CN101095707B - Use of bupleurum root total polyoses in the preparing of medicine for preventing and treating acute respiratory distress syndrome - Google Patents
Use of bupleurum root total polyoses in the preparing of medicine for preventing and treating acute respiratory distress syndrome Download PDFInfo
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- CN101095707B CN101095707B CN2006101460243A CN200610146024A CN101095707B CN 101095707 B CN101095707 B CN 101095707B CN 2006101460243 A CN2006101460243 A CN 2006101460243A CN 200610146024 A CN200610146024 A CN 200610146024A CN 101095707 B CN101095707 B CN 101095707B
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Abstract
The invention belong to Chinese traditional medicine field, relates to the application of bupleurum root polysaccharide for preparing medicine preventing and treating acute respiratory distress syndrome. The invention separates polysaccharide extract from bupleurum root, the average productivity is 3.32%, and the polysaccharide content is more than 68.2%-78.6%. It is showed by whole animal model test that the protective effect for lung injury caused by acute respiratory distress syndrome is distinctive, and it is proven that polysaccharide can prevent acute respiratory distress syndrome. The bupleurum root polysaccharide can also be used to produce medicine preventing and treating fowl influenza.
Description
Technical field
The invention belongs to the field of Chinese medicines, relate to the new pharmaceutical usage of bupleurum Chinese total polysaccharides.Be specifically related to the purposes of bupleurum root total polyoses in preparation treats acute respiratory distress medicine.
Background technology:
Adult respiratory distress syndrome (acute respiratory distress syndrome, ARDS) be meant acute, the respiratory failure of carrying out property anoxia that the inside and outside paathogenic factor of heart source property various lungs is in addition caused, its clinical characters is acute respiratory distress and refractory hypoxemia, be one of comparatively common clinically critical, serious symptom, case fatality rate is very high.Davidson TA (Davidson TA, Caldwell ES, et al.JAMA, 1999,281:354.) wait and to be reported in over the mortality rate of 10 years ARDS up to 30%-40%.Serious symptom atypical pneumonia (SARS) patient also respiratory distress can occur, and pulmonary's pathological change is similar to ARDS, mainly shows as the damage of alveolar.The concurrent ARDS mortality rate of SARS is high.Glucocorticoid, antibiotic, anti-cytokine therapy medicine etc. often are used to the treatment of ARDS, but up to the present still do not have a kind of specific medicament.Studies show that the immunity infringement is another important pathogenesis of ARDS.And the generation of the excessive activation of complement system and ARDS and prognosis have significantly and get in touch.Suppress complement constituent by specificity and activate the clinical efficacy that may improve treatment ARDS.Therefore the complement inhibitor of being badly in need of high-efficiency low-toxicity comes treats acute respiratory distress.
Radix Bupleuri is conventional Chinese medicine, is the root of Umbelliferae Bupleurum (Bupleurum) various plants, have evacuate bring down a fever, the effect of soothing liver-QI, yang invigorating, cure mainly diseases such as cold, fever, alternate attack of chill and fever, malaria, sternal rib pain, menoxenia and uterine prolapse.The chemical constituent of Radix Bupleuri is quite complicated, up to now, has reported and has contained Saponin, volatile oil, flavone, sterol, coumarin, saccharide, lignin, organic acid, polyacetylene and alkaloid etc.Effects such as that modern pharmacological research proof Radix Bupleuri has is analgesic, analgesia, calmness, antiinflammatory, immunomodulating, antibiotic, antiviral and antitumor.
Document (Zhang Xingquan, Chen Hongshan. Chinese J Pharmacol Toxicol, 1989,3 (1): 31.) report Radix Bupleuri (Bupleurum chinense DC) polysaccharide can significantly increase the spleen coefficient; Improve macrophage and natural killer cell function; Can improve the virus-specific antibodies titre; Can obviously increase lymhocyte transformation rate and SDH, show immunologic enhancement.From Radix Bupeuri Scorzonerfolii. (Bupleurum scorzonerifolium Willd) isolated flavone and saponin show good immunosuppressive activity (Wen JC, Li WC, Jenn HL, et al.Phytochmistry, 2003,64:1375.).Hemolytic reaction due to the complement system classical pathway that the Radix Bupleuri polysaccharide IIb that tells can suppress from other Radix Bupleuri (Bupleurum falcatum L) activates shows immunosuppressive action (Yamada H, Ra KS, Kiyohara H, et al.Carbohydr Res, 1989,189:209.).But do not relate to effect to adult respiratory distress syndrome.
Take a broad view of domestic and international research, there is no the treats acute respiratory distress drug effect and the medicine thereof of report bupleurum root total polyoses.
Summary of the invention
The purpose of this invention is to provide bupleurum root total polyoses in preparation treats acute respiratory distress medicine new pharmaceutical usage and the preparation method of bupleurum root total polyoses.
The present invention's separation and Extraction from bupleurum Chinese obtains total polysaccharide extractive.Described total polysaccharide extractive confirms to have strong anticomplementary activity through experiment in vitro, and the whole animal model test confirms that it has the experimental adult respiratory distress syndrome effect of very strong control.
Bupleurum Chinese of the present invention comprises Radix Bupleuri (Bupleurum chinense DC), Radix Bupeuri Scorzonerfolii. (Bupleurum scorzonerifolium Willd) and Radix Bupleuri parvifolii (Bupleurum smithiiWolff).
Purpose of the present invention realizes by following method:
1, preparation bupleurum root total polyoses:
The Radix Bupleuri medical material extracts with 95% ethanol merceration, filters, and medicinal residues dry in room temperature underlying ventilation, use hot water extraction then 3 times, filter merge extractive liquid,, concentrate, centrifugal, supernatant removes floating preteins with trichloroacetic acid, centrifugal, supernatant water dialysis 3 days, dialysis solution adds ethanol to containing alcohol amount 80% after being concentrated into small size, centrifugal, the precipitation lyophilization promptly gets total polysaccharide extractive, and yield reaches more than 3%, and polyoses content surpasses 65%.
2, bupleurum root total polyoses is used for anticomplementary and treats acute respiratory distress test:
1) anticomplementary activates classical pathway cell hemolytic test
1: 32 diluent of serum is as complement to adopt Cavia porcellus (available from laboratory animal portion of Fudan University), and the antigenic activation classical pathway of complement causes sheep red blood cell haemolysis, and the result shows, records bupleurum root total polyoses haemolysis capable of inhibiting cell.
Radix Bupleuri (Bupleurum chinense DC): IC
50=232 μ g.ml
-1(n=4).
Radix Bupeuri Scorzonerfolii. (Bupleurum scorzonerifolium Willd): IC
50=284 μ g.ml
-1(n=4).
Radix Bupleuri parvifolii (Bupleurum smithii Wolff): IC
50=162 μ g.ml
-1(n=4).
2) anticomplementary activates alternative pathway cell hemolytic test
Adopt 1: 10 diluent of healthy human serum as complement, the activating complement alternative pathway causes rabbit erythrocyte haemolysis, and the result shows, records bupleurum root total polyoses and can suppress haemolysis.
Radix Bupleuri (Bupleurum chinense DC): AP
50=1.336mg.ml
-1(n=4).
Radix Bupeuri Scorzonerfolii. (Bupleurum scorzonerifolium Willd): AP
50=1.156mg.ml
-1(n=4).
Radix Bupleuri parvifolii (Bupleurum smithii Wolff): AP
50=0.925mg.ml
-1(n=4).
3) treats acute respiratory distress in vivo test
Select the SD rat for use,, set up the adult respiratory distress syndrome model relevant with the complement excessive activation with ischemia-reperfusion+endotoxin (LPS) two-hit.The bupleurum root total polyoses gastric infusion.Total complement activity and carbon dioxide indicator in the serum of observation animal.
To test rat and finish the back sacrificed by exsanguination in experiment, and take out lung, paraffin is fixed, section, and microscopically is observed pathological change.After the result confirmed oral bupleurum root total polyoses, the injury of lung that the rat acute respiratory distress syndrome is caused had significant protective effect.
Confirm bupleurum root total polyoses to inhibition all being arranged through in vitro tests, the adult respiratory distress syndrome of rat in the body is had preventive and therapeutic effect at cell haemolysis classical and that the alternative pathway activation is caused.
Table 1 is OD value rate of change (%, X ± SD) (n=5) that the animal serum complement activity is measured.
Table 2 is animal serum CO
2Change rate of concentration (%, X ± SD) (n=5).
Table 1
Wherein, * and ARDS model group compare P＜0.05; * and ARDS model group be P＜0.01 relatively.
Table 2
Wherein, * and ARDS model group compare P＜0.05; * and ARDS model group be P＜0.01 relatively.
Description of drawings
Fig. 1 is the OD value rate of change that ARDS rat blood serum complement activity is measured
Fig. 2 is ARDS rat blood serum CO
2Change rate of concentration
Fig. 3 is an ARDS induced lung slice map.
Fig. 4 is a blank group induced lung slice map
Fig. 5 is pseudo-operation group induced lung slice map.
Fig. 6 is positive control (andrographolide) induced lung slice map.
Fig. 7 is a bupleurum root total polyoses administration group induced lung slice map.
The specific embodiment
Get Radix Bupleuri medical material 100g, extract with 95% ethanol merceration, medicinal residues dry in room temperature underlying ventilation, use hot water extraction then 3 times, filter merge extractive liquid,, concentrate, centrifugal, supernatant removes floating preteins with the S trichloroacetic acid, centrifugal, supernatant water dialysis 3 days, dialysis solution is concentrated into small size and adds ethanol to containing alcohol amount 80%, centrifugal, the precipitation lyophilization promptly gets total polysaccharides, and product yield reaches more than 3%, and polyoses content surpasses 65%.Table 3 is bupleurum root total polyoses yield and content.
Described Radix Bupleuri medical material can be selected from Radix Bupleuri (Bupleurum chinense DC), Radix Bupeuri Scorzonerfolii. (Bupleurumscorzonerifolium Willd) or Radix Bupleuri parvifolii (Bupleurum smithii Wolff).
Table 3
Embodiment 2
With Radix Bupleuri (Bupleurum chinense DC) total polysaccharides (about 3.0mg.mL
-1) usefulness barbitol buffer solution (barbitol buffer solution, BBS) two-fold dilution gets the solution of eight concentration, respectively get each concentration sample of 0.2ml and add 0.2ml complement (1: 32 diluent of guinea pig serum), 0.1ml 2% sheep red blood cell (sheep redblood cell, SRBC) and 0.1ml1: 1000 hemolysins (anti-SRBC serum), 37 ℃ of water-baths of every pipe are placed into low-temperature and high-speed centrifuge 2500G, 4 ℃ after 30 minutes centrifugal, gets every pipe supernatant 0.25mL behind the 20min respectively and measure absorbance down in microplate reader 405nm.Only each concentration sample solution of 0.2ml is mixed with 0.4mlBBS again, be placed into the low-temperature and high-speed centrifuge, get every pipe supernatant 0.25mL and measure absorbance down, as this colour of sample extinction in microplate reader 405nm with 2500G, 4 ℃ of centrifugal 10min.Again the sample titration absorbance of every kind of variable concentrations is deducted this colour of sample extinction under the respective concentration for adding the haemolysis absorbance behind the medicine, and the normal haemolysis pipe of additional complement calculates medicine and suppresses hemolytic half-inhibition concentration (IC as the full haemolysis contrast of system
50) be 232 μ g.ml
-1(n=4).
Embodiment 3
With Radix Bupeuri Scorzonerfolii. (Bupleurum scorzonerifolium Willd) total polysaccharides (about 3.0mg.mL
-1) usefulness barbitol buffer solution (barbitol buffer solution, BBS) two-fold dilution gets the solution of eight concentration, respectively get each concentration sample of 0.2ml and add 0.2ml complement (1: 32 diluent of guinea pig serum), 0.1ml 2% sheep red blood cell (sheep red blood cell, SRBC) and 0.1ml1: 1000 hemolysins (anti-SRBC serum), 37 ℃ of water-baths of every pipe are placed into low-temperature and high-speed centrifuge 2500G, 4 ℃ after 30 minutes centrifugal, gets every pipe supernatant 0.25mL behind the 20min respectively and measure absorbance down in microplate reader 405nm.Only each concentration sample solution of 0.2ml is mixed with 0.4mlBBS again, be placed into the low-temperature and high-speed centrifuge, get every pipe supernatant 0.25mL and measure absorbance down, as this colour of sample extinction in microplate reader 405nm with 2500G, 4 ℃ of centrifugal 10min.Again the sample titration absorbance of every kind of variable concentrations is deducted this colour of sample extinction under the respective concentration for adding the haemolysis absorbance behind the medicine, and the normal haemolysis pipe of additional complement calculates medicine and suppresses hemolytic half-inhibition concentration (IC as the full haemolysis contrast of system
50) be 284 μ g.ml
-1(n=4).
Embodiment 4
With Radix Bupleuri parvifolii (Bupleurum smithii Wolff) total polysaccharides (about 3.0mg.mL
-1) usefulness barbitol buffer solution (barbitol buffer solution, BBS) two-fold dilution gets the solution of eight concentration, respectively get each concentration sample of 0.2ml and add 0.2ml complement (1: 32 diluent of guinea pig serum), 0.1ml 2% sheep red blood cell (sheep red blood cell, SRBC) and 0.1ml1: 1000 hemolysins (anti-SRBC serum), 37 ℃ of water-baths of every pipe are placed into low-temperature and high-speed centrifuge 2500G, 4 ℃ after 30 minutes centrifugal, gets every pipe supernatant 0.25mL behind the 20min respectively and measure absorbance down in microplate reader 405nm.Only each concentration sample solution of 0.2ml is mixed with 0.4mlBBS again, be placed into the low-temperature and high-speed centrifuge, get every pipe supernatant 0.25mL and measure absorbance down, as this colour of sample extinction in microplate reader 405nm with 2500G, 4 ℃ of centrifugal 10min.Again the sample titration absorbance of every kind of variable concentrations is deducted this colour of sample extinction under the respective concentration for adding the haemolysis absorbance behind the medicine, and the normal haemolysis pipe of additional complement calculates medicine and suppresses hemolytic half-inhibition concentration (IC as the full haemolysis contrast of system
50) be 162 μ g.ml
-1(n=4).
Embodiment 5
With Radix Bupleuri (Bupleurum chinense DC) total polysaccharides (about 5.0mg.mL
-1) with containing Mg
2+Gelatin-barbitol buffer solution [GVB/Mg (10mM) EGTA] two-fold dilution get the solution of eight concentration, get each concentration sample of 0.15ml and add 0.15ml complement (1: 10 diluent of human serum), 0.2ml 2% rabbit erythrocyte, 37 ℃ of water-baths of every pipe are placed into low-temperature and high-speed centrifuge 2500G, 4 ℃ after 30 minutes centrifugal, gets every pipe supernatant 0.25mL behind the 20min respectively and measure absorbance down in microplate reader 405nm.Only each concentration sample solution of 0.15ml is mixed with 0.35mlGVB/Mg (10mM) EGTA again, be placed into the low-temperature and high-speed centrifuge with 2500G, 4 ℃ of centrifugal 10min, get every pipe supernatant 0.25mL and measure absorbance down, as this colour of sample extinction in microplate reader 405nm.Again the sample titration absorbance of every kind of variable concentrations is deducted this colour of sample extinction under the respective concentration for adding the haemolysis absorbance behind the medicine, and the normal haemolysis pipe of additional complement calculates medicine and suppresses hemolytic half-inhibition concentration (AP as the full haemolysis contrast of system
50) be 1.336mg.ml
-1(n=4).
Embodiment 6
With Radix Bupeuri Scorzonerfolii. (Bupleurum scorzonerifolium Willd) total polysaccharides (about 5.0mg.mL
-1) with containing Mg
2+Gelatin-barbitol buffer solution [GVB/Mg (10mM) EGTA] two-fold dilution get the solution of eight concentration, get each concentration sample of 0.15ml and add 0.15ml complement (1: 10 diluent of human serum), 0.2ml 2% rabbit erythrocyte, 37 ℃ of water-baths of every pipe are placed into low-temperature and high-speed centrifuge 2500G, 4 ℃ after 30 minutes centrifugal, gets every pipe supernatant 0.25mL behind the 20min respectively and measure absorbance down in microplate reader 405nm.Only each concentration sample solution of 0.15ml is mixed with 0.35ml GVB/Mg (10mM) EGTA again, be placed into the low-temperature and high-speed centrifuge with 2500G, 4 ℃ of centrifugal 10min, get every pipe supernatant 0.25mL and measure absorbance down, as this colour of sample extinction in microplate reader 405nm.Again the sample titration absorbance of every kind of variable concentrations is deducted this colour of sample extinction under the respective concentration for adding the haemolysis absorbance behind the medicine, and the normal haemolysis pipe of additional complement calculates medicine and suppresses hemolytic half-inhibition concentration (AP as the full haemolysis contrast of system
50) be 1.156mg.ml
-1(n=4).
Embodiment 7
With Radix Bupleuri parvifolii (Bupleurum smithii Wolff) total polysaccharides (about 5.0mg.mL
-1) with containing Mg
2 +Gelatin-barbitol buffer solution [GVB/Mg (10mM) EGTA] two-fold dilution get the solution of eight concentration, get each concentration sample of 0.15ml and add 0.15ml complement (1: 10 diluent of human serum), 0.2ml 2% rabbit erythrocyte, 37 ℃ of water-baths of every pipe are placed into low-temperature and high-speed centrifuge 2500G, 4 ℃ after 30 minutes centrifugal, gets every pipe supernatant 0.25mL behind the 20min respectively and measure absorbance down in microplate reader 405nm.Only each concentration sample solution of 0.15ml is mixed with 0.35ml GVB/Mg (10mM) EGTA again, be placed into the low-temperature and high-speed centrifuge with 2500G, 4 ℃ of centrifugal 10min, get every pipe supernatant 0.25mL and measure absorbance down, as this colour of sample extinction in microplate reader 405nm.Again the sample titration absorbance of every kind of variable concentrations is deducted this colour of sample extinction under the respective concentration for adding the haemolysis absorbance behind the medicine, and the normal haemolysis pipe of additional complement calculates medicine and suppresses hemolytic half-inhibition concentration (AP as the full haemolysis contrast of system
50) be 0.925mg.ml
-1(n=4).
Embodiment 8
25 of SD rats (250-300g) are divided into 5 groups (A, B, C, D, E groups) at random.A group is the endotoxin ARDS group of causing injury, and B organizes negative matched group, and C organizes positive matched group, and the D group is pseudo-operative control group, and the E group is Radix Bupleuri polysaccharide group.Except all treated animals of D group are 45 ± 5mmHg from neck arteries blood-letting to mean arterial pressure in 20min, feed back blood behind the shock 1h under the room temperature, in 1h subsequently, feed back all collection blood, give LPS200 μ g.kg then in the trachea
-1E group before giving LPS half an hour gastric infusion, dosage is 10mg/kg; The D group is only implemented operation techniques such as anesthesia and carotid artery intubate.It is identical with the E group that B organizes all the other operations, just substituted the Radix Bupleuri polysaccharide with normal saline, as negative control.C group then before giving LPS half an hour with 70mg/kg lumbar injection andrographolide as positive controls.A-E group is injected (T before the endotoxin respectively in trachea
0) and inject 0.5h (T behind the endotoxin
0.5), 1h (T
1), 1.5h (T
1.5), 2.5h (T
2.5) blood sample collection, measure CO in the blood respectively
2The hemolytic activity of concentration and classical pathway of complement.Give animal sacrificed by exsanguination behind the endotoxin 2.5h, take out lung, paraffin is fixed, section, and microscopically is observed pathological change.The result shows A group and B group blood sample CO
2Concentration presents rising by a relatively large margin, and complement activity descends by a small margin.C, E group blood sample CO
2The concentration ascensional range is less relatively, and complement activity descends significantly.D group blood sample CO
2Concentration and complement activity descend slightly, and be basicly stable.Pathological section is 3.67 by pulmonary lesion situation A group grading mean, and B group grading mean is 2.33, and the C group is 3.33, and the D group is 1.17, and the E group is 2.2.By the result as can be known, the Radix Bupleuri polysaccharide also has the effect that suppresses complement in vivo, and can improve endotoxin to some extent and cause injury and cause the inflammation of anoxia and lungs, and the ARDS of alleviation rat model injury of lung trend is arranged.
Claims (1)
1. the purposes of bupleurum root total polyoses in preparation treats acute respiratory distress medicine, described bupleurum root total polyoses prepares by following method and step:
The Radix Bupleuri medical material extracts with 95% ethanol merceration, filters, and medicinal residues dry in room temperature underlying ventilation, use hot water extraction then 3 times, filter merge extractive liquid,, concentrate, centrifugal, supernatant removes floating preteins with trichloroacetic acid, centrifugal, supernatant water dialysis 3 days, dialysis solution adds ethanol to containing alcohol amount 80% after being concentrated into small size, centrifugal, the precipitation lyophilization promptly gets total polysaccharide extractive, and yield reaches more than 3%, and polyoses content is 68.2-78.6%;
Described Radix Bupleuri medical material is selected from Radix Bupleuri (Bupleurum chinense DC), Radix Bupeuri Scorzonerfolii. (Bupleurumscorzonerifolium Willd) or Radix Bupleuri parvifolii (Bupleurum smithii Wolff).
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| CN105560265B (en) * | 2014-10-13 | 2018-08-14 | 复旦大学 | Purposes of the Bupleurum chinense polysaccharide in the drug for preparing prevention diabetic nephropathy |
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Non-Patent Citations (2)
| Title |
|---|
| Haruki Yamada.Characterization of anti-complementary neutralpolysaccharides from the roots of Bupleurum falcatum.Phytochemistry27 10.1988,27(10),3163-3168. |
| Haruki Yamada.Characterization of anti-complementary neutralpolysaccharides from the roots of Bupleurum falcatum.Phytochemistry27 10.1988,27(10),3163-3168. * |
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