 19112609 of the freeze-dried powder of the Qingjin Huatan decoction aqueous extract and 0.25 mug/mL-1Macrophage by LPS on RAW264.7The phagocytic function of the cells shows obvious promotion effect, and the mass concentration of the clear golden sputum-reducing decoction water extract freeze-dried powder is 500 mug.mL when the experimental results are combined-1The composition has obvious promotion effect on the phagocytic function of macrophages, and the phagocytic index is larger than 111%.
Step B2, repeatability monitoring
19112607 groups of QINGJINHUATAN decoction water extract lyophilized powder solution with final mass concentration of 500 μ g/mL-1And the effect on the phagocytic index of RAW264.7 macrophages is determined, the experiment is repeated for 6 times, the experimental result is shown in Table 6,
TABLE 6 Effect of the lyophilized powder of QINGJINHUATAN decoction on phagocytic function of macrophages (
n＝6)
| Group of | Mass concentration (. mu.g.mL)-1) | Phagocytosis index (%) | RSD/％ |
| Control group | - | 100.00 | - |
| Batch 19112607 | 500 | 124.77±4.06 | 3.25 |
The results show that the phagocytosis index is 124.77% + -4.06%, the RSD value is 3.25%, and the repeatability is good.
Step B3, intermediate precision monitoring
500 mu g/mL is adopted by different experimenters in the same laboratory-1The results of the freeze-dried powder of the Qingjin Huatan Tang aqueous extract acting on RAW264.7 macrophage are shown in Table 7,
TABLE 7 Effect of QINGJINHUATAN decoction water extract lyophilized powder on phagocytic function of RAW264.7 macrophage (
n＝3)
As can be seen from Table 7, the phagocytosis index is 126.00% +/-6.32%, and the RSD value is 5.02%, indicating that the method has better precision;
step B4, method suitability monitoring
After 19112610 batches acted on RAW264.7 macrophages according to the method of step B1, their phagocytic index was determined and the results are shown in table 8,
TABLE 8 Effect of QINGJINHUATAN decoction water extract lyophilized powder on phagocytic function of RAW264.7 macrophage: (
n＝3)
| Group of | Mass concentration (. mu.g.mL)-1) | Phagocytosis index% | RSD％ |
| Control group | - | 100 | - |
| Batch 19112610 | 500 | 123.18±2.86 | 2.32 |
As can be seen from table 8, the phagocytic index of 19112610 batch acted on RAW264.7 macrophage was 123.18% ± 2.86%, and RSD value was 2.32%, which was judged to be qualified;
and 6, data processing, namely carrying out statistical processing on experimental data by using IBM SPSS Statistics19.0 statistical software, wherein single-factor variance analysis is adopted for comparison among groups, LSD (least squares) test is adopted for comparison among groups, and P <0.05 has statistical significance.
In summary, the mass concentration of the clear gold phlegm-resolving decoction water extract freeze-dried powder is more than or equal to 125 mug.mL-1Inhibit LPS-induced IL-6 secretion from RAW264.7 cells, or more than or equal to 500 μ g/mL-1Enhance the phagocytic function of RAW264.7 cells, reach any one of the phagocytic functions and serve as a bioactive quality control index.
The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.
Claims (6)
1. A biological limit value measuring method of Qingjin Huatan Tang is characterized by comprising the following steps:
step 1, preparing a clear gold phlegm-resolving decoction water extract freeze-dried powder solution and an LPS solution, and culturing RAW264.7 macrophages;
step 2, monitoring the relation between the inoculation density of RAW264.7 macrophage and the incubation time;
step 3, monitoring the influence of the clear gold phlegm-resolving decoction water extract freeze-dried powder solution on the activity of RAW264.7 cells;
step 4, determining the bioactivity limit of the Qingjin Huatan decoction water extract freeze-dried powder based on the secretion function of RAW264.7 macrophages;
step 5, determining the bioactivity limit of the Qingjin Huatan decoction water extract freeze-dried powder based on the phagocytic function of RAW264.7 macrophages;
step 6, data processing, namely performing statistical processing on experimental data by utilizing IBMSPSSSstatics 19.0 statistical software;
wherein, the step 4 specifically comprises the following steps:
step A1, testing the influence of the freeze-dried powder of the water extract of the Qingjin Huatan decoction on the content of IL-1 beta and IL-6 secreted by LPS-induced RAW264.7 macrophages;
step A2, determining the limit dosage of the clear gold phlegm-resolving decoction water extract freeze-dried powder;
and step A3, performing repeatability and method suitability verification.
2. The method for determining biological limit of Qing jin Hua Tang according to claim 1, wherein the method of step 2 comprises:
taking RAW264.7 macrophage in logarithmic growth phase, adjusting cell suspension concentration to 1 × 105、3×105、5×105one/mL of the cells were inoculated into a 96-well plate at 100. mu.L/well, and then 100. mu.L of complete medium of LDMEM was added thereto, and the mixture was incubated at 37 ℃ with 5% CO2Culturing in an incubator, adding 20 mu LCCK-8 after culturing for 12h, 24h, 36h, 48h and 60h respectively, and measuring the absorbance value of the mixture under the condition of 450nm of an enzyme labeling instrument after 2 h.
3. The method for determining biological limit of Qing jin Hua Tang according to claim 1, wherein the specific method in step 3 is:
taking RAW264.7 macrophage in logarithmic growth phase, adjusting cell suspension concentration to 3 × 105one/mL, inoculated in a 96-well plate at 100. mu.L/well, placed at 37 ℃ with 5% CO2Culturing in incubator for 3 hr, and dividing into control group and QINGJINHUATANG water extract lyophilized powder group with final mass concentration of 62.5, 125, 250, 500, 1000 μ g/mL-13 multiple wells in each group, after administration, 5% CO at 37 ℃2Culturing in incubator for 24 hr, adding 20 μ LCCK-8 reagent into each well, measuring absorbance at 450nm wavelength of microplate reader after 2 hr, calculating proliferation rate (drug OD value-control OD value)/control OD value × 100%, and repeating the test for 3 times.
4. The method for determining biological limit of Qingjin Huatang Tang as claimed in claim 1, wherein the mass concentration of the lyophilized powder of Qingjin Huatang Tang water extract is 62.5-250 μ g-mL-1The proliferation of RAW264.7 cells is promoted.
5. The method for determining biological limit of Qingjin Huatang Tang according to claim 1, wherein the mass concentration of the lyophilized powder of Qingjin Huatang Tang water extract is not less than 500 μ g-mL-1When the composition is used, the phagocytic function of RAW264.7 cells is enhanced.
6. The method for determining biological limit of Qingjin Huatang Tang according to claim 1, wherein the mass concentration of the lyophilized powder of Qingjin Huatang Tang water extract is not less than 125 μ g-mL-1Inhibit LPS-induced IL-6 secretion from RAW264.7 cells, or more than or equal to 500 μ g/mL-1Enhance the phagocytic function of RAW264.7 cells, reach any one of the phagocytic functions and serve as a bioactive quality control index.
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| CN113984913A (en) * | 2021-09-03 | 2022-01-28 | 中国中医科学院中药研究所 | Biological limit evaluation method based on phosphodiesterase inhibitory activity |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100048885A1 (en) * | 2005-06-14 | 2010-02-25 | Jae-Kwan Hwang | Immunostimulating Polysaccharides Isolated From Curcuma Xanthorrhiza and Manufacturing Method Thereof |
| CN109142701A (en) * | 2018-07-28 | 2019-01-04 | 江西师范大学 | A kind of the anti-inflammatory activity detection method and application of Chinese azalea extract |
| CN110687287A (en) * | 2019-11-12 | 2020-01-14 | 吉林鑫水科技开发有限公司 | Analysis method of deer fetus oligopeptide for immunoregulation of macrophage |
-
2021
- 2021-05-19 CN CN202110547215.5A patent/CN113322298A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100048885A1 (en) * | 2005-06-14 | 2010-02-25 | Jae-Kwan Hwang | Immunostimulating Polysaccharides Isolated From Curcuma Xanthorrhiza and Manufacturing Method Thereof |
| CN109142701A (en) * | 2018-07-28 | 2019-01-04 | 江西师范大学 | A kind of the anti-inflammatory activity detection method and application of Chinese azalea extract |
| CN110687287A (en) * | 2019-11-12 | 2020-01-14 | 吉林鑫水科技开发有限公司 | Analysis method of deer fetus oligopeptide for immunoregulation of macrophage |
Non-Patent Citations (2)
| Title |
|---|
| 孟倩等: "清金化痰汤调节ERK/p38MAPK信号通路改善哮喘大鼠模型气道炎症的研究", 四川中医, vol. 37, no. 10, pages 51 - 55 * |
| 张琼玲等: "基于RAW264.7巨噬细胞吞噬功能的甘草水提物生物限值测定研究", 中国药物警戒, vol. 17, no. 10, pages 659 - 664 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113984913A (en) * | 2021-09-03 | 2022-01-28 | 中国中医科学院中药研究所 | Biological limit evaluation method based on phosphodiesterase inhibitory activity |
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