One, background technology
Baicalin (Baicalin) is a kind of flavone compound that extraction separation comes out from scutellariae,radix, has tangible antifungal activity, and especially to yeast type fungus tool selectively acting, minimum inhibitory concentration (MIC) is between 70-100 μ g/ml.But do not see that baicalin is used to treat cellularity pneumonia report.
Bacterial pneumonia is that institutes such as pneumonia infection diplococcus, staphylococcus, streptococcus, hemophilus influenza, pneumobacillus cause.Bacterial pneumonia accounts for 80% of all kinds of pathogen pneumonia of adult.Because of much more serious the pneumonia state of an illness due to the staphy lococcus infection is, present therapeutic scheme uses oxazacillin (benzylpencilline) and cloxacillin (cloxacillin) usually, alternative the 1st generation, the 2nd generation cephalosporin.But use the maximum shortcoming of antibiotic therapy to be to be prone to produce drug resistance, can make the treatment failure.Therefore need to seek to have no drug resistance and safe and reliable medicine.Baicalin derives from Chinese medicine, and the present invention studies and confirms it to bacterial pneumonia, and especially golden Portugal bacterium pneumonia infection has actively useful effect.
Three, the specific embodiment
1. hemolytic test
The alpha hemolysin of purification is done successive doubling dilution in 96 orifice plates, add 10 in each hole
7Individual rabbit erythrocyte, centrifugal after in 37 ℃, hatching 30h.Make haemoclastic highly diluted multiple be regarded as haemolysis titre (Hemolysis titre).The haemolysis titre such as the following table 1 that do not add alpha hemolysin behind baicalin and the baicalin that adds variable concentrations:
Table 1. baicalin is to the inhibitory action of alpha hemolysin hemolytic activity
Baicalin (μ g/ml) |
Haemolysis titre (Hemolysis titre) |
0 |
2048 |
16 |
32 |
8 |
128 |
4 |
256 |
2 |
1024 |
2. the protection test of people's pulmonary epithelial cells (A549) damage
The gold bacterium 8325-4 of Portugal is cultured to OD in the TSB culture medium
600nm=0.5, get the bacterial cultures of 5ml, centrifugal and be resuspended in the F12K culture medium of 10ml.The A549 cell in F12K culture medium (adding 10% hyclone) in 37 ℃, 5%CO
2Cultivate after 24 hours, with 1.5 * 10
4Individual cells/well is laid in the 96 porocyte culture plates, every hole 100 μ l.Behind the cell attachment, add 100 μ l gold Portugal bacteria suspension, and add the baicalin of variable concentrations, place cell culture incubator in 37 ℃, 5%CO
2Cultivate 8h altogether.Add live (green)/dead (red) reagent, the state of A549 cell is observed in the back under laser confocal microscope (LSCM), and it is green that living cells shows, and dead cell shows redness.
The result shows, people's pulmonary epithelial cells (A549) damage that baicalin can the bacterium alpha hemolysin mediation of protective money Portugal, and this effect presents dose dependent.
3. the experimental therapeutic research of mice gold Portugal bacterium pneumonia
3.1 mice gold Portugal bacterium pneumonia model
(male, 18-22g) behind etherization, per nasal gives 30 μ l gold Portugal's bacteria suspension (the golden bacterium 8325-4 of Portugal) to the C57BL/6J mice, and mice lies low until reviving, and sets up the model of mice gold Portugal bacterium pneumonia.The test that causes death is given with 4 * 10
8The golden Portugal bacterium of CFUs, and histopathology is investigated to 2 * 10
8The golden Portugal bacterium of CFUs.
3.2 protective rate test
The baicalin of 2h difference subcutaneous injection 100,50 and 25mg/kg (100 μ l) behind the mouse inoculation gold Portugal bacterium, every 6h is administered once.Not administration matched group is given the normal saline with 100 μ l, every group of 30 mices.By after the dosage regimen administration, write down the mouse infection gold bacterium 24h of Portugal respectively, 48h, the mortality rate behind the 72h.
The result shows, after baicalin is handled, significantly reduces the mortality rate (P<0.05) of mice gold Portugal bacterium pneumonia.Like table 2.
Table 2. baicalin is to the influence of mice gold Portugal bacterium pneumonia mortality rate