CN103191188A - Six-ingredient botanical composition for treating hepatic fibrosis and preparation method thereof - Google Patents
Six-ingredient botanical composition for treating hepatic fibrosis and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a six-ingredient botanical composition for treating hepatic fibrosis. In every kilogram by weight, the composition contains the following ingredients: 180-350 mg of crude cordyceps polysaccharide, 0.06-0.82 ml of fat-soluble cordyceps ingredient, 100-190 mg of salvianolic acid, 2-68 mg of gynostemma pentaphyllum saponin, 3-12 mg of gynostemma pentaphyllum polysaccharide and 0.1-4 mg of amygdalin. The method comprises the following steps of: (1) extracting the crude cordyceps polysaccharide, the fat-soluble cordyceps ingredient, the salvianolic acid, the amygdalin, the gynostemma pentaphyllum saponin and the gynostemma pentaphyllum polysaccharide; and (2) preparing water-soluble pellets which serve as a portion A from the salvianolic acid, the crude cordyceps polysaccharide and the gynostemma pentaphyllum polysaccharide, preparing solid dispersion pellets which serve as a portion B from the gynostemma pentaphyllum saponin, the fat-soluble cordyceps ingredient and the amygdalin, and forming a multiple drug release system and various clinical preparations according to the mass ratio of 8:0.3. The composition has remarkable clinical treatment effect improvement and few side effects, thereby being very worthy of recommendation.
Description
Technical field
The invention belongs to plant amedica components composition and the preparation thereof for the treatment of hepatic fibrosis, particularly a kind of 6 component plant amedica composition and method of making the sames for the treatment of hepatic fibrosis.
Background technology
China is the hotspot of hepatic disease such as chronic viral hepatitis B in the world, and chronic hepatopathy causes great harm to China people's health for a long time.But treating liver fibrosis still is the big difficult point in the chronic hepatopathy treatment up till now.From the eighties in last century, Shanghai Univ. of Traditional Chinese Medicine successfully formulates anti-hepatic fibrosis country new Chinese medicine " FUZHENG HUAYU JIAONANG " (the accurate word 20020073 of traditional Chinese medicines), this medicine is made up of Radix Salviae Miltiorrhizae, Cordyceps mycelium, Semen Persicae, Herb Gynostemmae Pentaphylli, Pollen Pini, Fructus Schisandrae Chinensis, and effect is blood circulation promoting and blood stasis dispelling, beneficial smart tonify deficiency.Studies confirm that for many years FUZHENG HUAYU JIAONANG can effectively improve chronic hepatitis Bhepatic fibrosis, liver tissue fibrosis reversion rate (than 1 phase of decline before the treatment or more than 1 phase) by stages reaches 52%, by the clinical observation in 2 years by a definite date, show that this medicine can reduce liver cirrhosis portal hypertension patient's upper gastrointestinal hemorrhage rate.
But the preparation of FUZHENG HUAYU JIAONANG is relatively backward at present, and 5 capsules of once taking medicine cause the reaction of part patient's gastrointestinal tract, though the tablet of having developed can reduce stomach discomfort, but still has further raising to treat the demand of hepatic fibrosis clinical efficacy." pharmacology " record, the amygdaloside in the Semen Persicae can produce a large amount of hydrocyanic acids after the intestinal digestive enzyme decomposes, hydrocyanism occurs.Though the patient was never taken place clinically because taking the event that the appearance of supporting vital QI and dispersing blood stasis compound recipe is poisoned, how to prevent that potential serious toxic and side effects was very necessary.Therefore, further secondary development supporting vital QI and dispersing blood stasis Chinese medicine compound improves curative effect again on the basis of existing reverse hepatic fibrosis 52%, and reducing toxic and side effects is the direction that efforts still need to be.
Summary of the invention
Technical problem to be solved by this invention provides a kind of 6 component plant amedica composition and method of making the sames for the treatment of hepatic fibrosis, has remarkable enhancing clinical efficacy, and side effect is little, is a kind of very recommendable clinical usage.
A kind of 6 component plant amedica compositionss for the treatment of hepatic fibrosis of the present invention, raw material is made up of Cordyceps mycelium, Radix Salviae Miltiorrhizae, Semen Persicae and Herb Gynostemmae Pentaphylli.
The compatibility dosage of described 6 component stock solutions is respectively by per kilogram of body weight: Cordyceps crude polysaccharides 180-350mg, Cordyceps fat-soluble ingredient 0.06～0.82ml, gypenoside 2-68mg, Herb Gynostemmae Pentaphylli polysaccharides 3-12mg, salvianolic acid 100-190mg, amygdaloside 0.1～4mg.
Preferably be respectively by per kilogram of body weight: Cordyceps crude polysaccharides 230～250mg, Cordyceps fat-soluble ingredient 0.1～0.2ml, gypenoside 10～30mg, Herb Gynostemmae Pentaphylli polysaccharides 7～8mg, salvianolic acid 120～140mg, amygdaloside 1～2mg.
Further preferably be respectively by per kilogram of body weight: Cordyceps crude polysaccharides 240mg, Cordyceps fat-soluble ingredient 0.1ml, gypenoside 20mg, Herb Gynostemmae Pentaphylli polysaccharides 7.3mg, salvianolic acid 130mg, amygdaloside 1mg.
A kind of 6 component plant amedica preparation of compositions methods for the treatment of hepatic fibrosis among the present invention comprise:
(1) extracts described 6 components: Cordyceps crude polysaccharides, Cordyceps fat-soluble ingredient, salvianolic acid, amygdaloside, gypenoside and Herb Gynostemmae Pentaphylli polysaccharides;
1. Cordyceps fat-soluble ingredient: get the Cordyceps mycelium powder, put into the extraction kettle of supercritical extraction instrument after granulating with the 20vol% ethanol water, extraction conditions: temperature is 45 ℃, and pressure is 30MPa, and the time is 2h, and flow is 35kg/h; Separation condition is: first order separating pressure is 10MPa, and temperature is 50 ℃, and second level separating pressure is 6MPa, and temperature is 40 ℃; Collect and separate the grease that parses, obtain the Cordyceps fat-soluble ingredient;
2. Cordyceps crude polysaccharides: will in 10 times of NaOH aqueous solutions of the Cordyceps mycelium slag behind the supercritical extraction at pH=8, boil 3 hours, totally 2 times, after the merging, centrifugal removing slag, supernatant concentration, adding ethanol to final concentration is 75vol%, refrigerator is placed, and separates out polysaccharide, filters, clean dry obtains the Cordyceps crude polysaccharides;
3. salvianolic acid: get red rooted salvia 1kg, add 10 times of amount deionized waters, boil 1h, medicinal liquid is inclined to, add 10 times of amount deionized waters in the medicinal residues again, boil 1h, medicinal liquid is inclined to, merge extracted twice liquid, leave standstill, filter.Be concentrated into 1.5L, the medicinal liquid ratio is 1.11 for 1:1.5(proportion), add 4.2L95vol% ethanol and be about 70vol% to concentration of alcohol, precipitate with ethanol spends the night, and is centrifugal, to go up solution decompression and be concentrated into nothing alcohol flavor, about 1.5L gets the good HZ-816 resin 2L of pretreatment, wet method dress post, do not distinguish the flavor of to there being alcohol with deionized water rinsing, with the flow velocity of 1/2 bed volume/hour (BV/h) with said extracted concentrated solution upper prop after, with the ethanol elution salvianolic acid of 2.5BV40%, flow velocity is 1BV/h again, collect 40% ethanol elution, drying under reduced pressure gets the yellow solid powder, after measured, the yield of salvianolic acid component is about 8%, and wherein the content of phenolic acid such as salvianolic acid B is about 25%;
4. amygdaloside: take by weighing Semen Persicae medical material 1kg, 50% ethanol that adds 10 times of amounts, reflux 90min filters, filtrate decompression is concentrated into solid-liquid ratio 1:1, get the good HZ-818 resin 250ml of pretreatment, wet method dress post is not distinguished the flavor of to there being alcohol with deionized water rinsing, with Semen Persicae extract concentrated solution with 1BV/h flow velocity upper prop after, with the 2BV deionized water rinsing, speed is 2BV/h, discards water wash liquid earlier, again with 3BV50vol% ethanol elution amygdaloside, speed is 2BV/h, collects 50% ethanol elution, concentrating under reduced pressure, dry, get the light yellow solid powder, after measured, the yield of Semen Armeniacae Amarum glycoside component is about 0.8%, and wherein amygdaloside content is about 63%;
5. gypenoside: take by weighing Herb Gynostemmae Pentaphylli medical material 1kg, add 60% ethanol of 20 times of amounts, reflux, extract, 2h, filter, filtrate decompression is concentrated into the extractum shape, adds 30% ethanol to solid-liquid ratio 1:3, ultrasonic dissolution 15min, centrifugal, supernatant is crossed macroporous adsorbent resin, gets the good HZ-816 resin 2L of pretreatment, wet method dress post, do not distinguish the flavor of to there being alcohol with deionized water rinsing, Herb Gynostemmae Pentaphylli extract with 1/4BV/h flow velocity upper prop, with the flushing of 2BV water, is discarded leacheate with the 1/2BV/h flow velocity, again under identical flow velocity, with the 2BV30% alcohol flushing, discard leacheate, at last with 3BV95% ethanol elution gypenoside, collect 95% ethanol elution, concentrating under reduced pressure, drying gets the faint yellow solid powder, after measured, the yield of gypenoside class component is about 1%, and wherein Herb Gynostemmae Pentaphylli total glycosides content is about 40%;
6. Herb Gynostemmae Pentaphylli polysaccharides: 5. the Herb Gynostemmae Pentaphylli medicinal residues dry naturally in the extraction process, take by weighing medicinal residues 500g, with 15 times of amount deionized water reflux, extract, 1h, filter, filtrate decompression is concentrated into solid-liquid ratio 1:1, adds 95% ethanol 900ml, about 70% to the alcohol amount of containing, precipitate with ethanol spends the night, and filters, filter cake is dried with 95% ethanol twice, pulverizes, get yellow Herb Gynostemmae Pentaphylli polysaccharides powder, after measured, the yield of Herb Gynostemmae Pentaphylli soap polysaccharide component is about 4%, and wherein Herb Gynostemmae Pentaphylli polysaccharides content is about 15%;
(2) by described per kilogram of body weight salvianolic acid, Cordyceps crude polysaccharides and Herb Gynostemmae Pentaphylli polysaccharides are made the water solublity micropill as the A part, gypenoside, the fat-soluble part of Cordyceps and amygdaloside are made the solid dispersion micropill as the B part, and A and B form polynary medicine-releasing system by mass ratio 8:0.3; Wherein, A part drug loading is that 33%, B part drug loading is 2.5%.
Described component stock solution is applied to the preparation of the specific part slow release of intestinal, and consumption can be used by one or many according to adjustment such as the type of route of administration, patient's age, body weight, the disease for the treatment of and the orders of severity.
Described plant amedica compositions can be made into solid preparation, as tablet or capsule.
Described plant amedica compositions can be made into microcapsule or dispersion micropill.
The every 100g body weight of described plant amedica compositions is irritated stomach dosage: A part 18.9～37.8mg, B part 0.709～1.418mg.
The every 100g body weight of described plant amedica compositions is irritated stomach dosage: A part 18.9mg, B part 0.709mg or A part 37.8mg, B part 1.418mg.
Through experiment and clinical research for many years, the mechanism of action of FUZHENG HUAYU JIAONANG anti-hepatic fibrosis and material base are relatively clearer and more definite.Discover that components such as Cordyceps polysaccharide, the amygdaloside in the Semen Persicae and the salviol acid A in the Radix Salviae Miltiorrhizae, B in the Cordyceps mycelium have remarkable effect of anti hepatic fibrosis.Can promote I, the degraded of III Collagen Type VI as Cordyceps polysaccharide, obviously reduce the content of immunological liver fibrosis rat liver tissue T GF-β 1 and TBRI thereof; Amygdaloside in the Semen Persicae can significantly suppress the propagation of HSC and the anabolism of collagen stroma composition, promotes its catabolism; Salvianolic acid B has significant anti-carbon tetrachloride (CCl
4) and N-nitrosodimethylamine (DMN) the rat liver fibrosis effect of inducing, hepatic stellate cell (HSC) propagation and collagenation suppress to go down to posterity, the HSC intracellular signal transduction that suppresses TGF-β 1, thus reduce the total amount of the HSC secretion TGF-B1 that goes down to posterity and suppress its activation.And salviol acid A can be synthetic by significant anti peroxidation of lipid damaging action inhibition collagen.Once with supporting vital QI and dispersing blood stasis side's treatment CCl
4The in type rat liver fibrosis of inducing finds that each herbal medicine in the supporting vital QI and dispersing blood stasis side mainly contains following characteristics: the 1) principal agent that collagen fiber are degraded in anti-hepatic fibrosis, the promotion liver in the Semen Persicae side of being; 2) Radix Salviae Miltiorrhizae is to improving liver function, and the synthetic reduction serum bilirubin level that reaches of short albumin plays a major role; 3) Cordyceps mycelium is the factor of influence that reduces serum total bilirubin content; 4) Herb Gynostemmae Pentaphylli is the principal agent of this side's protection hepatocyte, reduction serum ALT activities.
Because in the FUZHENG HUAYU JIAONANG anti-hepatic fibrosis, Pollen Pini and Fructus Schisandrae Chinensis do not play a major role, so the present invention has removed Pollen Pini and Fructus Schisandrae Chinensis among the former side of supporting vital QI and dispersing blood stasis, selected the Cordyceps with " setting upright " effect, the 4 flavor Chinese medicines such as Herb Gynostemmae Pentaphylli that have Radix Salviae Miltiorrhizae, the Semen Persicae of " blood circulation promoting and blood stasis dispelling " effect and have " heat-clearing and toxic substances removing " effect.Therefrom extract and purification 6 kinds of active components: salvianolic acid (Radix Salviae Miltiorrhizae), gypenoside, Herb Gynostemmae Pentaphylli polysaccharides (Herb Gynostemmae Pentaphylli), amygdaloside (Semen Persicae), Cordyceps crude polysaccharides, Cordyceps mycelium CO
2Supercritical extract (Cordyceps mycelium).Adopt the method for uniform Design, extract component to these 6 kinds and screen, by checking, the optimum compatibility dosage of active component group that filters out supporting vital QI and dispersing blood stasis side is: Cordyceps crude polysaccharides (240mgkg
-1), Cordyceps fat-soluble ingredient (0.1mlkg
-1), Herb Gynostemmae Pentaphylli polysaccharides (7.3mgkg
-1), gypenoside (20mgkg
-1), salvianolic acid (130mgkg
-1) and amygdaloside (1mgkg
-1).Finally select this 6 kinds of exploitations that active component enters polynary medicine-releasing system for use, wherein salvianolic acid, Cordyceps crude polysaccharides, Herb Gynostemmae Pentaphylli polysaccharides are made micropill (A part) to water solublity preferably, fat-soluble gypenoside preferably, the fat-soluble part of Cordyceps, Semen Persicae extract are made solid dispersion micropill (B part), two parts are formed supporting vital QI and dispersing blood stasis component compound multi-component medicine-releasing system jointly, and the drug loading of each several part is respectively: the A part is that the 100g micropill contains corresponding melange effect component 33g for 33%(); The B part is that the 100g micropill contains corresponding melange effect component 2.5g for 2.5%().Proportioning: A:B is that 8:0.3(is every 8g water-soluble portion micropill, carries out proportioning with 0.3g solid dispersion micropill).Research worker the experiment proved that FUZHENG HUAYU JIAONANG (containing extract 0.5g/ grain) and supporting vital QI and dispersing blood stasis compound recipe medicine-releasing system (0.415g/ grain, i.e. A part 0.4g, B part 0.015g) demonstrate close stripping behavior.
The present invention develops the novel form of supporting vital QI and dispersing blood stasis compound recipe, determine the compatibility of supporting vital QI and dispersing blood stasis compound recipe active component by the effect experiment of different levels, adopt the novel form development component herbal mixture of polynary medicine-releasing system, to realize compound recipe attenuation synergistic, safe and quality controllable.
Beneficial effect
The medicine of plant amedica components composition of the present invention confirms to have the fibrosis therapeutical effect of anti-hepatic fibrosis and other internal organs through zoopery, the more former compound recipe of curative effect has the trend of notable synergistic, behind the micropill drug administration, micropill can all be dispersed in the gastrointestinal tract, can avoid capsule and tablet to be trapped in a certain position because gastrointestinal tract transfer time is variant and discharge medicine and gastrointestinal tract mucosa is produced stimulate, have good potential applicability in clinical practice.
Description of drawings
Fig. 1 is normal group, model group, A group and B group rat liver HE colored graph (* 200), and the A group is the former side's extractum of supporting vital QI and dispersing blood stasis group, and the B group is the present invention;
Fig. 2 is normal group, model group, A group and the B group rat liver scarlet colored graph of sky wolf (* 100), and the A group is the former side's extractum of supporting vital QI and dispersing blood stasis group, and the B group is the present invention;
Fig. 3 is normal group, model group, A group and B group rat liver collagen deposition area change, and the A group is the former side's extractum of supporting vital QI and dispersing blood stasis group, and the B group is the present invention;
Fig. 4 is the changes of contents of normal group, model group, A group and B group liver tissues of rats Hyp, and the A group is the former side's extractum of supporting vital QI and dispersing blood stasis group, and the B group is the present invention;
Fig. 5 is normal group, model group, A group and B group rat blood serum ALT activity, and the A group is the former side's extractum of supporting vital QI and dispersing blood stasis group, and the B group is the present invention;
Fig. 6 is normal group, model group, A group and B group rat blood serum AST activity, and the A group is the former side's extractum of supporting vital QI and dispersing blood stasis group, and the B group is the present invention;
Fig. 7 is normal group, model group, A group and B group rat blood serum Alb content, and the A group is the former side's extractum of supporting vital QI and dispersing blood stasis group, and the B group is the present invention;
Fig. 8 is normal group, model group, A group and B group rat blood serum TBiL content, and the A group is the former side's extractum of supporting vital QI and dispersing blood stasis group, and the B group is the present invention;
Fig. 9 is normal group, model group, former side's group, high dose group and middle dosage group rat liver HE colored graph (* 200);
Figure 10 is normal group, model group, former side's group, high dose group and the middle dosage group rat liver scarlet colored graph of sky wolf (* 100);
Figure 11 is the percentage ratio that normal group, model group, former side's group, high dose group and the former depositional area of middle dosage group rat liver account for the gross area;
Figure 12 is the changes of contents of normal group, model group, former side's group, high dose group and middle dosage group liver tissues of rats Hyp;
Figure 13 is normal group, model group, former side's group, high dose group and middle dosage group rat blood serum ALT, AST activity;
Figure 14 is normal group, model group, former side's group, high dose group and middle dosage group rat blood serum Alb content;
Figure 15 is normal group, model group, former side's group, high dose group and middle dosage group rat blood serum TBiL content.
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The anti-hepatic fibrosis curative effect of supporting vital QI and dispersing blood stasis compound recipe active component compound recipe:
1.1 animal:
59 of Wistar male rats, the SPF level, body weight 150 ± 10g, Chinese Academy of Sciences's Shanghai Experimental Animal Center provides credit number: SCXK (Shanghai): 2007-0005.All rat feedings are in the Experimental Animal Center SPF of Shanghai Univ. of Traditional Chinese Medicine laboratory, and full diet is freely drunk water.
1.2 main agents:
N-nitrosodimethylamine (dimethylnitrosamine, DMN), available from Tokyo HuaCheng Industry Co., Ltd, lot number: MAL05; Citric acid, analytical pure, lot number: F20080916; Anhydrous sodium acetate, analytical pure, lot number: F20080714; Trisodium citrate (Tri-sodium citrate), analytical pure, lot number: F20070110; Paradimethylaminobenzaldehyde (4-Dimethylamino benzaldehyde), lot number: F20090516; Chlorine ammonia-T (chloramines-T), analytical pure, lot number: T20061130; Dehydrated alcohol, analytical pure, lot number: 20090716; Dimethylbenzene, analytical pure, lot number 20100123; Formaldehyde (Formal dehyde solution), lot number: 20071019; Isopropyl alcohol (Iso-propanol), analytical pure, lot number: T20070815; Concentrated hydrochloric acid, analytical pure, lot number: T20090312, more than all available from Shanghai chemical reagents corporation of Chinese Medicine group.Neutral gum, lot number: 60090715, available from the virtuous foreign Instr Ltd. in Shanghai.0.9% sodium chloride injection (Sodium Chloride Injection), lot number: 090903, available from Shuanghe Pharmaceutical Ind Co., Ltd., Anhui.Pentobarbital sodium (Pentobarbital Sodium, Lot.No.WS20090920, German packing.Hydroxyproline (hydroxyproline, Hyp) standard substance, analytical pure, Sigma-Aldrich Chemie GmbH, Germany product.Perchloric acid (Perchloric acid), analytical pure, lot number: 20070807 available from Tao Pu chemical plant, Shanghai.Serum liver function test kit, comprise glutamate pyruvate transaminase (alanine aminotransamine, ALT), glutamic oxaloacetic transaminase, GOT (aspartate aminotransferase, AST), albumin (albumin, Alb), total bilirubin (total bilirubin, T.Bil), all build up bio-engineering research institute available from Nanjing.
1.3 medicine:
Cordyceps crude polysaccharides, Cordyceps fat-soluble ingredient, salvianolic acid, amygdaloside, gypenoside, the same first of Herb Gynostemmae Pentaphylli polysaccharides.The extract dry powder of supporting vital QI and dispersing blood stasis compound recipe is provided by Shanghai Modern TCM Co., Ltd., lot number: 100306.Main quality control index (HPLC mensuration): danshensu sodium 10.48mg/g; Adenosine 2.5mg/g; Salvianolic acid B 12.4mg/g; Moisture content: 6.9%.Dilution is irritated gastric juice for the solution of 94.5mg/ml during application.
1.4 key instrument:
The FIM20 ice machine, Dutch Philips company product.The biochemical special refrigerator of last Pedicellus et Pericarpium Trapae is available from last marine Pedicellus et Pericarpium Trapae company.Cryogenic refrigerator (70 ℃), Forma scientific company product.Desk centrifuge (microfuge lite), Beckman company product.The AA-200 analytical balance, inverted microscope (37 * B) U.S. Denver instrument company products.Pure water system (HPLC/UP), Labconco company product.The CQX25-12 ultrasonic cleaner, Shanghai must ultrasonic company limited product.The roasting sheet machine of rotary microtome (RM2035), HI1220, HI1210 thermostat water bath, LEICA ASP300 automatic dehydration machine, LEICA EG1160 paraffin embedding machine are all available from German Leica company.Instant camera, Polariod company product.XW-80A type vortex mixer, its woods Bel instrument Manufacturing Co., Ltd of Haimen, Jiangsu.ML-902 time constant-temperature magnetic stirring apparatus, Shanghai Pujiang analytical tool factory produces.PH meter
, Beckmanwc company product.1000 μ l, 200 μ l, 20 μ l sample injectors, French Gilson company makes.The multi-functional microplate reader of M5, U.S. Molecular Devices company produces.The Precellys24 homogenizer, French Bertin company produces.
2.1 model preparation
59 of Wistar male rats after conforming 3 days, are divided into normal group (10) and modeling group (49) at random with the method for table of random number.The modeling group is pressed 2mlkg with 0.5%DMN
-1The body weight lumbar injection is injected 2/3 of full dosage first, injects rest 4d behind the 3d continuously every day 1 time, and totally 3 weeks, preceding 3 days of the 4th week are injected 0.5%DMN full dose, 2/3 amount and 1/2 amount respectively.Normal rats is given the sodium chloride injection of identical time and position lumbar injection equivalent.
2.2 administration
The 4th week of modeling, get 3 rats at random and put to death the hepatic lesions situation of observing, after confirmation has hepatic fibrosis to change remaining 46 rats are divided into 1 model group (18) and 2 treatment groups (A, B) with the method for table of random number, A is the former side's extractum of supporting vital QI and dispersing blood stasis group; B is the full constituent group, 14 every group.B treatment group is prepared dosage with corresponding respectively component aqueous solution according to table 1 respectively, with 5mlkg
-1Body weight is irritated stomach.The Cordyceps fat-soluble ingredient is because can not be fine soluble in water, and every 100g rat body weight expense is very little, so press 0.5mlkg with edible oil dilution back
-1Body weight is irritated stomach in addition.A group administration supporting vital QI and dispersing blood stasis extractum, dosage 5mlkg
-1Body weight, normal group and model group are irritated stomach with isodose drinking water and edible oil, and every day 1 time is totally 4 weeks.
Stomach component extract compatibility dosage is irritated in the grouping of table 1 Chinese drug-treated group
2.3 specimen is left and taken
Experiment finishes back rat fasting and can't help water, takes by weighing rat body weight behind the 24h, with 3% pentobarbital sodium 2mlkg
-1The dosage lumbar injection of body weight, postanesthetic rat is opened the abdominal cavity fully, if any ascites, takes by weighing ascites weight after claiming overweight dry cotton ball to blot liquid.Take a blood sample through postcava with the 10ml syringe then, leave standstill 2 hours under 4 ℃ after, 3000rpm, 15min is centrifugal, gets supernatant and is sub-packed in the 1.5ml centrifuge tube ,-70 ℃ of cryopreservation.Animal takes off liver and spleen and claims its weight record after getting blood execution, gets 2 of the big or small hepatic tissues of about 1.0cm * 0.8cm * 0.3cm then, and is fixing in 10% neutral formalin buffer.
2.4 learning, serum biochemistry detects
The active reitman-frankel method that adopts of ALT; The active reitman-frankel method that adopts of AST; Alb content adopts the bromocresol green method; TBiL content adopts sodium benzoate-caffeine colorimetry.
2.5 hepatic tissue hydroxyproline (Hydroxyproline, Hyp) assay
Adopt the JamallShi method to measure
2.6 liver histological is observed
2.6.1 dyeing
The hepatic tissue of formaldehyde fixed, dehydration of alcohol step by step behind the 24h, dimethylbenzene is transparent, 56 ℃ of paraffin embedding, the thick section of 4 μ m is used for HE, day scarlet dyeing of wolf, om observation.
2.6.2 hepatic tissue hemorrhagic necrosis grading standard
The hepatic injury of the rat model that DMN induces is based on hemorrhagic necrosis.Make standard by oneself, observe classification down at light microscopic (200 *).(seeing Table 2)
Table 2 hepatic tissue hemorrhagic necrosis grading
2.6.3 the semi-quantitative standards of hepatic tissue collagen fiber deposition degree
According to the semi-quantitative standards of pertinent literature, observe the hepatic fibrosis degree.
Table 3 hepatic tissue collagen fiber deposition degree by stages
2.6.4 hepatic tissue collagen fiber depositional area image is handled semi-quantitative analysis
Every scarlet staining section of sky wolf is got four jiaos and central authorities totally 5 position pictures taken respectively, adopts image-pro plus6.1 computed in software to go out every pictures collagen deposition area, averages.
2.7 statistical method
Adopt SPSS18.0 software statistics system to carry out statistical disposition.Measurement data with
Expression adopts the ANOVA program to carry out one factor analysis of variance, carries out homogeneity test of variance, and compares in twos with the LSD program; Ranked data are checked with the Ridit method.(be that notable level carry out statistical test with α=0.05).
3 results
3.1 rat ordinary circumstance
Each organizes rat modeling situation: 4 weeks of modeling do not have rats death when finishing.Normal rats did not have death when 8 weeks, experiment finished, dead 2 of model group, and A organizes dead 2, and B organizes dead 3, and the rat mental status is very poor when dead, and weight loss is obvious, and feed reduces, and may be liver failure death.Found when 8 weeks, experiment finished that 11 rats of model group have ascites, AS occurrence is 68.8%.(seeing Table 4)
Table 4 is respectively organized rats death and ascites, and a situation arises
3.2 respectively organize rat body weight, liver body ratio, spleen body frequently
Compare with normal group, the model group rat body weight significantly reduces, and spleen is heavy obviously to be increased, and the spleen body is than increase; Compare with model group, each organizes rat body weight all answer, and wherein the amplitude of A group is bigger, and significant difference is arranged.All the other each indexs change no significant difference between A, B group and model group.(seeing Table 5)
Rats'liver heavily on the same group, spleen is heavy, the liver body compares for table 5, the comparison of spleen body ratio
Compare with normal group:
#P＜0.05,
##P＜0.01; Compare with model group:
*P＜0.05
3.3 rat liver pathological observation
3.3.1 gross examination of skeletal muscle
Normal rats liver color is scarlet, and matter is tender soft, smooth surface, clear-cut margin; The spleen color is dark red, and matter is medium.Model group rat liver quality is harder, and color is dark red, and edge is blunt, and rough surface is compared liver volume with normal group and obviously dwindled, and spleen obviously increases.Each group for the treatment of is compared liver size, color and luster and quality with model group improvement is arranged, and spleen has dwindling in various degree.
The observation 3.3.2 hepatic tissue HE dyes
Normal rats lobules of liver clear in structure, hepatocyte along the liver rope by central vein to around be radial arrangement, no degeneration necrosis, sinus hepaticus do not see narrow and the expansion.Compare with normal group, the model group normal configuration is destroyed, and the portal area spoke gathers; The hepatocyte arrangement disorder though hepatocellular degeneration is not serious, has 5 examples that tangible hemorrhagic necrosis companion inflammatory cell infiltration is arranged, and has 2 examples to also have obvious silt gallbladder; Sinus hepaticus is narrow.Above-mentioned various pathological change also has existence in each treatment group, but than model group alleviating is in various degree arranged.Relatively, A group sinus hepaticus is narrow, the hepatocellular degeneration degree of necrosis is lighter between each treatment group, and the extent of hemorrhage of B group is lighter.(seeing Fig. 1, table 6)
Table 6 is respectively organized liver tissues of rats hemorrhagic necrosis grading
3.3.3 the scarlet dyeing of hepatic tissue sky wolf is observed
Observed result shows that the normal rats liver only has a small amount of collagen fiber deposition around portal area and central vein.Model group rat portal area enlarges liver collagen fiber deposition, and the fibrous septum obviously forms, and part holds the formation pseudolobuli.Compare with model group, each treatment group collagen fiber hypertrophy, deposition degree obviously alleviate.(see figure 2)
3.4 respectively organize rat liver collagen deposition semi-quantitative analysis
Each organizes the rat liver fibrosis by standard sxemiquantitative classification, and analyzes demonstration with Ridit, compares with normal group, and model group significance (P＜0.01) in the treatment group, is compared no significant difference with model group.(seeing Table 7)
Table 7 is respectively organized rat liver collagen deposition sxemiquantitative Ridit and is analyzed
Compare with normal group:
##P＜0.01.
3.5 respectively organizing rat liver collagen deposition area graph picture handles
Model group rat collagen deposition area has been compared tangible increase (P＜0.01) with normal group, B organizes to have significantly and alleviates, and (P＜0.05) (sees Table 8, Fig. 3)
Table 8 component prescription is to the influence of rat liver collagen deposition
Compare with normal group:
##P＜0.01; Compare with model group:
*P＜0.05
3.6 the liver tissues of rats hydroxyproline content changes
With the same period normal group compare, the Hyp content of model group liver tissues of rats obviously increases (P＜0.01); Compare with model group, the Hyp content of A group liver tissues of rats obviously reduces (P＜0.01); The B group is compared with model group, and the Hyp content of liver tissues of rats has obvious reduction (P＜0.05).(see Table 9, Fig. 4)
Table 9 component prescription is to the influence of rat Hyp
Compare with normal group:
##P＜0.01; Compare with model group:
*P＜0.05,
*P＜0.01.
3.7 rat liver biochemical indicator
3.7.1 the component prescription is to the influence of rats with liver cirrhosis Serum ALT, AST activity
With the same period normal group compare, model group rat blood serum ALT is active significantly to raise (P＜0.01); Each group of Drug therapy is compared with model group, active significantly reduce (P＜0.05, P＜0.01) of A group rat blood serum ALT.(see Table 10, Fig. 5).With the same period normal group compare, model group rat blood serum AST is active significantly to raise (P＜0.05); The influence that each treatment group and the same period, model group compared rat blood serum AST activity does not have remarkable change.(see Table 10, Fig. 6)
Table 10 component prescription is to the influence of rats'liver function ALT, AST
Compare with normal group:
#P＜0.05;
##P＜0.01; Compare with model group:
*P＜0.05.
3.7.2 the component prescription is to the influence of rats with liver cirrhosis serum Alb, TBiL activity
With the same period normal group compare active significantly reduce (P＜0.01) of model group rat blood serum Alb; Each group of giving Drug therapy with the same period model group compare the improvement that has in various degree, wherein A group and B organize remarkable statistical significance (P＜0.01).Compare with normal group, model group rat blood serum TBiL is active significantly to raise (P＜0.05); Each group of giving Drug therapy with the same period model group compare the improvement that has in various degree, wherein A group, B group rat blood serum TBiL activity all significantly reduce (P＜0.01).(seeing Table 11, Fig. 7,8).
Table 11 component prescription is to the influence of rats'liver function Alb, TBiL activity
Compare with normal group:
#P＜0.05,
##P＜0.01; Compare with model group:
*P＜0.05,
*P＜0.01 conclusion:
Cordyceps crude polysaccharides, Cordyceps mycelium CO
2The component compatibility of supercritical extract, Herb Gynostemmae Pentaphylli polysaccharides, gypenoside, salvianolic acid and amygdaloside can reappear the former side's of supporting vital QI and dispersing blood stasis anti-hepatic fibrosis effect.
Embodiment 2
The curative effect of supporting vital QI and dispersing blood stasis component compound multi-component medicine-releasing system anti-hepatic fibrosis
1.1 material
1.1.1 animal
79 of Sprague-Dawley (SD) male rats, the SPF level, body weight 150 ± 10g, Chinese Academy of Sciences's Shanghai Experimental Animal Center provides credit number: SCXK (Shanghai): 2007-0005.All rat feedings are in the Experimental Animal Center SPF of Shanghai Univ. of Traditional Chinese Medicine laboratory, and full diet is freely drunk water.
1.1.2 main agents
N-nitrosodimethylamine (dimethylnitrosamine, DMN), available from Tokyo HuaCheng Industry Co., Ltd, lot number: KWM6890; Citric acid, analytical pure, lot number: T20100811; Anhydrous sodium acetate, analytical pure, lot number: F20100707; Trisodium citrate (Tri-sodium citrate), analytical pure, lot number: 20100915; Paradimethylaminobenzaldehyde (4-Dimethylamino benzaldehyde), lot number: 20000428; Chlorine ammonia-T (chloramines-T), analytical pure, lot number:; Dehydrated alcohol, analytical pure, lot number: 20120104; Dimethylbenzene, analytical pure, lot number 20120112; Formaldehyde (Formal dehyde solution), lot number: 20071019; Isopropyl alcohol (Iso-propanol), analytical pure, lot number: 100625; Concentrated hydrochloric acid, analytical pure, lot number: T20090312, more than all available from Shanghai chemical reagents corporation of Chinese Medicine group.Neutral gum, lot number: 20080715, available from the virtuous foreign Instr Ltd. in Shanghai.0.9% sodium chloride injection (Sodium Chloride Injection), lot number: G1108023 is available from Shuanghe Pharmaceutical Ind Co., Ltd., Anhui.Pentobarbital sodium (Pentobarbital Sodium, Lot.No.WS20090920, German packing.Hydroxyproline (hydroxyproline, Hyp) standard substance, analytical pure, Sigma-Aldrich Chemie GmbH, Germany product.Perchloric acid (Perchloric acid), analytical pure, lot number: 20100428, available from Tao Pu chemical plant, Shanghai.Serum liver function test kit, comprise glutamate pyruvate transaminase (alanine aminotransamine, ALT), glutamic oxaloacetic transaminase, GOT (aspartate aminotransferase, AST), albumin (albumin, Alb), total bilirubin (total bilirubin, T.Bil), all available from Shanghai Kehua Bio-engineering Co., Ltd.
1.1.3 medicine
The extract dry powder of supporting vital QI and dispersing blood stasis compound recipe is provided by Shanghai Modern TCM Co., Ltd., and every gram contains the 7.3g crude drug, lot number: 110202.Main quality control index (HPLC mensuration): danshensu sodium 10.48gkg
-1Adenosine 2.5gkg
-1Salvianolic acid B 12.4gkg
-1Moisture content: 6.9%.Dilution is 34.52gkg during application
-1Solution irritate gastric juice.
Supporting vital QI and dispersing blood stasis component compound multi-component medicine-releasing system preparation provides lot number by the modern preparation technique of Shanghai Univ. of Traditional Chinese Medicine's Chinese medicine Ministry of Education Engineering Research Center: 111001.
1.1.4 key instrument
The FIM20 ice machine, Dutch Philips company product.The biochemical special refrigerator of last Pedicellus et Pericarpium Trapae is available from last marine Pedicellus et Pericarpium Trapae company.Cryogenic refrigerator (70 ℃), Forma scientific company product.Desk centrifuge (microfuge lite), Beckman company product.The AA-200 analytical balance, inverted microscope (37 * B) U.S. Denver instrument company products.Pure water system (HPLC/UP), Labconco company product.The CQX25-12 ultrasonic cleaner, Shanghai must ultrasonic company limited product.The roasting sheet machine of rotary microtome (RM2035), HI1220, HI1210 thermostat water bath, LEICA ASP300 automatic dehydration machine, LEICA EG1160 paraffin embedding machine are all available from German Leica company.Instant camera, Polariod company product.XW-80A type vortex mixer, its woods Bel instrument Manufacturing Co., Ltd of Haimen, Jiangsu.ML-902 time constant-temperature magnetic stirring apparatus, Shanghai Pujiang analytical tool factory produces.PH meter
, Beckmanwc company product.1000 μ l, 200 μ l, 20 μ l sample injectors, French Gilson company makes.The multi-functional microplate reader of M5, U.S. Molecular Devices company produces.The Precellys24 homogenizer, French Bertin company produces.Remarkable 450 full automatic biochemical apparatus, Shanghai Kehua Bio-engineering Co., Ltd produces.The automatic slide scanner of Leica Scn400 is available from German Leica company.
1.2 method
1.2.1 model preparation
The SD rat conforms behind the 3d, is divided into normal group (10) and modeling group (69) with the table of random number method.The modeling group is pressed 2mlkg with 0.5%DMN
-1The body weight lumbar injection is injected 2/3 of full dosage first, every day 1 time, injects rest 4d, totally 4 weeks behind the 3d continuously.Preceding 3 days of the 4th week are injected 0.5%DMN full dose, 2/3 amount and 2/3 amount respectively.Normal rats is given the normal saline of identical time and position lumbar injection equivalent.
1.2.2 administration
The supporting vital QI and dispersing blood stasis extract dry powder 1g that Shanghai Modern TCM Co., Ltd. provides contains crude drug amount 7.3g, supposes that people's clinical dosage is Amgkg
-1, the dose,equivalent of rat=6.3 * A.
After the 4th week of modeling finishes, 1 rats death, remaining rat is got 2 rats at random with the method for drawing lots and puts to death the hepatic lesions situation of observing, confirmation is divided into model group (21), the former side's extractum of supporting vital QI and dispersing blood stasis group, the high dose group of supporting vital QI and dispersing blood stasis component compound multi-component medicine-releasing system, middle dosage group, 15 every group with remaining 66 modeling rats with the table of random number method after having hepatic fibrosis to change at random.Each administration group is pressed 10mlkg with the aqueous solution of matched doses respectively for every group
-1Body weight is irritated stomach, and the former side's extractum of supporting vital QI and dispersing blood stasis group dosage is 34.52gkg
-1(crude drug content is equivalent to the body 60kg normal adult consumption of every day), 2 groups of dosages of supporting vital QI and dispersing blood stasis component compound multi-component medicine-releasing system see Table 12, and every day 1 time is totally 4 weeks.Model group and normal group are given the drinking water of equivalent and are irritated stomach.
The dosage of each group of table 12 supporting vital QI and dispersing blood stasis component compound multi-component medicine-releasing system treatment group
1.2.3 specimen is left and taken
Experiment finishes back rat fasting and can't help water, takes by weighing rat body weight behind the 24h, with 3% pentobarbital sodium 2mlkg
-1The dosage lumbar injection of body weight, postanesthetic rat is opened the abdominal cavity fully, if any ascites, takes by weighing ascites weight after claiming overweight dry cotton ball to blot liquid.Take a blood sample through postcava with the 10ml syringe then, leave standstill 3h under 4 ℃ after, 3000rpm, 15min is centrifugal, gets supernatant and is sub-packed in the 1.5ml centrifuge tube ,-70 ℃ of cryopreservation.Animal takes off liver and spleen and claims its weight record after getting blood execution, gets 2 of the big or small hepatic tissues of 1.0cm * 0.8cm * 0.3cm then approximately, and is fixing in 10% neutral formalin buffer.
1.2.4 specimen detects, observes and analyzes
All with embodiment 1.
1.2.5 statistical method
Adopt SPSS18.0 software statistics system to carry out statistical disposition.Measurement data with
Expression adopts the ANOVA program to carry out one factor analysis of variance, carries out homogeneity test of variance, and compares in twos with the LSD program, and ranked data are checked with the Ridit method.(be that notable level carry out statistical test with α=0.05)
2 results
2.1 respectively organizing the rat ordinary circumstance changes
Each organizes rat modeling situation: rats death was 1 when 4 weeks of modeling finished.Normal rats did not have death when 8 week experiments finished, dead 2 of model group, each dead 1 of former side's group, high dose group, middle dosage group, the rat mental status is very poor before dead, and weight loss is obvious, and feed reduces, dissecting and find that flatulence is obvious, may be liver failure death.8 weeks found altogether that 11 rats had ascites when experiment finishes, and wherein model group is 3, and AS occurrence is 15.8%.4 of high dose group.(seeing Table 13)
Table 13 is respectively organized rats death and ascites, and a situation arises
2.2 respectively organize rat body weight, liver body ratio, spleen body frequently
Compare with normal group, the model group rat body weight significantly reduces, heavy obvious decline of liver, and heavy obvious the increasing of spleen, liver body ratio reduces, spleen body ratio increases; Compare with model group, former side's group, high dose group, middle dosage group rat body weight all have answer, and wherein the amplitude of middle dosage group is bigger, and statistical significance is arranged.The liver of middle dosage group is heavy, liver body ratio all gos up to some extent, and significant difference is arranged.(seeing Table 14)
Table 14 is respectively organized the rats'liver weight, spleen is heavy, the liver body compares, the comparison of spleen body ratio
Compare with normal group:
##P＜0.01; Compare with model group,
*P＜0.05,
*P＜0.01.
2.3. rat liver pathological observation
2.3.1 gross examination of skeletal muscle
Normal rats liver color is scarlet, tender soft, the smooth surface of matter, clear-cut margin; The spleen color is dark red, and matter is medium.The model group rat liver obviously dwindles, quality is hard, color is dark red, edge is blunt, rough surface; Spleen obviously increases.Each group for the treatment of is compared liver size, color and luster and quality to be had in various degree and improves with model group, and spleen has dwindling in various degree.
The observation 2.3.2 hepatic tissue HE dyes
Normal rats lobules of liver clear in structure, hepatocyte form the liver rope by central vein to around be radial arrangement, sinus hepaticus is not seen narrow and is expanded.Compare with normal group, the model group leaflet structure destroys, and the portal area enlarges; The hepatocyte arrangement disorder, the loose or hydropic degeneration of a large amount of hepatocyte endochylemas has 5 examples that the hemorrhage and inflammatory cell infiltration of tangible hepatic necrosis companion is arranged; Sinus hepaticus is narrow.The every above-mentioned pathological change of each treatment group has alleviating in various degree than model group.Relatively, former side organizes, middle dosage group hepatocellular degeneration degree is lighter between each treatment group, and there is the hemorrhage and hepatic necrosis of 1 example in former side's group, and middle dosage group does not have hemorrhagic necrosis; Then leaflet structure disorder of high dose group, sinus hepaticus is narrow, hepatocellular degeneration is obvious.(see figure 9)
2.3.3 the scarlet dyeing of hepatic tissue sky wolf is observed
Observed result shows that the normal rats liver only has a small amount of collagen fiber deposition around portal area and central vein.Model group rat liver portal area enlarges, the collagen fiber deposition, and the fibrous septum forms, and partly cuts apart to hold hepatic tissue formation pseudolobuli.Compare with model group, each treatment group collagen fiber hypertrophy, deposition degree obviously alleviate, and be wherein obvious with middle dosage group, rare complete fibrous septum, and the interval that has becomes elongated, and is slim.(see figure 10)
2.4 respectively organize the semi-quantitative analysis of rat liver collagen deposition
Each organizes the rat liver fibrosis by the semi-quantitative standards classification, and analyzes with Ridit.The result shows that compare with normal group, the model group collagen deposition is obvious, and there were significant differences (P＜0.01).Compare with model group, in the treatment group only in the degree of dosage group collagen deposition alleviate and remarkable statistical significance arranged (P＜0.01).All the other compare no significant difference with model group.(seeing Table 15)
Table 15 is respectively organized rat liver collagen deposition sxemiquantitative Ridit and is analyzed
Compare with normal group:
##P＜0.01; Compare with model group,
*P＜0.01
2.5 respectively organizing the image of rat liver area of collagen handles
Model group rat area of collagen has been compared tangible increase (P＜0.01) with normal group, each treatment group is compared with model group, and the area of collagen of former side's group, high dose group, middle dosage group all has significant minimizing, and statistical significance (P＜0.01) is all arranged.(see Table 16, Figure 11)
Table 16 is respectively organized the comparison of rat liver area of collagen
Compare with normal group:
##P＜0.01; Compare with model group,
*P＜0.01
2.6 respectively organizing the liver tissues of rats hydroxyproline content changes
With the same period normal group compare, the Hyp content of model group liver tissues of rats obviously increases (P＜0.01); Compare with model group, the Hyp content of former side's group, middle dosage group liver tissues of rats has obvious reduction (P＜0.05), and is though high dose group liver Hyp content is also lower, more not obvious than difference with model group.(see Table 17, Figure 12)
Table 17 is respectively organized liver tissues of rats Hyp content relatively
Compare with normal group:
##P＜0.01; Compare with model group:
*P＜0.05
2.7 respectively organize the variation of rat liver biochemical indicator
2.7.1 respectively organize the variation of rat blood serum ALT, AST activity
With the same period normal group compare, model group rat blood serum ALT and AST are active significantly to raise, (all P＜0.01); Each treatment group is compared with model group, only the active significantly reduction of former side's group, middle dosage group rat blood serum ALT and AST (all P＜0.05).(see Table 18, Figure 13)
Table 18 is respectively organized rat blood serum ALT, AST specific activity
Compare with normal group:
##P＜0.01; Compare with model group:
*P＜0.05
2.7.2 respectively organize the comparison of rat blood serum Alb, TBiL content
With the same period normal group compare, model group rat blood serum Alb content significantly reduces, (P＜0.01); Each treatment group serum Alb content is compared the raising that has in various degree with model group, wherein middle dosage group has remarkable statistical significance (P＜0.01), the also significantly rising (P＜0.05) of former side's group, high dose group rat blood serum Alb content.
Compare with normal group, model group rat blood serum TBiL content significantly raises, (P＜0.05); Compare with model group, middle dosage group rat blood serum TBiL content significantly reduces (P＜0.01), and former side's group also has obvious reduction (P＜0.05); The serum T BiL content of high dose group changes then than the model group no difference of science of statistics.(seeing Table 19, Figure 14,15).
Table 19 is respectively organized the comparison of rat blood serum Alb and TBiL content
Compare with normal group:
##P＜0.01; Compare with model group,
*P＜0.05,
*P＜0.05
Claims (9)
1. 6 component plant amedica compositionss for the treatment of hepatic fibrosis are respectively by per kilogram of body weight: Cordyceps crude polysaccharides 180-350mg, Cordyceps fat-soluble ingredient 0.06～0.82ml, salvianolic acid 100-190mg, gypenoside 2-68mg, Herb Gynostemmae Pentaphylli polysaccharides 3-12mg, amygdaloside 0.1～4mg.
2. a kind of 6 component plant amedica compositionss for the treatment of hepatic fibrosis according to claim 1, it is characterized in that: by per kilogram of body weight, described Cordyceps crude polysaccharides 230～250mg, Cordyceps fat-soluble ingredient 0.1～0.2ml, salvianolic acid 120～140mg, gypenoside 10～30mg, Herb Gynostemmae Pentaphylli polysaccharides 7～8mg, the amygdaloside 1～2mg of being respectively.
3. a kind of 6 component plant amedica compositionss for the treatment of hepatic fibrosis according to claim 1, it is characterized in that: by per kilogram of body weight, described Cordyceps crude polysaccharides 240mg, Cordyceps fat-soluble ingredient 0.1ml, salvianolic acid 130mg, gypenoside 20mg, Herb Gynostemmae Pentaphylli polysaccharides 7.3mg, the amygdaloside 1mg of being respectively.
4. 6 component plant amedica preparation of compositions methods for the treatment of hepatic fibrosis comprise:
(1) extracts described 6 components: Cordyceps crude polysaccharides, Cordyceps fat-soluble ingredient, salvianolic acid, amygdaloside, gypenoside and Herb Gynostemmae Pentaphylli polysaccharides;
1. Cordyceps fat-soluble ingredient: get the Cordyceps mycelium powder, put into the extraction kettle of supercritical extraction instrument after granulating with the 20vol% ethanol water, extraction conditions: temperature is 45 ℃, and pressure is 30MPa, and the time is 2h, and flow is 35kg/h; Separation condition is: first order separating pressure is 10MPa, and temperature is 50 ℃, and second level separating pressure is 6MPa, and temperature is 40 ℃; Collect and separate the grease that parses, obtain the Cordyceps fat-soluble ingredient;
2. Cordyceps crude polysaccharides: will in 10 times of NaOH aqueous solutions of the Cordyceps mycelium slag behind the supercritical extraction at pH=8, boil 3 hours, totally 2 times, after the merging, centrifugal removing slag, supernatant concentration, adding ethanol to final concentration is 75vol%, refrigerator is placed, and separates out polysaccharide, filters, clean dry obtains the Cordyceps crude polysaccharides;
3. salvianolic acid: get red rooted salvia 1kg, add 10 times of amount deionized waters, boil 1h, medicinal liquid is inclined to, add 10 times of amount deionized waters in the medicinal residues again, boil 1h, medicinal liquid is inclined to, merge extracted twice liquid, leave standstill, filter; Be concentrated into 1.5L, medicinal liquid is than being 1:1.5, add 4.2L95vol% ethanol and be about 70vol% to concentration of alcohol, precipitate with ethanol spends the night, and is centrifugal, to go up solution decompression and be concentrated into nothing alcohol flavor, 1.5L, get the good HZ-816 resin 2L of pretreatment, wet method dress post, do not distinguish the flavor of to there being alcohol with deionized water rinsing, with 1/2 bed volume/hour flow velocity with said extracted concentrated solution upper prop after, with the ethanol elution salvianolic acid of 2.5BV40%, flow velocity is 1BV/h again, collect 40% ethanol elution, drying under reduced pressure gets the yellow solid powder, after measured, the yield of salvianolic acid component is 8%, and wherein the content of phenolic acid such as salvianolic acid B is 25%;
4. amygdaloside: take by weighing Semen Persicae medical material 1kg, 50% ethanol that adds 10 times of amounts, reflux 90min filters, filtrate decompression is concentrated into solid-liquid ratio 1:1, get the good HZ-818 resin 250ml of pretreatment, wet method dress post is not distinguished the flavor of to there being alcohol with deionized water rinsing, with Semen Persicae extract concentrated solution with 1BV/h flow velocity upper prop after, with the 2BV deionized water rinsing, speed is 2BV/h, discards water wash liquid earlier, again with 3BV50vol% ethanol elution amygdaloside, speed is 2BV/h, collects 50% ethanol elution, concentrating under reduced pressure, dry, get the light yellow solid powder, after measured, the yield of Semen Armeniacae Amarum glycoside component is about 0.8%, and wherein amygdaloside content is about 63%;
5. gypenoside: take by weighing Herb Gynostemmae Pentaphylli medical material 1kg, add 60% ethanol of 20 times of amounts, reflux, extract, 2h, filter, filtrate decompression is concentrated into the extractum shape, adds 30% ethanol to solid-liquid ratio 1:3, ultrasonic dissolution 15min, centrifugal, supernatant is crossed macroporous adsorbent resin, gets the good HZ-816 resin 2L of pretreatment, wet method dress post, do not distinguish the flavor of to there being alcohol with deionized water rinsing, Herb Gynostemmae Pentaphylli extract with 1/4BV/h flow velocity upper prop, with the flushing of 2BV water, is discarded leacheate with the 1/2BV/h flow velocity, again under identical flow velocity, with the 2BV30% alcohol flushing, discard leacheate, at last with 3BV95% ethanol elution gypenoside, collect 95% ethanol elution, concentrating under reduced pressure, drying gets the faint yellow solid powder, after measured, the yield of gypenoside class component is about 1%, and wherein Herb Gynostemmae Pentaphylli total glycosides content is 40%;
6. Herb Gynostemmae Pentaphylli polysaccharides: will 5. extract the Herb Gynostemmae Pentaphylli medicinal residues and dry naturally, and take by weighing medicinal residues 500g, with 15 times of amount deionized water reflux, extract, 1h, filter, filtrate decompression is concentrated into solid-liquid ratio 1:1, adds 95% ethanol 900ml, to containing alcohol amount 70%, precipitate with ethanol spends the night, and filters, filter cake is dried with 95% ethanol twice, pulverizes, get yellow Herb Gynostemmae Pentaphylli polysaccharides powder, after measured, the yield of Herb Gynostemmae Pentaphylli soap polysaccharide component is 4%, and wherein Herb Gynostemmae Pentaphylli polysaccharides content is 15%;
(2) by described per kilogram of body weight salvianolic acid, Cordyceps crude polysaccharides and Herb Gynostemmae Pentaphylli polysaccharides are made the water solublity micropill as the A part, gypenoside, the fat-soluble part of Cordyceps and amygdaloside are made the solid dispersion micropill as the B part, and A and B press mass ratio 8:0.3; Wherein, A part drug loading is that 33%, B part drug loading is 2.5%.
5. a kind of 6 component plant amedica compositionss for the treatment of hepatic fibrosis according to claim 1, it is characterized in that: the preparation of making the specific part that is applied to intestinal, consumption is according to type and the order of severity adjustment of patient's age, body weight, the disease for the treatment of, and one or many is used.
6. a kind of 6 component plant amedica compositionss for the treatment of hepatic fibrosis according to claim 1 or 5, it is characterized in that: described preparation is tablet or capsule.
7. a kind of 6 component plant amedica compositionss for the treatment of hepatic fibrosis according to claim 6, it is characterized in that: described capsule is made microcapsule or dispersion micropill.
8. a kind of 6 component plant amedica compositionss for the treatment of hepatic fibrosis according to claim 7 is characterized in that: every 100g body weight filling stomach dosage: A part 18.9～37.8mg, B part 0.709～1.418mg.
9. a kind of 6 component plant amedica compositionss for the treatment of hepatic fibrosis according to claim 8 is characterized in that: every 100g body weight filling stomach dosage: A part 18.9mg, B part 0.709mg or A part 37.8mg, B part 1.418mg.
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| CN107126452A (en) * | 2016-02-26 | 2017-09-05 | 上海中医药大学附属曙光医院 | Gypenosides and preparation method thereof |
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