CN109925342B - New application of phlegm-heat clearing medicine in preparation of brain glioma treatment medicine - Google Patents
New application of phlegm-heat clearing medicine in preparation of brain glioma treatment medicine Download PDFInfo
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- CN109925342B CN109925342B CN201711372132.7A CN201711372132A CN109925342B CN 109925342 B CN109925342 B CN 109925342B CN 201711372132 A CN201711372132 A CN 201711372132A CN 109925342 B CN109925342 B CN 109925342B
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Abstract
The invention discloses a new application, in particular to a new application of phlegm-heat clearing in preparing a medicament for treating brain glioma. The invention provides an application of phlegm-heat clearing in preparing a medicament for treating brain glioma, an application of phlegm-heat clearing in preparing a medicament for inhibiting glioma cell proliferation, and an application of phlegm-heat clearing in preparing a medicament for killing glioma cells. The inventor of the invention discovers through experiments that the injection of phlegm-heat-clearing has a remarkable inhibiting effect on glioma cells, and a large number of components (chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, seco-loglycoside, ursodeoxycholic acid, chenodeoxycholic acid, baicalin, chrysin glycoside, wogonoside and oroxylin glycoside) in the phlegm-heat-clearing can penetrate through a blood brain barrier, so that the phlegm-heat-clearing can be used for treating brain glioma.
Description
Technical Field
The invention relates to a new application of phlegm-heat clearing, in particular to a new application of phlegm-heat clearing in preparing a medicament for treating brain glioma.
Background
Brain gliomas are the most common primary craniocerebral tumors arising from brain and spinal glioblastomas canceration. The annual incidence is about 3-8 people per 10 million people. Like other tumors (diseases), gliomas are also caused by the interaction of innate genetic high risk factors and environmental carcinogens. Some known genetic diseases, such as neurofibromatosis (type I) and tuberculous sclerosis, are genetic predisposing factors for brain gliomas. Patients with these diseases have a much higher chance of developing brain gliomas than the general population. In addition, some environmental carcinogens may also be associated with the development of gliomas. Studies have shown that electromagnetic radiation, such as the use of a mobile phone, may be associated with the development of gliomas.
The phlegm-heat clearing injection is one of the common injections in the clinical traditional Chinese medicine, is scientifically prepared from scutellaria baicalensis, bear gall powder, goat horns, honeysuckle, forsythia and the like, and has the effects of clearing heat, removing toxicity, reducing phlegm, relieving spasm, inhibiting bacteria, resisting viruses, relieving fever, regulating immunity and the like. The Chinese medicinal composition can be mainly used for treating respiratory diseases, liver and gall diseases, digestive system diseases and the like in clinic.
Disclosure of Invention
The invention aims to provide a new application of phlegm-heat clearing, in particular to a new application of the phlegm-heat clearing in preparing a medicament for treating brain glioma.
The invention provides application of phlegm-heat clearing in preparation of a medicament for treating brain glioma. The brain glioma may specifically be a brain glioma caused by glioma cells. The glioma cell may be a brain glioma cell. The glioma cell may be a human glioma cell. The glioma cell may specifically be a U87 cell.
The invention also provides a medicament for treating brain glioma, and the active ingredient of the medicament is phlegm heat clearing. The brain glioma may specifically be a brain glioma caused by glioma cells. The glioma cell may be a brain glioma cell. The glioma cell may be a human glioma cell. The glioma cell may specifically be a U87 cell.
The invention also protects the application of the phlegm heat clearing medicament in preparing the medicament for inhibiting the proliferation of glioma cells. The glioma cell may be a brain glioma cell. The glioma cell may be a human glioma cell. The glioma cell may specifically be a U87 cell.
The invention also provides a medicament for inhibiting glioma cell proliferation, and the active ingredient of the medicament is phlegm heat clearing. The glioma cell may be a brain glioma cell. The glioma cell may be a human glioma cell. The glioma cell may specifically be a U87 cell.
The invention also protects the application of the phlegm heat clearing medicament in preparing the medicament for killing the glioma cells. The glioma cell may be a brain glioma cell. The glioma cell may be a human glioma cell. The glioma cell may specifically be a U87 cell.
The invention also provides a medicament for killing glioma cells, and the active ingredient of the medicament is phlegm heat clearing. The glioma cell may be a brain glioma cell. The glioma cell may be a human glioma cell. The glioma cell may specifically be a U87 cell.
The invention also protects the application of the brain-entering component of the phlegm-heat clearing medicine in preparing the medicine for treating the brain glioma. The brain glioma may specifically be a brain glioma caused by glioma cells. The glioma cell may be a brain glioma cell. The glioma cell may be a human glioma cell. The glioma cell may specifically be a U87 cell.
The invention also provides a medicament for treating brain glioma, and the active ingredient of the medicament is the brain-entering ingredient of the phlegm-heat clearing medicament. The brain glioma may specifically be a brain glioma caused by glioma cells. The glioma cell may be a brain glioma cell. The glioma cell may be a human glioma cell. The glioma cell may specifically be a U87 cell.
The invention also protects the application of the brain-entering component of the phlegm-heat clearing medicine in preparing the medicine; the drug is a drug with the functions of inhibiting glioma cell proliferation and/or killing glioma cells. The glioma cell may be a brain glioma cell. The glioma cell may be a human glioma cell. The glioma cell may specifically be a U87 cell.
The invention also protects a medicament, the active ingredient of which is the brain-entering ingredient of the phlegm-heat clearing medicine; the drug is a drug with the functions of inhibiting glioma cell proliferation and/or killing glioma cells. The glioma cell may be a brain glioma cell. The glioma cell may be a human glioma cell. The glioma cell may specifically be a U87 cell.
Any one of the above phlegm-heat-clearing drugs can be specifically a phlegm-heat-clearing injection, such as a phlegm-heat-clearing injection produced by Shanghai Kaibao pharmaceutical industry Co., Ltd.
Any one of the brain-entering components of the phlegm-heat-clearing medicine comprises the following 10 components: chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, seco-oxysaponin, ursodeoxycholic acid, chenodeoxycholic acid, baicalin, chrysin, wogonoside and oroxylin. Any one of the above-mentioned phlegm-heat-clearing brain-entering components is 10 kinds as follows: chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, seco-oxysaponin, ursodeoxycholic acid, chenodeoxycholic acid, baicalin, chrysin, wogonoside and oroxylin.
The inventor of the invention discovers through experiments that the injection of phlegm-heat-clearing has obvious inhibiting effect on glioma cells, and a large amount of components in the phlegm-heat-clearing can permeate blood brain barriers, so that the phlegm-heat-clearing can be used for treating brain glioma.
Drawings
FIG. 1 shows the results of the first test in example 1.
FIG. 2 shows the results of the second test in example 1.
FIG. 3 shows the results of a third test in example 1.
FIG. 4 is a graph of the detection of chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid in example 2.
FIG. 5 is a graph of the detection of loganin oxide in example 2.
FIG. 6 is a graph showing the detection of ursodeoxycholic acid and chenodeoxycholic acid in example 2.
FIG. 7 is a map of detection of baicalin in example 2.
FIG. 8 is a map of the detection of chrysin glycoside in example 2.
FIG. 9 is a graph showing the detection of wogonoside and oroxylin in example 2.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. Tanreqing injection (batch No. 1606122), Shanghai Kaibao pharmaceutical industry Co., Ltd.
Example 1 Effect of phlegm Heat clearing on glioma cell proliferation
U87 cells (human glioma cell line): the cell resource center of the Chinese medical academy of sciences.
The cell concentrations in the examples were obtained by counting live cells using a cell counting plate.
The cell culture solutions in the examples were MEM/EBSS culture solutions containing 10% (by volume) fetal bovine serum.
The cells in the examples were cultured at 37 ℃ in the presence of 5% (by volume) CO2In an incubator according to (1).
1. Taking U87 cells, firstly carrying out pancreatin digestion, then suspending in cell culture solution to obtain the cell concentration of 5 × 104Cell suspension per ml.
2. A96-well plate was taken, 100. mu.l of the cell suspension prepared in step 1 was added to each well, cultured for 24 hours, and then the supernatant was aspirated.
3. After completion of step 2, the 96-well plate was taken, 100. mu.l of the cell treatment solution was added to each well, cultured for 24 hours, and then the supernatant was aspirated.
The cell treatment fluid consists of cell culture fluid and sputum heat clearing injection, and the concentration of the sputum heat clearing injection is respectively set as: each milliliter of the cell treatment solution contains 32 mul, 16 mul, 8 mul, 4 mul or 2 mul of the injection solution of the phlegmathermal clear. Four replicate wells were set for each concentration. Controls were set without the addition of the TZ injection (i.e., 0. mu.l TZ per ml of cell treatment fluid).
4. After completion of step 3, the 96-well plate was taken, and 100. mu.l of fresh cell culture solution and 10. mu.l of CCK-8 reagent were added to each well and cultured for 45 min.
5. And (4) after the step 4 is finished, taking the 96-pore plate, measuring the absorbance of each pore under the wavelength of 450nm of an enzyme labeling instrument, and calculating the cell survival rate.
Cell viability ═ absorbance of test wells ÷ absorbance of control wells × 100%.
Three replicates were performed.
The results of the first test are shown in table 1 and figure 1 (note: compared to the control,*P<0.05，**P<0.01)。
TABLE 1
| Content of phlegm-heat clearing injection in per milliliter of cell treatment fluid | 0μl | 2μl | 4μl | 8μl | 16μl | 32μl |
| Average value of cell survival (%) | 100 | 89.09 | 79.58 | 68.68 | 35.68 | 18.52 |
| Standard deviation of | 4.24 | 6.42 | 9.9 | 5.21 | 6.766 | 0.4 |
The results of the second test are shown in table 2 and figure 2 (note: compared to the control,*P<0.05，**P<0.01)。
TABLE 2
| Content of phlegm-heat clearing injection in per milliliter of cell treatment fluid | 0μl | 2μl | 4μl | 8μl | 16μl | 32μl |
| Average value of cell survival (%) | 100 | 86.87 | 79.25 | 69.22 | 32.58 | 15.42 |
| Standard deviation of | 3.32 | 1.65 | 9.26 | 6.58 | 5.50 | 5.045 |
The results of the third trial are shown in table 3 and figure 3 (note: compared to the control,*P<0.05，**P<0.01)。
TABLE 3
| Content of phlegm-heat clearing injection in per milliliter of cell treatment fluid | 0μl | 2μl | 4μl | 8μl | 16μl | 32μl |
| Average value of cell survival (%) | 100 | 88.06 | 84.32 | 64.30 | 31.28 | 16.49 |
| Standard deviation of | 12.08 | 12.56 | 18.17 | 3.54 | 6.94 | 3.66 |
The result shows that the injection can obviously inhibit the proliferation of U87 cells, kill U87 cells and have a dosage effect.
Example 2 test of phleclearing across the blood brain Barrier
The rats used in the examples were male SD rats, SPF grade, 6-7 weeks old, purchased from experimental animal technology ltd, viton, beijing, license number: SCXK (Kyoto) 2016-. Rats were housed in IVC cages (free-standing cages, 461 × 274 × 229mm long × height, Tecniplast, italy), 3 per cage, maintaining the laboratory ambient temperature at 22 ℃ and humidity at about 50%. Adaptive feeding was carried out for 20 days before the test.
10% chloral hydrate (chloral hydrate): national pharmaceutical group chemical reagent, ltd, lot number 20130201.
The blood collection tubes used in the examples were CBAS-C30 heparinized blood collection tubes (CBAS-C30Heparin-coated polyurethane reagents): solomon scientific, USA.
First, maximum tolerated dose test
1. Femoral vein intubation operation
Rats were fasted for 12-16h before testing and had free access to water. After weighing the rats, the rats were anesthetized with 10% chloral hydrate by intraperitoneal injection, then fixed in the supine position, the right inguinal part was cut, sterilized with iodine, deiodinated with 75% alcohol, then a small opening of about 1.5cm in length was cut, connective tissue, muscle and nerves were separated bluntly to expose the femoral vein to the visual field, then a small opening was cut, a venous indwelling catheter was carefully inserted, and ligation was performed. The venous indwelling catheter was pulled out from the back of the rat and through the rat waistcoat, and the IVC cage was raised in a single cage overnight.
2. Maximum tolerated dose test
Rats were divided into 3 groups of 5 rats each, and the second day after surgery for femoral vein cannulation, the maximum tolerated dose test was performed. In the maximum tolerated dose test, administration was via a venous indwelling catheter inserted into the femoral vein.
The first group of rats was administered multiple times, each time within 1min, and the interval between two times was 5 min. The dosage of the first administration of the injection of the phlegm heat clearing is 5mL/kg of body weight, and then the dosage of each administration of the injection of the phlegm heat clearing is 2.5mL/kg until the cumulative total dosage of the injection of the phlegm heat clearing reaches 20mL/kg of body weight. On constant observation, each rat was silent and did not die within 4h after the last dose.
The second group of rats, which were administered multiple times, each time within 1min, with 5min intervals between each two administrations. The dosage of the first administration of the injection of phlegm heat clearing is 10mL/kg of body weight, and the dosage of each subsequent administration of the injection of phlegm heat clearing is 2.5 mL/kg. When the accumulated total dose of the phlegm-heat clearing injection reaches 15mL/kg body weight, the rats have symptoms of foot-back arch, convulsion, paralysis, trembling of the tail root, face scratching and the like, but do not die within 4 hours.
The third group of rats was administered a single injection of sputum fever reducing agent at a dose of 15mL/kg body weight within 1 min. After the administration, the rats have symptoms of foot-back reverse bow, convulsion, paralysis, trembling of tail root, etc., but the rats recover to normal after 2-3min, and do not die within 4 h.
According to the conditions of reaction, non-death and the like of rats in the maximum tolerated dose test, the administration dose of the phlereqing injection is determined at 15mL/kg body weight.
Second, test for phlegm-heat clearing penetrating blood brain barrier
1. Femoral vein intubation operation
The same as step 1.
2. Grouping and administering drugs
Rats were divided into 4 groups of 4-6 rats each, and the test was performed the next day after the femoral vein cannulation surgery.
The doses administered in the second to fourth groups were all 15mL/kg body weight, and were all single administration, which was performed through a venous indwelling catheter inserted into the femoral vein.
The first group of rats was a blank control group, which was killed by blood sampling from abdominal aorta (centrifugation at 20 ℃ C. and 2500r/min for 15min, plasma sampling) without drug administration and anesthesia. Stripping the muscles of the head pillow part, exposing the macropore of the occiput, and extracting clean cerebrospinal fluid.
The second group of rats was anesthetized 15min after administration and killed by blood sampling from the abdominal aorta (centrifugation at 20 ℃ for 15min at 2500r/min, plasma was sampled). Stripping the muscles of the head pillow part, exposing the macropore of the occiput, and extracting clean cerebrospinal fluid.
The rats in the third group were anesthetized 30min after administration, and were killed by blood sampling from the abdominal aorta (centrifugation at 20 ℃ for 15min at 2500r/min, plasma was sampled). Stripping the muscles of the head pillow part, exposing the macropore of the occiput, and extracting clean cerebrospinal fluid.
The rats in the fourth group were anesthetized 60min after administration, and were killed by blood sampling from the abdominal aorta (centrifugation at 20 ℃ for 15min at 2500r/min, plasma was sampled). Stripping the muscles of the head pillow part, exposing the macropore of the occiput, and extracting clean cerebrospinal fluid.
3. Detection of cerebrospinal fluid samples (Studies of phlegm-heat clearing injections into brain)
Chlorogenic acid standard, ursodeoxycholic acid standard, chenodeoxycholic acid standard and wogonoside standard were purchased from the institute of food and drug testing. The secoisolaricoside standard, chrysin standard and oroxylin standard were purchased from Doctorbai Biotech Co., Ltd. The cryptochlorogenic acid standard, the neochlorogenic acid standard and the baicalin standard are purchased from Shanghai-sourced leaf Biotechnology GmbH.
And (4) taking the cerebrospinal fluid obtained in the step (2) for LC-MS detection.
Agilent RRLC liquid chromatograph and Agilent6320Trap MS mass spectrum were used.
The preparation method of the cerebrospinal fluid sample comprises the following steps: taking 100 mu l of cerebrospinal fluid, precipitating with 3 times volume of chromatographic pure methanol, swirling, centrifuging, sucking supernatant, blowing dry with nitrogen flow, dissolving with 50 mu l of 50% methanol, swirling, and centrifuging.
The injection for clearing phlegm heat is diluted to 100 times of volume by 50% methanol and used as a TRQ injection sample.
Liquid phase conditions chromatography column Agilent Zorbax SB C18(4.6 mm. times.150 mm,5 μm). The mobile phase is liquid A or liquid B or the mixed liquid of liquid A and liquid B. The solution A is an aqueous solution containing 0.1% (volume ratio) of formic acid. And the liquid B is obtained by mixing 9 parts by volume of acetonitrile and 1 part by volume of methanol. Gradient elution process: the liquid B accounts for 5 percent of the volume fraction of the mobile phase within 0 to 5 min; the volume fraction of the liquid B in the mobile phase is linearly increased from 5% to 90% in 5-45 min; and (4) 45-55min, wherein the liquid B accounts for 90% of the volume fraction of the mobile phase. The flow rate of the mobile phase is 1.0 mL/min-1And 4:1 flow division after the column, wherein the flow velocity of the gas entering the mass spectrum is 0.2 mL/min-1. The amount of sample was 10. mu.L. The column temperature was 30 ℃.
Mass spectrum conditions comprise ESI ion source and positive ion detection. The ion source conditions were a drying gas temperature (gas temp) of 350 deg.C and a drying gas flow rate of 10L min-1Atomizer pressure 40psi, capillary voltage (capillary) 4000V. Scanning range: m/z is 50 to 1000.
The chromatogram for detecting chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid is shown in figure 4. The detected spectrum of secologanin is shown in FIG. 5. The detected patterns of ursodeoxycholic acid and chenodeoxycholic acid are shown in figure 6. The detection spectrum of baicalin is shown in FIG. 7. The map for detecting chrysin glycoside is shown in figure 8. The map for detecting wogonoside and oroxylin is shown in FIG. 9.
The results of the detection of chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, secologlycoside, ursodeoxycholic acid, chenodeoxycholic acid, baicalin, chrysin, wogonoside, oroxylin are shown in Table 4.
The results show that 10 components in the phlegm-heat-clearing injection can penetrate through a blood brain barrier and enter cerebrospinal fluid, and the 10 components are respectively chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid, secologlycoside, ursodeoxycholic acid, chenodeoxycholic acid, baicalin, chrysin glycoside, wogonoside and orophyllin glycoside.
TABLE 4
| Chromatographic peak | Retention time (min) | Excimer | Ingredient identification | |
| 1 | 4.6 | 353[M-H]- | |
|
| 2 | 6.0 | 353[M-H]- | Cryptochlorogenic acid | |
| 3 | 9.5 | 353[M-H]- | Novel chlorogenic acid | |
| 4 | 12.2 | 403[M-H]- | Secoisolaricoside | |
| 5 | 16.8 | 445[M-H]- | Baicalin | |
| 6 | 17.8 | 429[M-H]- | Chrysin glycoside | |
| 7 | 18.1 | 459[M-H]+ | |
|
| 8 | 18.5 | 459[M-H]- | Oroxylin | |
| 9 | 28.6 | 391[M-H]- | |
|
| 10 | 33.7 | 391[M-H]- | Chenodeoxycholic acid |
Claims (3)
1. The application of the phlegm-heat clearing injection in preparing the medicine for treating the brain glioma.
2. The application of the phlegm-heat clearing injection in preparing the medicine for inhibiting the proliferation of glioma cells.
3. The application of the phlegm-heat clearing injection in preparing the medicine for killing glioma cells.
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Non-Patent Citations (4)
| Title |
|---|
| 汉黄芩素对胶质瘤U87 细胞增殖和侵袭的抑制作用及机制;曾志青等;《细胞与分子免疫学杂志》;20151231;第31卷(第3期);第302-306页 * |
| 综合性医院3年94例中药注射剂不良反应报告分析;柳青等;《中成药》;20091031;第31卷(第10期);第1605-1607页 * |
| 黄芩及黄酮类成分防治肿瘤作用的研究进展;王晓瑜;《华西医学》;20110228;第26卷(第2期);第297-300页 * |
| 黄芩的化学成分与药理作用研究进展;郑勇凤等;《中成药》;20160131;第38卷(第1期);第141-147页 * |
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