The specific embodiment
The present invention is described further below in conjunction with embodiment.
Solid among each embodiment in the related solid mixture, the liquid in the liquid, and the solid percentage ratio in the liquid is respectively to calculate with wt/wt, vol/vol, wt/vol, except as otherwise noted.
Embodiment 1: the preparation of Herba Hyperici Japonici total flavones
It is an amount of to get the Herba Hyperici Japonici medical material, and cutting decocts three times with 12 times, 10 times, 8 times water gagings respectively, and each 1 hour, filter, filtrate merges, and is evaporated to contain Herba Hyperici Japonici medical material 0.5g/ml; Join on the preprepared polyamide column, it is light at first to be eluted to eluent color color with distilled water, till adding 60% ethanol elution to hydrochloric acid-magnesium powder reaction again and being negative, collect pure washing liquid, being concentrated into relative density is 1.08~1.10(60 ℃) concentrated solution, in inlet temperature is 100~200 ℃, and leaving air temp is that spray drying promptly gets Herba Hyperici Japonici total flavones under 50~150 ℃ the condition.As stated above, select for use five batches of Herba Hyperici Japonici medical materials to extract altogether, obtain five batches of hypericum japonicum total flavone extractions.
Precision takes by weighing Quercitroside reference substance 10mg, puts in the 50ml measuring bottle, adds dissolve with methanol, is diluted to scale, shakes up, and promptly gets (containing Quercitroside 200 μ g among every 1ml).Precision is measured Quercitroside reference substance solution 0.5ml, 1.0ml, 1.5ml, 2.0ml, 2.5ml, put respectively in the 10ml measuring bottle, respectively add methanol to 2.5ml, add 5% sodium nitrite solution 0.4ml, mixing, placed 6 minutes, add 10% aluminum nitrate solution 0.4ml, shake up, placed 6 minutes, hydro-oxidation sodium test solution 4ml, add water to scale again, shake up, placed 15 minutes, do blank with corresponding reagent, according to ultraviolet visible spectrophotometry (V A of Pharmacopoeia of the People's Republic of China version in 2005), measure absorbance at 500nm wavelength place, be vertical coordinate with the absorbance, concentration is abscissa, the drawing standard curve.Get every crowd of about 50mg of aforementioned Herba Hyperici Japonici total flavones, the accurate title, decide, and puts in the 50ml measuring bottle, and it is an amount of to add methanol, and supersound process (power 360W, frequency 35kHz) 10 minutes adds methanol and is diluted to scale, shakes up, as need testing solution.Precision is measured need testing solution 2ml, puts in the 10ml measuring bottle, adds methanol and is diluted to scale, shakes up, precision is measured 2ml, puts in the 10ml measuring bottle, and the method under the sighting target directrix curve preparation is from " adding methanol to 2.5ml ", measure absorbance in accordance with the law, calculate according to standard curve, promptly.Press dry product and calculate, flavonoid content is with Quercitroside (C in five batches of Herba Hyperici Japonici total flavoness
21H
20O
10) meter, measurement result such as table 1:
Table 1: flavonoid content in five batches of Herba Hyperici Japonici total flavoness
Batch |
Flavonoid content |
1 |
56.7% |
2 |
60.8% |
3 |
63.5% |
4 |
64.1% |
5 |
66.4% |
Choose batches 3 Herba Hyperici Japonici total flavones and carry out the experiment of following examples.
Embodiment 2: Herba Hyperici Japonici total flavones is to the effect of common bile duct caused by ligature rat liver fibrosis
1. laboratory animal:
The SD rat, the SPF level, male and female half and half are provided by Guangdong Medical Lab Animal Center, the animal quality certification number: SCXK(Guangdong) 2008-0002.
Medicine grouping and processing:
Normal control group: distilled water.
Model control group: adopt the common bile duct ligation to cause the rat liver fibrosis model, give the equal-volume distilled water.
The Herba Hyperici Japonici total flavones low dose group: get Herba Hyperici Japonici total flavones (general flavone content is 63.5%) before the experiment, it is 0.9 mg/ml suspension that adding distil water is mixed with concentration.
Dosage group in the Herba Hyperici Japonici total flavones: get Herba Hyperici Japonici total flavones (general flavone content is 63.5%) before the experiment, it is 1.8 mg/ml suspensions that adding distil water is mixed with concentration.
The Herba Hyperici Japonici total flavones high dose group: get Herba Hyperici Japonici total flavones (general flavone content is 63.5%) before the experiment, it is 3.6 mg/ml suspensions that adding distil water is mixed with concentration.
Experimental technique:
Get the SD rat and be divided into 5 groups at random, be i.e. dosage group, Herba Hyperici Japonici total flavones high dose group in normal control group, model control group, Herba Hyperici Japonici total flavones low dose group, the Herba Hyperici Japonici total flavones.It is that 10% chloral hydrate solution is anaesthetized by the dosage lumbar injection concentration of 4ml/kg that rat is respectively organized in modeling, expose bile duct under the liver, ligation liver and bile duct intersection and bile duct and pancreas intersection wipe out the intermediary bile duct in two places to cause complete bile duct obstruction rat model; The processing that do not operate of normal control group.From modeling same day, the basic, normal, high dosage group of Herba Hyperici Japonici total flavones is according to dosage irritated stomach by the 1ml/100g body weight respectively and give Herba Hyperici Japonici total flavones 1 time, continuous 28 days every day.Behind the last administration 1h, each is organized rat and slightly anaesthetizes with ether, and the heart blood sampling is got serum and adopted test kit to detect TBIL, DBIL, ALT, AST, HA, LN, PC-III, TNF-alpha levels; Opening the abdominal cavity then takes out hepatic tissue rapidly and clean the detection of employing test kit SOD, MDA, GSH-Px, Hyp after the homogenate, employing ELISA method detection MMP-1 and the proteic expression of TIMP-1 under normal saline; Other cuts rat liver, and with neutral 10% formalin fixed, wherein lobus sinister carries out HE dyeing, and observing hepatocyte structure and degree of hepatic fibrosis, lobus dexter is used for immunohistochemical method (SABC method) and detects (the expression of α-SMA) of hepatic tissue α-Ji Dongdanbai.
Experimental result:
(1) Herba Hyperici Japonici total flavones is to the influence of liver function index TBIL, DBIL, ALT, AST in the serum
By table 2 as seen, compare with the normal control group, TBIL, DBIL, ALT and AST level all significantly raise (P<0.01) in the model control group rat blood serum, point out the obstruction of biliary tract of this rat model obvious, and hepatocyte are obviously destroyed.The level of TBIL, DBIL, ALT and AST and model control group compare in the basic, normal, high dosage group of the Herba Hyperici Japonici total flavones rat blood serum, and difference all has statistical significance (P<0.01 or 0.05), and is dose-effect relationship.Illustrate that Herba Hyperici Japonici total flavones can reduce the jaundice level and the hepatic injury degree of common bile duct caused by ligature hepatic fibrosis rats.
Table 2 Herba Hyperici Japonici total flavones to the influence of liver function index TBIL, DBIL, ALT, AST in the serum (
)
Annotate: compare with the normal control group:
##P<0.01; Compare with model control group:
*P<0.05
*P<0.01.
(2) Herba Hyperici Japonici total flavones is to the influence of hepatic fibrosis index PC-III, HA, LN, Hyp
By table 3 as seen, compare with the normal control group, all significantly risings (P<0.01) of Hyp content in PC-III, HA, LN content and the hepatic tissue are pointed out this rat model to have tangible liver cirrhosis pathology and are changed in the model control group rat blood serum.Hyp content and model control group comparison in PC-III, HA, LN content and the hepatic tissue in the basic, normal, high dosage group of the Herba Hyperici Japonici total flavones rat blood serum, difference all has statistical significance (P<0.01 or 0.05), and be dose-effect relationship, the prompting Herba Hyperici Japonici total flavones is inhibited to common bile duct caused by ligature rat liver fibrosis.
Table 3 Herba Hyperici Japonici total flavones to the influence of hepatic fibrosis index PC-III, HA, LN, Hyp (
)
Annotate: compare with the normal control group:
##P<0.01; Compare with model control group:
*P<0.05
*P<0.01.
(3) Herba Hyperici Japonici total flavones is to the influence of TNF-α in hepatic tissue SOD, GSH-Px, MDA and the serum
By table 4 as seen, compare with the normal control group, model control group liver tissues of rats SOD, GSH-Px are active significantly to reduce (P<0.01), and MDA content obviously improves (P<0.01), the antioxidant levels of prompting Liver Fibrosis Model rat liver significantly reduces, and oxidative damage is obvious.In addition, can participate in the also significantly rising (P<0.01) of important cytokine TNF-α of liver fibrosis process regulation and control by number of mechanisms.And TNF-α and model control group comparison in the basic, normal, high dosage group of Herba Hyperici Japonici total flavones liver tissues of rats SOD, GSH-Px, MDA and the serum, difference all has statistical significance (P<0.01 or 0.05), and is dose-effect relationship.Illustrate that Herba Hyperici Japonici total flavones can improve the oxidation resistance of Liver Fibrosis Model rat liver, significantly reduce the oxidative damage of liver, also can suppress the secretion of TNF-α, thereby suppress the development of hepatic fibrosis.
Table 4 Herba Hyperici Japonici total flavones to the influence of TNF-α in hepatic tissue SOD, GSH-Px, MDA and the serum (
)
Annotate: compare with the normal control group:
##P<0.01; Compare with model control group:
*P<0.05
*P<0.01.
(4) Herba Hyperici Japonici total flavones is to the influence of hepatic tissue MMP-1 and TIMP-1 protein expression
By table 5 as seen, compare with the normal control group, model control group liver tissues of rats TIMP-1 protein expression significantly increases (P<0.01), and hepatic tissue MMP-1 does not have significant change (P>0.05), the TIMP-1 protein expression of prompting Liver Fibrosis Model rat liver increases, and extracellular matrix decomposes minimizing in the liver.And the basic, normal, high dosage group of Herba Hyperici Japonici total flavones liver tissues of rats TIMP-1 protein expression and model control group are relatively, and difference all has statistical significance (P<0.01 or 0.05), and is dose-effect relationship.Illustrate that Herba Hyperici Japonici total flavones can suppress the TIMP-1 protein expression of Liver Fibrosis Model rat liver, significantly promotes the decomposition of extracellular matrix in the liver.
Table 5 Herba Hyperici Japonici total flavones to the influence of hepatic tissue MMP-1 and TIMP-1 protein expression (
)
Annotate: compare with the normal control group:
##P<0.01; Compare with model control group:
*P<0.05
*P<0.01.
(5) Herba Hyperici Japonici total flavones is to the influence of hepatic tissue pathology variation
Hepatic tissue HE coloration result is seen Fig. 1.The result shows: normal control group lobules of liver clear in structure, and hepatocyte is normal substantially, and the portal area clear in structure is not seen little bile duct and the obvious hypertrophy of fibrous tissue around the portal area reaches; Model control group sees that the lobules of liver structure is unintelligible, the hepatocyte edema is obvious in the lobule, focal hepatic necrosis is more, the remarkable hypertrophy of little bile duct, and being that the center is radial with the portal area extends in liver parenchyma, outgrowth bile duct is the garland sample, and the hypertrophy bile duct is on every side with obvious proliferation of fibrous tissue and cell infiltration; In the dosage Herba Hyperici Japonici total flavones organize visible lobules of liver clear in structure, little bile duct is slightly hypertrophy only of center with the portal area, accompanies slight proliferation of fibrous tissue around the hypertrophy bile duct.
(6) Herba Hyperici Japonici total flavones is to the influence of hepatic tissue α-SMA protein expression
Hepatic tissue α-SMA protein expression immunohistochemical staining the results are shown in Figure 2.The result shows: liver tissues of rats α-SMA protein expression is colored as pale brown color based on cell cytoplasm, mainly is distributed in the portal area.The only visible a small amount of positive expression of hepatic tissue portal area α-SMA of normal control group; Visible α-the SMA of model control group area of positive expression in hepatic tissue significantly increases; The area of middle dosage Herba Hyperici Japonici total flavones group α-SMA positive expression in hepatic tissue obviously is less than model control group, is tending towards normal.
Embodiment 3: Herba Hyperici Japonici total flavones causes the effect of rat immunity hepatic fibrosis to porcine blood serum
1. laboratory animal:
The SD rat, the SPF level, male and female half and half are provided by Guangdong Medical Lab Animal Center, the animal quality certification number: SCXK(Guangdong) 2008-0002.
, medicine grouping with handle:
Normal control group: distilled water.
Model control group: adopt porcine blood serum to cause the rat immunity Liver Fibrosis Model, give the equal-volume distilled water.
The Herba Hyperici Japonici total flavones low dose group: get Herba Hyperici Japonici total flavones (general flavone content is 63.5%) before the experiment, it is 0.9 mg/ml suspension that adding distil water is mixed with concentration.
Dosage group in the Herba Hyperici Japonici total flavones: get Herba Hyperici Japonici total flavones (general flavone content is 63.5%) before the experiment, it is 1.8 mg/ml suspensions that adding distil water is mixed with concentration.
The Herba Hyperici Japonici total flavones high dose group: get Herba Hyperici Japonici total flavones (general flavone content is 63.5%) before the experiment, it is 3.6 mg/ml suspensions that adding distil water is mixed with concentration.
Experimental technique:
Get the SD rat and be divided into 5 groups at random, be i.e. dosage group, Herba Hyperici Japonici total flavones high dose group in normal control group, model control group, Herba Hyperici Japonici total flavones low dose group, the Herba Hyperici Japonici total flavones.All the other respectively organize the rats by intraperitoneal injection porcine blood serum except that the normal control group, every injection 0.5 ml, 2 times weekly, the time be Tuesday, the mornings 8 Saturday point, the modeling time was 12 weeks.The normal control group is pressed the same manner injecting normal saline.From modeling same day, the basic, normal, high dosage group of Herba Hyperici Japonici total flavones is according to dosage irritated stomach respectively and give Herba Hyperici Japonici total flavones 1 time, continuous 12 weeks every day; Normal control group and model control group gavage the equal-volume distilled water.Behind the last administration 1h, each is organized rat and slightly anaesthetizes with ether, in the ventral aorta blood sampling, gets serum and adopts test kit to detect ALT, AST, ALB, TP, HA, LN, PC-III level; Open the abdominal cavity then and take out hepatic tissue rapidly, in normal saline, clean, adopt test kit to detect Hyp after the homogenate, adopt the ELISA method to detect MMP-1 and the proteic expression of TIMP-1; Other cuts rat liver, and with neutral 10% formalin fixed, wherein lobus sinister carries out HE dyeing, and observing hepatocyte structure and degree of hepatic fibrosis, lobus dexter is used for immunohistochemical method (SABC method) and detects (the expression of α-SMA) of hepatic tissue α-Ji Dongdanbai.
Experimental result:
(1) Herba Hyperici Japonici total flavones is to the influence of liver function index ALT, AST, ALB/GLB in the serum
By table 6 as seen, compare with the normal control group, ALT, AST level significantly raise (P<0.01) in the model control group rat blood serum, and the ALB/GLB value significantly reduces (P<0.01), points out the hepatocyte of this rat model obviously to damage, and liver function significantly changes.The level of ALT, AST and ALB/GLB value and model control group compare in the basic, normal, high dosage group of the Herba Hyperici Japonici total flavones rat blood serum, and difference all has statistical significance (P<0.01 or 0.05), and is dose-effect relationship.Illustrate that Herba Hyperici Japonici total flavones can reduce the hepatic injury degree of hepatic fibrosis rats due to the lumbar injection porcine blood serum.
Table 6 Herba Hyperici Japonici total flavones to the influence of liver function index ALT, AST, ALB/GLB in the serum (
)
Annotate: compare with the normal control group:
##P<0.01; Compare with model control group:
*P<0.05
*P<0.01.
(2) Herba Hyperici Japonici total flavones is to the influence of hepatic fibrosis index PC-III, HA, LN, Hyp
By table 7 as seen, compare with the normal control group, all significantly risings (P<0.01) of Hyp content in PC-III, HA, LN content and the hepatic tissue are pointed out this rat model to have tangible liver cirrhosis pathology and are changed in the model control group rat blood serum.Hyp content and model control group comparison in PC-III, HA, LN content and the hepatic tissue in the basic, normal, high dosage group of the Herba Hyperici Japonici total flavones rat blood serum, difference all has statistical significance (P<0.01 or 0.05), and be dose-effect relationship, the prompting Herba Hyperici Japonici total flavones is inhibited to rat immunity hepatic fibrosis due to the lumbar injection porcine blood serum.
Table 7 Herba Hyperici Japonici total flavones to the influence of hepatic fibrosis index PC-III, HA, LN, Hyp (
)
Annotate: compare with the normal control group:
##P<0.01; Compare with model control group:
*P<0.05,
*P<0.01.
(3) Herba Hyperici Japonici total flavones is to hepatic tissue MMP-1 and the proteic influence of TIMP-1
By table 8 as seen, compare with the normal control group, model control group liver tissues of rats TIMP-1 protein expression significantly increases (P<0.01), and hepatic tissue MMP-1 does not have significant change (P>0.05), the TIMP-1 protein expression of prompting Liver Fibrosis Model rat liver increases, and extracellular matrix decomposes minimizing in the liver.And the basic, normal, high dosage group of Herba Hyperici Japonici total flavones liver tissues of rats TIMP-1 protein expression and model control group are relatively, difference all has statistical significance (P<0.01 or 0.05), and be dose-effect relationship, illustrate that Herba Hyperici Japonici total flavones can suppress the TIMP-1 protein expression of immunological liver fibrosis rat model liver due to the lumbar injection porcine blood serum, significantly promotes the decomposition of extracellular matrix in the liver.
Table 8 Herba Hyperici Japonici total flavones to the influence of hepatic tissue MMP-1 and TIMP-1 protein expression (
)
Annotate: compare with the normal control group:
##P<0.01; Compare with model control group:
*P<0.05
*P<0.01.
(4) Herba Hyperici Japonici total flavones is to the influence of hepatic tissue pathology variation
Hepatic tissue HE coloration result is seen Fig. 3.The result shows: hepatocyte is normal in the normal control group lobules of liver clear in structure, lobule, and the portal area clear in structure is not seen little bile duct and proliferation of fibrous tissue around portal area and the portal area; Model control group is seen the lobules of liver structural fuzzy, hepatocyte edema in the lobule, focal hepatic necrosis, the a large amount of hypertrophy of little bile duct, being that the center is radial with the portal area extends in liver parenchyma, and outgrowth bile duct is the garland sample, accompanies proliferation of fibrous tissue and cell infiltration around the hypertrophy bile duct; It is more clear that middle dosage Herba Hyperici Japonici total flavones is organized visible lobules of liver structure, and little bile duct is the slight hypertrophy in center with the portal area, accompanies slight proliferation of fibrous tissue around the hypertrophy bile duct.
(5) Herba Hyperici Japonici total flavones is to the influence of hepatic tissue α-SMA protein expression
Hepatic tissue α-SMA protein expression immunohistochemical staining the results are shown in Figure 2.The result shows: liver tissues of rats α-SMA protein expression is colored as pale brown color based on cell cytoplasm, mainly is distributed in the portal area.Visible α-the SMA of normal control group is at a small amount of positive expression in hepatic tissue portal area; Visible α-the SMA of model control group area of positive expression in hepatic tissue increases greatly; The area of middle dosage Herba Hyperici Japonici total flavones group α-SMA positive expression in hepatic tissue obviously is less than model control group, is tending towards normal.
Embodiment 4: Herba Hyperici Japonici total flavones is to CCl
4The influence of rat liver fibrosis due to the complex factors
1, laboratory animal:
The SD rat, the SPF level, male and female half and half are provided by Guangdong Medical Lab Animal Center, the animal quality certification number: SCXK(Guangdong) 2008-0002.
, medicine grouping with handle:
Normal control group: distilled water.
Model control group: adopt CCl
4Complex factors cause the rat liver fibrosis model, give the equal-volume distilled water.
The Herba Hyperici Japonici total flavones low dose group: get Herba Hyperici Japonici total flavones (general flavone content is 63.5%) before the experiment, it is 0.9 mg/ml suspension that adding distil water is mixed with concentration.
Dosage group in the Herba Hyperici Japonici total flavones: get Herba Hyperici Japonici total flavones (general flavone content is 63.5%) before the experiment, it is 1.8 mg/ml suspensions that adding distil water is mixed with concentration.
The Herba Hyperici Japonici total flavones high dose group: get Herba Hyperici Japonici total flavones (general flavone content is 63.5%) before the experiment, it is 3.6 mg/ml suspensions that adding distil water is mixed with concentration.
, experimental technique:
Get the SD rat, except that 10 of normal control groups, all the other the 1st weeks of rat (first) are used pure CCl
45 ml/kg are to the rat back subcutaneous injection, and the 2nd~6 week was used 40% CCl
4-olive oil 3 ml/kg are to the rat back subcutaneous injection, 2 times/week.In the 1st~6 week simultaneously, and with the unique drinking-water of 20% ethanol as rat.In continuous 6 weeks, duplicate the rat liver fibrosis model.To duplicate CCl
4Liver Fibrosis Model to the 6 week and the rat of survival is divided into 4 groups at random by sex and body weight, 10 every group, add the normal control group, amount to 5 groups.Give medicine or distilled water then respectively: the basic, normal, high dosage group of Herba Hyperici Japonici total flavones is according to dosage irritated stomach respectively and give Herba Hyperici Japonici total flavones 1 time, continuous 12 weeks every day; Normal control group and model control group gavage the equal-volume distilled water.Behind the last administration 1h, each is organized rat and slightly anaesthetizes with ether, in the ventral aorta blood sampling, gets serum and adopts test kit to detect TBIL, DBIL, ALT, AST, HA, LN, PC-III, TNF-alpha levels; Opening the abdominal cavity then takes out hepatic tissue rapidly and clean the detection of employing test kit SOD, MDA, GSH-Px, Hyp after the homogenate, employing ELISA method detection MMP-1 and the proteic expression of TIMP-1 under normal saline; Other cuts rat liver, and with neutral 10% formalin fixed, wherein lobus sinister carries out HE dyeing, and observing hepatocyte structure and degree of hepatic fibrosis, lobus dexter is used for immunohistochemical method (SABC method) and detects (the expression of α-SMA) of hepatic tissue α-Ji Dongdanbai.
, experimental result:
(1) Herba Hyperici Japonici total flavones is to the influence of liver function index TBIL, DBIL, ALT, AST in the serum
By table 9 as seen, compare with the normal control group, TBIL, DBIL, ALT and AST level all significantly raise (P<0.01) in the model control group rat blood serum, point out the obstruction of biliary tract of this rat model obvious, and hepatocyte are obviously destroyed.The level of TBIL, DBIL, ALT and AST and model control group are relatively in the basic, normal, high dosage group of the Herba Hyperici Japonici total flavones rat blood serum, difference all has statistical significance (P<0.01 or 0.05), and be dose-effect relationship, illustrate that Herba Hyperici Japonici total flavones can reduce the jaundice level and the hepatic injury degree of common bile duct caused by ligature hepatic fibrosis rats.
Table 9 Herba Hyperici Japonici total flavones to the influence of liver function index TBIL, DBIL, ALT, AST in the serum (
)
Annotate: compare with the normal control group:
##P<0.01; Compare with model control group:
*P<0.05
*P<0.01.
(2) Herba Hyperici Japonici total flavones is to the influence of hepatic fibrosis index PC-III, HA, LN, Hyp
By table 10 as seen, compare with the normal control group, all significantly risings (P<0.01) of Hyp content in PC-III, HA, LN content and the hepatic tissue are pointed out this rat model to have tangible liver cirrhosis pathology and are changed in the model control group rat blood serum.Hyp content and model control group comparison in PC-III, HA, LN content and the hepatic tissue in the basic, normal, high dosage group of the Herba Hyperici Japonici total flavones rat blood serum, difference all has statistical significance (P<0.01 or 0.05), and be dose-effect relationship, the prompting Herba Hyperici Japonici total flavones is to CCl
4Complex factors cause rat liver fibrosis inhibited.
Table 10 Herba Hyperici Japonici total flavones to the influence of hepatic fibrosis index PC-III, HA, LN, Hyp (
)
Annotate: compare with the normal control group:
##P<0.01; Compare with model control group:
*P<0.05
*P<0.01.
(3) Herba Hyperici Japonici total flavones is to the influence of TNF-α in hepatic tissue SOD, GSH-Px, MDA and the serum
By table 11 as seen, compare with the normal control group, model control group liver tissues of rats SOD, GSH-Px are active significantly to reduce (P<0.01), and MDA content obviously improves (P<0.01), the antioxidant levels of prompting Liver Fibrosis Model rat liver significantly reduces, and oxidative damage is obvious.In addition, can participate in the also significantly rising (P<0.01) of important cytokine TNF-α of liver fibrosis process regulation and control by number of mechanisms.And TNF-α and model control group comparison in the basic, normal, high dosage group of Herba Hyperici Japonici total flavones liver tissues of rats SOD, GSH-Px, MDA and the serum, difference all has statistical significance (P<0.01 or 0.05), and be dose-effect relationship, illustrate that Herba Hyperici Japonici total flavones can improve CCl
4The oxidation resistance of the Liver Fibrosis Model rat liver that complex factors cause significantly reduces the oxidative damage of liver, also can suppress the secretion of TNF-α.
Table 11 Herba Hyperici Japonici total flavones to the influence of TNF-α in hepatic tissue SOD, GSH-Px, MDA and the serum (
)
Annotate: compare with the normal control group:
##P<0.01; Compare with model control group:
*P<0.05
*P<0.01.
(4) Herba Hyperici Japonici total flavones is to the influence of hepatic tissue MMP-1 and TIMP-1 protein expression
By table 12 as seen, compare with the normal control group, model control group liver tissues of rats TIMP-1 protein expression significantly increases (P<0.01), and hepatic tissue MMP-1 does not have significant change (P>0.05), the TIMP-1 protein expression of prompting Liver Fibrosis Model rat liver increases, and extracellular matrix decomposes minimizing in the liver.And the basic, normal, high dosage group of Herba Hyperici Japonici total flavones liver tissues of rats TIMP-1 protein expression and model control group are relatively, and difference all has statistical significance (P<0.01 or 0.05), and is dose-effect relationship, illustrates that Herba Hyperici Japonici total flavones can suppress CCl
4Complex factors cause the TIMP-1 protein expression of Liver Fibrosis Model rat liver, significantly promote the decomposition of extracellular matrix in the liver.
Table 12 Herba Hyperici Japonici total flavones to the influence of hepatic tissue MMP-1 and TIMP-1 protein expression (
)
Annotate: compare with the normal control group:
##P<0.01; Compare with model control group:
*P<0.05
*P<0.01.
(5) Herba Hyperici Japonici total flavones is to the influence of hepatic tissue pathology variation
Hepatic tissue HE coloration result is seen Fig. 5.The result shows: normal control group lobules of liver clear in structure, and hepatocyte is normal in the lobule, portal area clear in structure, portal area and do not see little bile duct and proliferation of fibrous tissue on every side; Model control group is seen the lobules of liver structural fuzzy, hepatocyte edema in the lobule, and focal necrosis, a large amount of hypertrophy of little bile duct are that the center is radial with the portal area extends in liver parenchyma, and outgrowth bile duct is obvious, and on every side with proliferation of fibrous tissue and cell infiltration; It is more clear that middle dosage Herba Hyperici Japonici total flavones is organized visible lobules of liver structure, and little bile duct is the slight hypertrophy in center with the portal area, accompanies slight proliferation of fibrous tissue around the hypertrophy bile duct.
(6) Herba Hyperici Japonici total flavones is to the influence of hepatic tissue α-SMA protein expression
Hepatic tissue α-SMA protein expression immunohistochemical staining the results are shown in Figure 6.The result shows: liver tissues of rats α-SMA protein expression is colored as sundown based on cell cytoplasm, mainly is distributed in the portal area.Visible α-the SMA of normal control group has a small amount of positive expression in the hepatic tissue portal area; Visible α-the SMA of model control group area of positive expression in hepatic tissue increases greatly; The area of middle dosage Herba Hyperici Japonici total flavones group α-SMA positive expression in hepatic tissue obviously is less than model control group, is tending towards normal.
Embodiment 5:SD rat oral gives the Herba Hyperici Japonici total flavones acute toxicity test
1, laboratory animal:
24 of SPF level SD rats, male and female half and half, body weight 130~150g, west, Shanghai pul must provide (production licence number: SCXK(Shanghai)-2003-0002) by triumphant laboratory animal company limited.
, experimental technique:
EXPERIMENTAL DESIGN administration group and blank group, every group of 12 animals, male and female half and half.Administration group dosage is 4.44g/kg, by Cmax 222mg/ml, the administration volume 20ml/kg gastric infusion that can pass through to irritate the stomach pin.Matched group is irritated stomach and is given the equal-volume sterilized water for injection.
The outward appearance of observation every day and record rat, the mental status, breathing, skin are by ordinary circumstance and other poisoning manifestations and death condition such as hair, fecaluria, eye, ear, nose, oral cavity, genitals.Measured rat body weight once in per two days, and measured the rat food ration weekly once, observed continuously 14 days.
, experimental result:
(1) general situation and death condition are observed
Until the 14th day, each animal spirit and behavioral activity were in order, skin is normal by hair cleaning, defecation, also do not see other toxic reaction symptoms after the administration.Observe end back gross anatomy and do not see that internal organs are obviously unusual.
(2) body weight
Female, great and mighty or powerful Mus is compared in female, the great and mighty or powerful Mus body weight of each time point body weight and sterilized water for injection matched group, and the difference not statistically significant (P〉0.05), think that Herba Hyperici Japonici total flavones per os gastric infusion does not have obvious influence to rat body weight.See table 13 for details.
Table 13 Herba Hyperici Japonici total flavones single oral gastric infusion to the influence of SD rat body weight (
)
In the last table, " matched group " is meant " blank group ", and " administration group " is meant " Herba Hyperici Japonici total flavones administration group ".
(3) food ration
Herba Hyperici Japonici total flavones per os gastric infusion does not have obvious influence to the average food ration of female, male Mus.See table 14 for details.
Table 14 Herba Hyperici Japonici total flavones single oral gastric infusion is to the influence of the average food ration of SD rat.
, conclusion:
In sum, under this laboratory condition, the SD rat is with Cmax 222mg/ml, maximum volume 20ml/kg single oral is irritated the Herba Hyperici Japonici total flavones of stomach 4.44g/kg, in 14 day observation period, death does not appear in rat, and spirit and behavioral activity are in order, skin is normal by hair cleaning, defecation, body weight gain is normal, the food ration no abnormality seen.Observe end back gross anatomy and do not see that internal organs are unusual.