Summary of the invention
The present invention is based on heat clearing away, blood stasis dispelling, removing food stagnancy, and double is the treatment rule with the invigorating the spleen and benefiting QI, makes the evil clearly of dampness-heat in the liver and gallbladder, and stasis of blood mass resulting from the blood stasis is long-pending as to disappear, and spleen and stomach function recovers, and the suitable then spleen fortune of gas is to reach liver-protective purpose.The inventor is through a large amount of animal and clinical trial, and screening has obtained the sure Chinese prescription of curative effect, i.e. liver heat removing blood stasis dispelling pharmaceutical composition, thus finished the present invention.
Primary and foremost purpose of the present invention provides a kind of medicine for the treatment of hepatic fibrosis, and next provides the preparation method of this Chinese medicine composition.
A kind of medicine for the treatment of hepatic fibrosis of the present invention, also claim liver heat removing blood stasis dispelling medicine, mainly contain Radix Gentianae, Rhizoma Curcumae, Radix Scutellariae, rhizoma sparganic (vinegar system), Radix Salviae Miltiorrhizae, the Rhizoma Atractylodis Macrocephalae, the Radix Astragali, their parts by weight are: 10~30 parts of Radix Gentianae, 20~40 parts of Rhizoma Curcumae (vinegar system), 30~50 parts of Radix Scutellariaes, 15~35 parts of rhizoma sparganic, 25~45 parts of Radix Salviae Miltiorrhizaes, 15~35 parts of the Rhizoma Atractylodis Macrocephalaes, 10~30 parts of the Radixs Astragali.
The medicine of treatment hepatic fibrosis also can contain Pericarpium Citri Reticulatae, Rhizoma Polygoni Cuspidati, and their parts by weight are: 10~30 parts of Pericarpium Citri Reticulataes, 10~30 parts of Rhizoma Polygoni Cuspidati.
The medicine of treatment hepatic fibrosis also can contain Radix Glycyrrhizae, and its parts by weight are: 5~15 parts.
Medicine of the present invention can adopt the conventional method of Chinese medicine preparation to be prepared into the oral formulations of any routine, but in order to make the Chinese crude drug in this medicine bring into play better drug effect, is necessary medical material is carried out the extraction of active component.
When the medicine of treatment hepatic fibrosis contained Radix Gentianae, Rhizoma Curcumae, Radix Scutellariae, rhizoma sparganic (vinegar system), Radix Salviae Miltiorrhizae, the Rhizoma Atractylodis Macrocephalae, the Radix Astragali at least, its preparation method was as follows:
A, Rhizoma Curcumae (vinegar system), the Rhizoma Atractylodis Macrocephalae extract with steam distillation, collect volatile oil, and standby, volatile oil also can be with standby behind the beta-cyclodextrin inclusion compound, and the aqueous solution after distillation device is in addition collected;
B, Radix Scutellariae, rhizoma sparganic (vinegar system), the Radix Astragali, Radix Salviae Miltiorrhizae add the water reflux, extract,, and merge extractive liquid, filters, and the aqueous solution that filtrate and step a collect merges, and concentrates, and adds ethanol, and cold preservation is spent the night, and filters, and supernatant is standby;
C, Radix Gentianae alcohol heating reflux, extracting solution filters, and the supernatant of filtrate and step b preparation merges, and reclaims ethanol and does not extremely have the alcohol flavor, and be condensed into clear paste, is spray dried to dry extract;
D, with step a preparation volatile oil or the Benexate Hydrochloride of volatile oil and the dry extract of step c preparation merge, promptly make treatment hepatic fibrosis medicines of the present invention.
When also containing Pericarpium Citri Reticulatae, Rhizoma Polygoni Cuspidati in the medicine of treatment hepatic fibrosis, its preparation method is as follows:
A, Rhizoma Curcumae (vinegar system), the Rhizoma Atractylodis Macrocephalae and Pericarpium Citri Reticulatae extract with steam distillation, collect volatile oil, and standby, volatile oil also can be with standby behind the beta-cyclodextrin inclusion compound, and the aqueous solution after distillation device is in addition collected;
B, Radix Scutellariae, rhizoma sparganic (vinegar system), Rhizoma Polygoni Cuspidati, the Radix Astragali, Radix Salviae Miltiorrhizae add the water reflux, extract,, and merge extractive liquid, filters, and the aqueous solution that filtrate and step a collect merges, and concentrates, and adds ethanol, and cold preservation is spent the night, and filters, and supernatant is standby;
C, Radix Gentianae alcohol heating reflux, extracting solution filters, and the supernatant of filtrate and step b preparation merges, and reclaims ethanol and does not extremely have the alcohol flavor, and be condensed into clear paste, is spray dried to dry extract;
D, the volatile oil of step a preparation or the Benexate Hydrochloride of volatile oil and the dry extract of step c preparation are merged, promptly make treatment hepatic fibrosis medicines of the present invention.
When also containing Radix Glycyrrhizae in the treatment hepatic fibrosis medicines, its preparation method is as follows:
A, Rhizoma Curcumae (vinegar system), the Rhizoma Atractylodis Macrocephalae and Pericarpium Citri Reticulatae extract with steam distillation, collect volatile oil, and standby, volatile oil also can be with standby behind the beta-cyclodextrin inclusion compound, and the aqueous solution after distillation device is in addition collected;
B, Radix Scutellariae, rhizoma sparganic (vinegar system), Rhizoma Polygoni Cuspidati, the Radix Astragali, Radix Salviae Miltiorrhizae, Radix Glycyrrhizae add the water reflux, extract,, and merge extractive liquid, filters, and the aqueous solution that filtrate and step a collect merges, and concentrates, and adds ethanol, and cold preservation is spent the night, and filters, and supernatant is standby;
C, Radix Gentianae alcohol heating reflux, extracting solution filters, and the supernatant of filtrate and step b preparation merges, and reclaims ethanol and does not extremely have the alcohol flavor, and be condensed into clear paste, is spray dried to dry extract;
D, with step a preparation volatile oil or the Benexate Hydrochloride of volatile oil and the dry extract of step c preparation merge, promptly make treatment hepatic fibrosis medicines of the present invention.
In treatment hepatic fibrosis medicines of the present invention, can add the required various conventional adjuvant of preparation different dosage form, as disintegrating agent, lubricant, binding agent etc., method of Chinese medicinal with routine is prepared into any peroral dosage form commonly used, as pill, powder, tablet, capsule, oral liquid etc.
Prescription analysis: we are monarch drug with Radix Gentianae, Rhizoma Curcumae, and the former liver heat removing gallbladder is damp and hot, latter's removing blood stasis circulation of qi promoting removing food stagnancy, " being the medicine of Liver Channel blood system, the blood in the energy dispelling the stagnated QI ".Two medicines share the effect that promptly reaches the liver heat removing blood stasis dispelling.Help Rhizoma Curcumae with the blood stasis dispelling removing food stagnancy with the auxilliary Radix Gentianae liver heat removing dampness of Radix Scutellariae, rhizoma sparganic; Join the Radix Salviae Miltiorrhizae activating blood circulation to dissipate blood stasis and dredge the collateral, the wonderful of " removing blood stasis with potent drugs blood, tissue regeneration promoting blood " arranged; With the Radix Astragali, Rhizoma Atractylodis Macrocephalae invigorating the spleen and benefiting QI, the body resistance strengthening and constitution consolidating, in order to the eliminating evil of monarch drug and just do not hinder, the above five tastes are ministerial drug altogether, with the monarch drug liver heat removing blood stasis dispelling that matches, eliminating pathogenic factor for supporting vital QI.Rhizoma Polygoni Cuspidati can be helped the monarch and his subjects' medicine, an energy clearing away heat-damp and promoting diuresis, but two blood circulation promoting and blood stasis dispelling; Pericarpium Citri Reticulatae can make monarch drug " eliminating evil and just do not hinder " with the merit of its invigorating the spleen and regulating the stomach, with the effect of its dampness of regulating the flow of vital energy, makes the ministerial drug Radix Astragali, the Rhizoma Atractylodis Macrocephalae " set upright and do not hold back heresy ", and the two is adjuvant drug altogether.Make with Radix Glycyrrhizae slow and that monarch drug is eliminating evil is high, the merit that association's ministerial drug is set upright, but coordinating the actions of various ingredients in a prescription again.Full side's compatibility is rigorous, and monarch is orderly, plays the merit of liver heat removing dehumidifying, blood circulation promoting and blood stasis dispelling, invigorating the spleen and benefiting QI altogether.Damp and hot going, then catharsis is smooth; Blood stasis is logical, and then mass in the abdomen disappears; Taste are strong, and then fortuneization is strong.For the physical fatigue and lassitude of spirit that liver cirrhosis occurs, dim complexion, the icteric sclera yellowish body, hepatosplenomegaly, the gastral cavity abdomen is tired to expand or the both sides of the chest distending pain, and the indigestion and loss of appetite grade of bitter taste disease must be played the therapy rehabilitation effect.
The function of liver heat removing blood stasis dispelling pharmaceutical composition of the present invention is liver heat removing blood stasis dispelling, invigorating the spleen and benefiting QI.
Subject range: be used for the treatment of hepatic fibrosis and early stage liver cirrhosis, disease sees that dampness-heat in the liver and gallbladder, the liver blood stasis of blood stagnate, dim complexion, icteric sclera yellowish body due to the weak deficiency of vital energy of spleen, gastral cavity abdomen painful abdominal mass expands or two side of body distending pains, food postemphasis, and bitter taste is indigestion and loss of appetite, physical fatigue and lassitude of spirit, yellowish or reddish urine, tongue are dark violet stasis of blood electricity or ecchymosis, yellow fur, stringy and thready pulse or a stringy and rolling pulse.
The specific embodiment
Further set forth the preparation method of medicine of the present invention by the following examples
Embodiment one: preparation treatment hepatic fibrosis medicines (liver heat removing blood stasis dispelling medicine)
1, prescription: 20 parts of Radix Gentianae, 30 parts of Rhizoma Curcumae (vinegar system), 40 parts of Radix Scutellariaes, 25 parts of rhizoma sparganic (vinegar system), 35 parts of Radix Salviae Miltiorrhizaes, 25 parts of the Rhizoma Atractylodis Macrocephalaes, 20 parts of the Radixs Astragali, 20 parts of Pericarpium Citri Reticulataes, 20 parts of Rhizoma Polygoni Cuspidati, 10 parts in Radix Glycyrrhizae.
2, preparation method:
A, Rhizoma Curcumae (vinegar system), the Rhizoma Atractylodis Macrocephalae and Pericarpium Citri Reticulatae add 8 times of water gagings, extract 6 hours with steam distillation, collect volatile oil, and with standby behind the beta-cyclodextrin inclusion compound, the aqueous solution after distillation device is in addition collected;
B, Radix Scutellariae, rhizoma sparganic (vinegar system), Rhizoma Polygoni Cuspidati, the Radix Astragali, Radix Salviae Miltiorrhizae, Radix Glycyrrhizae add 10 times of water gaging reflux, extract, three times (each 2 hours), merge extractive liquid,, filter, the aqueous solution that filtrate and step a collect merges, be evaporated to 1: 1, and added ethanol and make and contain alcohol amount and reach 60%, cold preservation is spent the night, filter, supernatant is standby;
C, Radix Gentianae are with 70% alcohol heating reflux three times (each 1 hour), and merge extractive liquid, filters, and filtrate merges with the supernatant of step b preparation, reclaims ethanol and does not distinguish the flavor of to there being alcohol, and be condensed into clear paste, is spray dried to dry extract;
D, the Benexate Hydrochloride of the volatile oil of step a preparation and the dry extract of step c preparation are merged, promptly make treatment hepatic fibrosis medicines of the present invention.
Embodiment two: the hard capsule of preparation treatment hepatic fibrosis medicines
On the basis of embodiment one, carry out the preparation of capsule preparations
Treatment hepatic fibrosis medicines 387g
Micropowder silica gel 10g
Magnesium stearate 2g
With each several part mixing in the above-mentioned prescription, fill is made 1000 capsules, promptly.Every 0.4g obeys 15 every day.
Embodiment three: the soft capsule of preparation treatment hepatic fibrosis medicines
On the basis of embodiment one, carry out the preparation of capsule preparations
Treatment hepatic fibrosis medicines 383g
Medicinal soybean oil 258g
Glyceryl monostearate 0.4g
Get 1 part in gelatin, add 1 part in water, 0.6 part of glycerol, 70-80 ℃ melt glue after, move into 55-65 ℃ of heat preservation for standby use in the insulation jar, it is an amount of with Oleum Glycines, glyceryl monostearate to get it filled, heating, 70 ℃ make miscible, put coldly, the active component of above-mentioned liver heat removing blood stasis dispelling medicine is added stirring and evenly mixing, grind with colloid mill, make it form the suspension of homogeneous.With carrying out pelleting in the medicinal liquid adding feed tank for preparing, make 1000.Soft capsule setting under 25 ℃, the condition of relative humidity 30% moved in the drying machine and carries out drying after 20 hours, and drying condition is 30 ℃, and relative humidity is 20%, and wind speed is 0.6m/s, and the time is 8h.Dried soft capsule is washed ball, is chosen and wash, after the check promptly.Every 0.65g obeys 12 every day.
Embodiment four: preparation treatment hepatic fibrosis medicines (liver heat removing blood stasis dispelling medicine)
1, prescription: 26 parts of Radix Gentianae, 32 parts of Rhizoma Curcumae (vinegar system), 32 parts of Radix Scutellariaes, 25 parts of rhizoma sparganic (vinegar system), 35 parts of Radix Salviae Miltiorrhizaes, 25 parts of the Rhizoma Atractylodis Macrocephalaes, 20 parts of the Radixs Astragali.
2, preparation method
A, Rhizoma Curcumae (vinegar system), the Rhizoma Atractylodis Macrocephalae add 8 times of water gagings, extract 6 hours with steam distillation, collect volatile oil, and with standby behind the beta-cyclodextrin inclusion compound, the aqueous solution after distillation device is in addition collected;
B, Radix Scutellariae, rhizoma sparganic (vinegar system), the Radix Astragali, Radix Salviae Miltiorrhizae add 10 times of water gaging reflux, extract, three times (each 2 hours), and merge extractive liquid, filters, the aqueous solution that filtrate and step a collect merges, and is evaporated to 1: 1, adds ethanol and makes and contain the alcohol amount and reach 60%, cold preservation is spent the night, and filters, and supernatant is standby;
C, Radix Gentianae are with 70% alcohol heating reflux three times (each 1 hour), and merge extractive liquid, filters, and filtrate merges with the supernatant of step b preparation, reclaims ethanol and does not distinguish the flavor of to there being alcohol, and be condensed into clear paste, is spray dried to dry extract;
D, the Benexate Hydrochloride of the volatile oil of step a preparation and the dry extract of step c preparation are merged, liver heat removing blood stasis dispelling medicine of the present invention.
Embodiment five: preparation treatment hepatic fibrosis medicines hard capsule
On the basis of embodiment four, carry out the preparation of capsule preparations
Liver heat removing blood stasis dispelling medicine 356g
Lactose 30g
Micropowder silica gel 12g
Magnesium stearate 2g
With each several part mixing in the above-mentioned prescription, fill is made 1000 capsules, promptly.Every 0.4g obeys 15 every day.
Embodiment six: preparation treatment hepatic fibrosis medicines (liver heat removing blood stasis dispelling medicine)
1, prescription: 30 parts of Radix Gentianae, 40 parts of Rhizoma Curcumae (processed with vinegar), 40 parts of Radix Scutellariaes, 30 parts of rhizoma sparganic (processed with vinegar), 40 parts of Radix Salviae Miltiorrhizaes, 25 parts of the Rhizoma Atractylodis Macrocephalaes, 25 parts of the Radixs Astragali, 25 parts of Pericarpium Citri Reticulataes, 25 parts of Rhizoma Polygoni Cuspidati.
2, preparation method
A, Rhizoma Curcumae (vinegar system), the Rhizoma Atractylodis Macrocephalae and Pericarpium Citri Reticulatae add 8 times of water gagings, extract 6 hours with steam distillation, collect volatile oil, and with standby behind the beta-cyclodextrin inclusion compound, the aqueous solution after distillation device is in addition collected;
B, Radix Scutellariae, rhizoma sparganic (vinegar system), Rhizoma Polygoni Cuspidati, the Radix Astragali, Radix Salviae Miltiorrhizae add 10 times of water gaging reflux, extract, three times (each 2 hours), and merge extractive liquid, filters, the aqueous solution that filtrate and step a collect merges, and is evaporated to 1: 1, adds ethanol and makes and contain the alcohol amount and reach 60%, cold preservation is spent the night, and filters, and supernatant is standby;
C, Radix Gentianae are with 70% alcohol heating reflux secondary (each 1 hour), and merge extractive liquid, filters, and filtrate merges with the supernatant of step b preparation, reclaims ethanol and does not distinguish the flavor of to there being alcohol, and be condensed into clear paste, is spray dried to dry extract;
D, the Benexate Hydrochloride of the volatile oil of step a preparation and the dry extract of step c preparation are merged, liver heat removing blood stasis dispelling medicine of the present invention.
The present invention treats the effect of hepatic fibrosis medicines to hepatic injury for explanation, by setting up animal model, has investigated the therapeutical effect to hepatic fibrosis.
Test one, liver heat removing blood stasis dispelling capsule (treatment hepatic fibrosis medicines) is damaging to rat immunity
The effect of hepatic fibrosis
1 test objective
Adopt the intravenous injection bovine serum albumin to duplicate the damaging Liver Fibrosis Model of rat immunity, observe the capsular therapeutical effect of liver heat removing blood stasis dispelling.
2 test materials
2.1 medicine
Press the liver heat removing blood stasis dispelling hard capsule of embodiment two preparations, i.e. the present invention treats hepatic fibrosis medicines, and medicated powder is rufous, and the suspension that is made into suitable concn with 0.5%CMC uses.
FUFANG BIEJIA RUANGAN PIAN: commercially available, lot number 20021010, the suspension that is made into suitable concn with 0.5%CMC uses.
Colchicine: Serva company product, lot number C9754, the solution that is made into suitable concn with double distilled water uses.
2.2 reagent
Bovine serum albumin (BSA): Tianjin Hao ocean biological product company limited, lot number A-7030
Lanoline: Shanghai International Automobile City Tourist Festival Huating lanoline factory, lot number 000917
Liquid paraffin: Tianjin chemical reagent two factory's products, lot number 980425
Incomplete Freund: lanoline 120g adds liquid paraffin 240ml, and at 65 ℃ of stirring in water bath mixings, the rearmounted 4 ℃ of refrigerators of autoclave sterilization are preserved standby.
The Coomassie brilliant blue protein determination kit: bio-engineering research institute, lot number 20030715 are built up in Nanjing
The hydroxyproline determination test kit: bio-engineering research institute, lot number 20030715 are built in Nanjing
Sialic acid is measured test kit: bio-engineering research institute, lot number 20030707 are built in Nanjing
2.3 animal
Rat: the Wistar kind, the secondary animal is provided by institute of section of army four, the quality certification number: medical officer moves word B98014
The raising condition: sub-cage rearing, every cage 5-6 only freely drink waters, are ingested, and room temperature is controlled at 24 ± 2 ℃ 2 by central air-conditioning, and humidity is 50 ± 15%, and illumination 12 hours is bright, 12 hours secretly at 6 (light the morning six, in afternoon secretly).
3 test methods
3.1 model copy
Select 100 of healthy Wistar kind rats for use, body weight 130-150g, male and female half and half, except that normal control group (each 5 of male and female), all the other 90 rats all with the each 0.5ml/ of 9mg/ml BSA incomplete Freund suspension only, carry out the multiple spot subcutaneous injection with sensitized animal, continuous 5 times.1st, 2 weeks of 2 minor ticks, all the other each times are 1 week of interval all.In 1 week after the last sensitization, the equal eye socket vein of all animals is got blood, measures serum BSA antibody with capillary tube method.The animal tail vein of getting antibody positive is attacked BSA normal saline solution 0.4ml/ of injection variable concentrations, 2 times weekly, dosage from 2.0mg/0.4ml/ only, be incremented to 3.0mg/0.4ml/ only (at every turn increasing progressively 0.1mg) one by one, the 12nd time is 3.4mg/0.4ml/, only increase progressively 0.2mg to 4.0mg/0.4ml/ later on, totally 15 times, normal rats is carried out tail vein injection with physiologic saline for substitute BSA at every turn.Last is attacked back 3 days of injection, gets hematometry ALT, AST, TP, ALB, A/G (albumins/globulins) ratio, sialic acid; Get the part liver, measure hydroxyproline content after the homogenate; Other gets part liver 10% formalin fixed, carries out HE dyeing and Masson dyeing, and light microscopic is observed degree of hepatic fibrosis down.
3.1.2 group technology
Except that the normal control group, all the other animals are all after attack injection 4 times, be divided into 6 groups at random by body weight, model control group, liver heat removing blood stasis dispelling capsule 0.9,1.8,3.6g medicated powder/kg dosage group, positive drug FUFANG BIEJIA RUANGAN PIAN 2g medicated powder/kg dosage group, positive drug colchicine 0.25mg/kg dosage group, add the normal control group, totally 7 groups.
3.1.3 administration time
Each treated animal carries out gastric infusion by the filling stomach volume of 1ml/100g body weight all after attacking injection 4 times, and normal control group, model control group are irritated stomach and capacity 0.5%CMC suspension, every day 1 time, 7 times weekly, continuous 6 weeks such as given.
3.1.4 data statistics
Test data represents that with x ± SD two groups of means adopt statistical analysis-t value method, have the employing rank test of hierarchical relationship to carry out statistical analysis.
4 result of the tests
4.1 ordinary circumstance
After the sensitization 5 times, totally 87 of the rats of serum BSA antibody positive (having 3 male rats not meet test requirements document), after attacking injection, allergy shock sample reaction in various degree takes place in rat, rapid breathing, walk and unstablely, prostrate not rise, in addition dead, and dead animal is dissected immediately, each internal organs of perusal are not found obviously unusual, and it is normal that surviving animals was recovered in about 30 minutes.The dead animal major part appears at attacks injection the 1-3 time, and from attacking injection the 4th, idol has animal that the reaction of irritated shock sample takes place.After the attack injection 4 times, be total to dead 16 (10 of male and female, male 6), 71 of surviving animals, surviving animals is divided into 6 groups at random by body weight, model control group, liver heat removing blood stasis dispelling 0.9g medicated powder/kg dosage group, liver heat removing blood stasis dispelling 1.8g medicated powder/kg dosage group, liver heat removing blood stasis dispelling medicated powder 3.6g medicated powder/kg dosage group, FUFANG BIEJIA RUANGAN PIAN 2g medicated powder/kg dosage group all 12 every group (female, male each 6), 11 (female 5 of colchicine 0.25mg/kg dosage groups, male 6), add that 10 of normal control groups are (female, male each 5), totally 7 groups (81), carry out gastric infusion, continuous 6 weeks.In process of the test, gastric infusion is strayed into trachea and causes 1 of rats death (female liver heat removing blood stasis dispelling 3.6g medicated powder/kg), because of the rat of moulding death is 7, wherein: model control group 2 (female 1, male 1), liver heat removing blood stasis dispelling 0.9g medicated powder/kg dosage group 2 (female, male each 1), liver heat removing blood stasis dispelling 1.8g medicated powder/kg dosage group 1 (female), FUFANG BIEJIA RUANGAN PIAN 2g medicated powder/kg dosage group 1 (female), 1 of group of colchicine 0.25mg/kg dosage (male).
4.2 the mensuration of liver, spleen organ coefficient
After the last administration, rat fasting 15 hours is weighed in, and takes out liver, spleen, the weighing weight in wet base, and, spleen recast heavy with the liver of 100g body weight is organ coefficient, relatively the difference of each dosage group the results are shown in Table 1.
Table 1 liver heat removing blood stasis dispelling capsule is to the influence of immunologic injury hepatic fibrosis rats organ coefficient (x ± SD)
Group | Dosage (g medicated powder/kg) | Number of animals (only) | The liver coefficient | The spleen coefficient |
Normal group model control group clearing liver Huayu Capsule clearing liver Huayu Capsule clearing liver Huayu Capsule FUFANG BIEJIA RUANGAN PIAN colchicin | 0.9 1.8 3.6 2.0 0.25mg | 10 10 10 11 11 11 10 | 2.776±0.242 3.744±0.631## 3.348±0.406 3.224±0.370
* 3.187±0.303
* 3.645±0.491 3.217±0.414
* | 0.253±0.035 0.366±0.037 0.318±0.069 0.321v0.057
* 0.309±0.046
** 0.327±0.047
* 0.317±0.050
* |
##:p<0.01 (comparing) with the normal control group
*: p<0.05
*P<0.01 (comparing) with model control group
Table 1 is the result show, compares with the normal control group, and the liver of model control group rat, spleen organ coefficient obviously increase; Compare with model control group, liver heat removing blood stasis dispelling capsule 1.8,3.6g medicated powder/kg dosage group liver, spleen organ coefficient obviously reduce, and positive drug also has similar action.
4.3 blood biochemical is learned index determining
To the rat after the fasting, use the pentobarbital sodium intraperitoneal anesthesia, abdominal aortic blood, supernatant is drawn in centrifugal back, on Olympus Au640 automatic clinical chemistry analyzer, measure ALT, AST, TP, ALB, G, A/G, build up sialic acid (SA) the mensuration test kit description that bio-engineering research is produced, measure serum sialic acid content by Nanjing, relatively the difference of each dosage group the results are shown in Table 2.
Table 2 liver heat removing blood stasis dispelling capsule to the influence of immunologic injury hepatic fibrosis rats blood parameters (x ± SD, n)
Group | Dosage (g medicated powder/kg) | ALT (U/L) | AST (U/L) | TP (g/L) | ALB (g/L) | G (g/L) | A/G | SA (mmol/L) |
Normal control model contrast clearing liver Huayu Capsule clearing liver Huayu Capsule clearing liver Huayu Capsule compound turtle shell liver sheet colchicin | 0.9 1.8 3.6 2.0 0.25mg | 34.9±7.22 37.1±8.82 56.9±39.1 40.2±13.7 34.7±12.2 44.5±19.8 45.2±22.3 | 105.9±28.9 123.4±26.4 155.3±57.2 107.8±54.9 109.8±33.9 124.9±43.1 126.2±35.9 | 59.0±3.39 58.6±3.24 59.3±3.19 60.7±2.44 59.3±2.30 59.8±3.06 61.2±2.71 | 30.2±2.65 29.0±2.85 29.4±1.80 30.3±1.80 29.009±1.79 29.8±2.14 30.6±1.92 | 28.8±1.58 29.6±2.17 29.8±2.69 30.3±2.94 2.66±309 30.0±2.03 30.6±2.97 | 1.05±0.07 0.99±0.13 0.99±0.12 1.01±0.14 1.03±0.13 1.0±0.09 1.01±0.14 | 3.34±0.38 3.79±0.47# 3.63±0.41 3.62±0.69 3.40±0.40 3.46±0.50 3.55±0.68 |
#:p<0.05 (comparing) with the normal control group
*: p<0.05 (comparing) with model control group
N: number of animals, with table 1.
Table 2 is the result show: compare with the normal control group, the serum sialic acid content of model control group rat obviously raises, all the other every index no significant differences.Compare with model control group, liver heat removing blood stasis dispelling capsule 3.6g medicated powder/kg dosage group serum sialic acid content obviously reduces the equal no significant difference of all the other every indexs.
4.4 the liver hydroxyproline content is measured
Get the liver organization of about 1g, the normal saline that adds 9 times carries out homogenate, and 3500 changeed part centrifugal 10 minutes after the homogenate, get supernatant and build up the hydroxyproline test kit description that bio-engineering research is produced by Nanjing, measure the liver hydroxyproline content, relatively the difference of each dosage group the results are shown in Table 3.
Table 3 liver heat removing blood stasis dispelling capsule is to the influence of immunologic injury hepatic fibrosis rats liver hydroxyproline content (x ± SD)
Group | Dosage (g medicated powder/kg) | Number of animals (only) | Hydroxyproline (μ g/mgprot) |
Normal group model control group clearing liver Huayu Capsule clearing liver Huayu Capsule clearing liver Huayu Capsule FUFANG BIEJIA RUANGAN PIAN colchicin | 0.9 1.8 3.6 2 0.25mg | 10 10 10 11 11 11 10 | 0.609±0.151 0.903±0.121## 0.767±0.190 0.726±0.163
* 0.689±0.131
** 0.732±0.153
* 0.727±0.158
* |
##:p<0.001 (comparing) with the normal control group
*: p<0.05
*P<0.01 (comparing) with model control group
Table 3 is the result show: compare with the normal control group, model control group liver hydroxyproline content obviously raises, and shows to form hepatic fibrosis.Compare with model control group, liver heat removing blood stasis dispelling capsule 1.8,3.6g medicated powder/kg dosage group obviously reduce hydroxyproline content, and increase with dosage, and effect strengthens, showing has tangible preventive and therapeutic effect to hepatic fibrosis, and positive control drug FUFANG BIEJIA RUANGAN PIAN, colchicine also have similar action.
4.5 histopathologic examination
Get liver, use 10% formalin fixed, after routine is drawn materials, the dehydration of ethanol gradient, the embedding of the automatic paraffin embedding machine of FisherModel 266Mp, the film-making of Leica RM2135 microtome, two kinds of dyeing of HE, Masson, the Olympus microscopically carries out pathological study by following grade scale and takes a picture, relatively the difference of each dosage group.
Grade scale: list of references, degree of hepatic fibrosis is divided into level Four:
"-": hepatic tissue is normal, no fibrosis hamartoplasia
"+": there is proliferation of fibrous tissue in portal area and hole crack.
" ++ ": the lobules of liver structure has destruction, and proliferation of fibrous tissue forms the fibrous septum, has a small amount of pseudolobuli to form trend.
" +++": the lobules of liver structural deterioration, proliferation of fibrous tissue is obvious, and pseudolobuli extensively forms.
The results are shown in Table 4, table 5.
Table 4 liver heat removing blood stasis dispelling capsule is to the influence (HE dyeing) of immunologic injury hepatic fibrosis rats degree of hepatic fibrosis
Group | Dosage (the g crude drug/kg) | Number of animals (only) | HE dyeing, degree of hepatic fibrosis | p |
- | + | ++ | +++ |
Normal group model control group clearing liver Huayu Capsule clearing liver Huayu Capsule clearing liver Huayu Capsule FUFANG BIEJIA RUANGAN PIAN colchicin | 0.9 1.8 3.6 2 0.25mg | 10 10 10 11 11 11 10 | 10 1 3 7 8 5 4 | 0 3 1 1 1 2 3 | 0 1 1 1 0 3 2 | 0 5 5 2 2 1 1 | <0.01 >0.05 <0.05 <0.05 <0.05 <0.05 |
Annotate: the number under the different degree of hepatic fibrosis is the number of animals that each dosage group reaches this degree, and table 5 is with table 4
Table 5 liver heat removing blood stasis dispelling capsule is to the influence (Masson dyeing) of immunologic injury hepatic fibrosis rats degree of hepatic fibrosis
Group | Dosage (the g crude drug/kg) | Number of animals (only) | Masson dyeing, degree of hepatic fibrosis | p |
- | + | ++ | +++ |
Normal group model control group clearing liver Huayu Capsule clearing liver Huayu Capsule clearing liver Huayu Capsule FUFANG BIEJIA RUANGAN PIAN colchicin | 0.9 1.8 3.6 2 0.25mg | 10 10 10 11 11 11 10 | 10 1 3 7 8 5 4 | 0 3 1 1 1 2 3 | 0 1 1 1 0 3 2 | 0 5 5 2 2 1 1 | <0.01 >0.05 <0.05 <0.05 <0.05 <0.05 |
Table 4, table 5 show: compare with the normal control group, the model control group degree of hepatic fibrosis obviously increases, and shows the modeling success.Compare with model control group, liver heat removing blood stasis dispelling capsule 1.8,3.6g medicated powder/kg dosage group degree of hepatic fibrosis obviously alleviate, and showing has tangible preventive and therapeutic effect to hepatic fibrosis, and increase with dosage, and effect strengthens.Positive control drug FUFANG BIEJIA RUANGAN PIAN, colchicine also have obvious effect.
5 conclusion (of pressure testing)s
Adopt quiet notes bovine serum albumin to duplicate the damaging Liver Fibrosis Model of rat immunity, the result shows, compare with the normal control group, model control group rat liver hydroxyproline content obviously increases, and obvious outgrowth fibrous tissue is divided into pseudolobuli widely with hepatic tissue; Liver heat removing blood stasis dispelling capsule 1.8,3.6g medicated powder/kg dosage group liver hydroxyproline content obviously reduce, and degree of hepatic fibrosis obviously alleviates, and increase with dosage, and effect strengthens, and shows that liver heat removing blood stasis dispelling capsule has tangible preventive and therapeutic effect to the immunological liver fibrosis.
Test two, liver heat removing blood stasis dispelling medicine (treatment hepatic fibrosis medicines) are to acute and chronic
The preventive and therapeutic effect of hepatic injury
1. experiment material:
Liver heat removing blood stasis dispelling capsule: press the hard capsule of embodiment two preparations, i.e. the present invention treats hepatic fibrosis medicines, and content is the rufous powder.
Bifendate, the Beijing XieHe medicine Factory produces, lot number 970419.
Animal: Kunming mouse and SD rat, provide by Hebei province's Experimental Animal Center, the quality certification number is respectively 04056 and No. 04057, and the solid feed that provides with this center is provided during the laboratory observation, freely drinks water.
2. test method
2.1 influence to the mouse carbon tetrachloride acute liver damage
Method: get 60 of mices, male and female half and half, body weight 20-25g is divided into 6 groups at random, 10 every group.Blank group and model control group give water; Liver heat removing blood stasis dispelling capsule divides three dosage group: 0.9g, 1.8g and 3.6g/kg; Bifendate 0.2g/kg.Below respectively organize equal gastric infusion, volume 0.2ml/10g body weight, once a day, continuous 7 days.In administration after the 6th day, equal lumbar injection 1% carbon tetrachloride oil solution 0.1ml/10g body weight except that the blank group.
1 hour (give carbon tetrachloride after 20 hours) respectively organizes the equal arterial blood extracting of mice after the last administration, separation of serum, and reitman-frankel method is measured ALT (ALT).
With every zootomy, get same leaf, same position liver organization one fritter then, be fixed in 10% formalin solution, do pathological section and carry out histological examination, (Liu Ting is fast etc., new Chinese medicine and clinical pharmacology 1999 by following standard; 10 (4): 213-215) estimate the hepatic injury degree:
-: normal liver cell
+: hepatocellular degeneration or spotty necrosis
++: hepatocellular degeneration or focal necrosis, or the hepatocyte of pathological changes is less than 1/3 of lobules of liver
+++: lobules of liver 1/3-2/3 hepatic necrosis
++ ++: lobules of liver surpasses 2/3 hepatic necrosis
Observe the difference (Ridit check) of blank group and model control group and model control group and each administration group ATL difference (t check) and hepatic injury degree.
The result: the ALT of model control group is higher than blank group (P<0.01) very significantly, shows the model success.Liver heat removing blood stasis dispelling capsule 1.8g/kg can significantly reduce ALT (P<0.05), and bifendate has very significant reduction effect (P<0.01) to ALT, and experimental result sees Table 1.
Pathologic finding as seen, model group lobules of liver 1/3-2/3 even surpass 2/3 hepatic necrosis, liver heat removing blood stasis dispelling capsule 1.8 and 3.6g/kg group lobules of liver necrocytosis are light (P>0.05) than model, the hepatic necrosis of bifendate group or degeneration, obviously be less than model control group (P<0.05), experimental result sees Table 2.
Table 1: liver heat removing blood stasis dispelling capsule is to mouse carbon tetrachloride acute liver damage model transaminase's influence
Group | Dosage (g/kg) | Number of animals (only) | ALT(x±s)(u) |
Blank model contrast liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule bifendate | - - 0.9 1.8 3.6 0.2 | 10 10 10 10 10 10 | 48.8±38.5
** 293.9±9.6 292.0±12.9 285.3±7.0
* 288.7±11.6 225.1±72.5
** |
*P<0.05,
*P<0.01 and model contrast ratio
Table 2: liver heat removing blood stasis dispelling capsule is to the influence of mouse carbon tetrachloride acute liver damage model pathological change
Group | Dosage (g/kg) | Number of animals (only) | The hepatic injury degree | P value<0.01 |
- | + | ++ | +++ | ++++ |
Blank model contrast liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule bifendate | - - 0.9 1.8 3.6 0.2 | 8 10 10 10 10 10 | 8 | 1 2 2 5 | 9 9 8 8 5 | 1 | | >0.05 >0.05 >0.05 <0.05 |
2.2 effect to mice D-galactosamine acute hepatic function damage
Method: get 60 of mices, male and female half and half, body weight 20-25g is divided into 6 groups at random, 10 every group.Blank group and model control group give water; Liver heat removing blood stasis dispelling capsule divides three dosage group: 0.9g, 1.8g and 3.6g/kg; Bifendate 0.2g/kg.Below respectively organize equal gastric infusion, volume 0.2ml/10g body weight, once a day, continuous 10 days.
After the last administration 1 hour, except that the blank group, the equal lumbar injection D-galactosamine of every Mus 650mg/kg, 20 hours artery sacrificed by exsanguination mices, collect blood separation serum, press reitman-frankel method and measure ALT (ALT) and aspartic acid aminotransferase (AST) content.The significance that compares blank group and each administration group and model control group differences with the t check.
The result: the ALT of model group and AST content all significantly increase than the blank group, show the model success.Liver heat removing blood stasis dispelling capsule 0.9g, 1.8g and 3.6g/kg group ALT content significantly are low (P<0.05) than model control group all; 1.8g/kg still can significantly reduce AST content (P<0.05).Bifendate group ALT content is low (P<0.05, table 3) than model control group significantly also.
Table 3: liver heat removing blood stasis dispelling capsule is to the effect of mice D-galactosamine acute hepatic function damage
Group | Dosage (g/kg) | Number of animals (only) | ALT(x±s) (u) | AST(x±s)(u) |
Blank model contrast liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule bifendate | - - 0.9 1.8 3.6 0.2 | 10 10 10 10 10 10 | 55±17
** 233±53 188±40
* 183±47
* 180±49
* 190±24
* | 355±51
** 605±196 519±52 462±58
* 473±86 603±69
|
*P<0.05,
*P<0.01 and model contrast ratio
2.3 influence to rat carbon tetrachloride chronic hepatic injury
Method: get the SD rat, male 110, body weight 225-349g is divided into 6 groups at random, except that 10 of blank groups, and all the other every group equal 20.Blank group and model group give water; Liver heat removing blood stasis dispelling capsule divides three dosage group: 0.9g, 1.8g and 3.6g/kg; Positive control drug bifendate 0.1g/kg.Equal gastric infusion, volume 1ml/100g body weight.Every day 1 time, continuous 10 weeks.Except that blank, 2 subcutaneous injection 40% carbon tetrachloride peanut oil solution 3ml/kg weekly, continuous 10 weeks.
Next day after the last administration, get blood, press reitman-frankel method and measure aspartic acid aminotransferase (AST) and ALT (ALT), biuret method is measured total protein (TP), and the bromocresol green method is measured albumin (ALB) and calculated albumins/globulins ratio (A/G).Put to death animal then, get liver one fritter, measure the liver hydroxyproline content, use the formaldehyde fixed liver at last, paraffin embedding, Masson dyeing, according to following grading of pathological histology standard marking (Wang Baoen etc.: the hepatopathy magazine, 1993:1:69-72).
0 grade: hepatic tissue is normal
The I level: collagen fiber from the portal area or central vein stretch out
The II level: collagen fiber obviously extend, and do not hold whole lobules of liver but interconnect as yet
The III level: collagen fiber extend connection, hold whole lobules of liver
The IV level: collagen fiber hold cuts apart lobules of liver, and the normal hepatocytes leaflet structure is destroyed, and pseudolobuli forms, but based on big, square pseudolobuli
V level: be covered with little garden shape pseudolobuli in the liver, form large square and little garden shape pseudolobuli and respectively account for 50%
VI level: be covered with little garden shape pseudolobuli in the liver, thick hypertrophy collagen fiber are arranged between pseudolobuli
Pathological grading is checked with Ridit, and other index is all with t check, the relatively significance of each administration group and model control group difference.
Result: 1, survival rats and body weight: inject 40% carbon tetrachloride 3ml/kg first, employing be lumbar injection, rats death appears after 3 days, after change subcutaneous injection into, still have part death, dead at most with model control group, the death of middle dosage group is less.Body weight change: heavy dose of group body weight is than blank group light (P<0.05), with the model control group indistinction.See Table 4.
Table 4, liver heat removing blood stasis dispelling capsule give the influence of carbon tetrachloride survival number and body weight to rat
Group | Dosage (g/kg) | Raw animal number (only) | Number of animals (only) after 10 weeks | Original body weight (g) | Body weight (g) after 10 weeks |
Blank model contrast liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule bifendate | - - 0.9 1.8 3.6 0.1 | 10 20 20 20 20 20 | 10 10 12 15 13 13 | 281±28 289±28 286±27 290±25 287±25 285±31 | 358±38 339±42 338±35 347±26 325±26 331±31 |
*P<0.05 and blank ratio
2, to changing the influence of enzyme: the AST that liver heat removing blood stasis dispelling capsule 1.8g and 3.6g/kg all can make carbon tetrachloride raise reduces (P<0.05).3.6g/kg can significantly reduce ALT (P<0.05), bifendate can make AST and ALT significantly reduce (P<0.01).Experimental result sees Table 5.
Table 5, liver heat removing blood stasis dispelling capsule are changed the influence of enzyme to rat carbon tetrachloride chronic hepatic injury serum
Group | Dosage (g/kg) | Number of animals (only) | ALT(x±s) (u) | AST(x±s)(u) |
Blank model contrast liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule bifendate | - - 0.9 1.8 3.6 0.1 | 10 10 12 15 13 13 | 380±55
** 589±49 545±71 525±74
* 530±47
* 506±49
** | 44±9
** 138±34 146±43 149±52 109±21
* 66±12
** |
*P<0.05,
*P<0.01 and model contrast ratio
3, to the influence of serum protein content: each dosage liver heat removing blood stasis dispelling capsule and bifendate all do not have obvious influence (P>0.05) to total serum protein (TP) content; 3.6g/kg remarkable raising albumin content (P<0.01), A/G ratio also obviously raises (P<0.05), and bifendate does not have influence to ALB and A/G ratio, and experimental result sees Table 6.
Table 6, liver heat removing blood stasis dispelling capsule are to the influence of rat carbon tetrachloride chronic hepatic injury protein content
Group | Dosage (g/kg) | Number of animals (only) | TP(g/l) | ALB(g/l) | A/G ratio |
Blank model contrast liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule bifendate | - - 0.9 1.8 3.6 0.1 | 10 20 12 15 13 13 | 69.1±5.8 69.6±7.8 62.8±8.4 64.9±9.6 69.3±6.2 68.1±8.8 | 44.1±2.7 42.3±2.6 41.9±4.2 44.7±4.3 47.5±2.2
** 41.6±1.7
| 1.84±0.41 1.61±0.46 2.59±1.68 2.33±1.50 2.40±0.91
* 1.84±1.13
|
*P<0.05,
*P<0.01 and model contrast ratio
4, to the influence of hepatic tissue hydroxyproline content: liver heat removing blood stasis dispelling capsule 3.6g/kg can significantly reduce the hepatic tissue hydroxyproline content (P<0.05) that is increased by carbon tetrachloride, bifendate also can significantly reduce liver hydroxyproline content (P<0.05), and experimental result sees Table 7.
Table 7, liver heat removing blood stasis dispelling capsule are to the influence of rat carbon tetrachloride chronic hepatic injury liver hydroxyproline content
Group | Dosage (g/kg) | Number of animals (only) | Liver hydroxyproline content (x ± s) (μ g/kg) |
Blank model contrast liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule bifendate | - - 0.9 1.8 3.6 0.2 | 10 10 12 15 13 13 | 34±8
** 66±18 58±16 56±17 52±13
* 50±12
* |
*P<0.05,
*P<0.01 and model contrast ratio
5, hepatic tissue pathology inspection: the visible lobules of liver structure of model control group is destroyed fully, forms large square and garden shape pseudolobuli and respectively accounts for half approximately.Liver heat removing blood stasis dispelling capsule 1.8g and 3.6g/kg organize visible liver collagen fiber and extend, and or mutually do not hold whole lobules of liver as yet in succession, and the collagen fiber that have hold cuts apart lobules of liver, and the normal hepatocytes leaflet structure is destroyed, and form pseudolobuli.Press the marking of histological grade standard, and check its significance by Ridit.Compare with the model contrast, blank group, 1.8g and 3.6g/kg have significance,statistical (P<0.05 and 0.01), and experimental result sees Table 8.
Table 8, liver heat removing blood stasis dispelling capsule are to the fractionated influence of rat carbon tetrachloride liver injury pathology
Group | Dosage (g/kg) | Number of animals (only) | The pathology classification | The P value |
0 | I | II | III | IV | V | VI |
Blank model contrast liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule liver heat removing blood stasis dispelling capsule bifendate | - - 0.9 1.8 3.6 0.1 | 10 12 15 13 13 | 10 | 3 1 | 2 3 2 1 | 1 4 4 2 | 2 7 4 5 6 | 6 4 1 1 4 | | <0.01 >0.05 <0.05 <0.05 >0.05 |
2.4 conclusion: to the mouse carbon tetrachloride acute hepatic function damage, liver heat removing blood stasis dispelling capsule 1.8g/kg can reduce ALT (P<0.05), and liver tissue injury is slightly alleviated effect.To D-galactosamine chmice acute liver dysfunction, liver heat removing blood stasis dispelling capsule 0.9g, 1.8g and 3.6g/kg can reduce ALT (P<0.05); 1.8g/kg can reduce AST (P<0.05).To the chronic liver dysfunction of rat carbon tetrachloride, liver heat removing blood stasis dispelling capsule 1.8g and 3.6g/kg can reduce AST (P<0.05), and 3.6g/kg can reduce ALT (P<0.05); Liver heat removing blood stasis dispelling capsule 3.6g/kg can improve serum ALB content (P<0.05) and A/G ratio (P<0.05); Reduce hepatic tissue hydroxyproline content (P<0.05); To hepatic pathology credit level, liver heat removing blood stasis dispelling capsule 1.8g and 3.6g/kg can obviously alleviate lesion degree (P<0.05).