CN103191173B - Five-ingredient botanical composition for treating hepatic fibrosis and preparation method thereof - Google Patents
Five-ingredient botanical composition for treating hepatic fibrosis and preparation method thereof Download PDFInfo
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- CN103191173B CN103191173B CN201310111665.5A CN201310111665A CN103191173B CN 103191173 B CN103191173 B CN 103191173B CN 201310111665 A CN201310111665 A CN 201310111665A CN 103191173 B CN103191173 B CN 103191173B
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- cordyceps
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- herb gynostemmae
- gynostemmae pentaphylli
- polysaccharides
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- JWBPVFVNISJVEM-UHFFFAOYSA-M sodium caffeine benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1.CN1C(=O)N(C)C(=O)C2=C1N=CN2C JWBPVFVNISJVEM-UHFFFAOYSA-M 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000000528 statistical test Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- 239000003440 toxic substance Substances 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
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Abstract
The invention relates to a five-ingredient botanical composition for treating hepatic fibrosis and a preparation method thereof. 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, 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 five ingredients, namely, the crude cordyceps polysaccharide, the fat-soluble cordyceps ingredient, the amygdalin, the gynostemma pentaphyllum saponin and the gynostemma pentaphyllum polysaccharide; and (2) preparing original ingredient liquid and various clinical preparations according to combination doses. The five-ingredient botanical composition has the same role as that of a composition of cordyceps mycelia, red sage root and gynostemma pentaphyllum and can obviously improve the treatment effect and reduce side effects, thereby having good clinical application prospects.
Description
Technical field
The invention belongs to plant amedica components composition for the treatment of hepatic fibrosis and preparation method thereof, particularly a kind of 5 component botanical drug compositions for the treatment of hepatic fibrosis and preparation method thereof.
Background technology
China is the hotspot of the hepatic disease such as chronic viral hepatitis B in the world, and the health of chronic hepatopathy to our people causes significant damage for a long time.But, current treating liver fibrosis or chronic hepatopathy treatment in a large difficult point.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.Research for many years confirms that FUZHENG HUAYU JIAONANG effectively can improve chronic hepatitis Bhepatic fibrosis, liver tissue fibrosis reversion rate by stages (more than comparatively decline before treatment 1 phase or 1 phase) reaches 52%, by the clinical observation of 2 years by a definite date, show the upper gastrointestinal hemorrhage rate that this medicine can reduce Cirrhotic Patients.
But the preparation of FUZHENG HUAYU JIAONANG is relatively backward at present, and 5 capsules of once taking medicine, cause some patients gastrointestinal reaction, although the tablet developed can reduce stomach discomfort, but still there is the demand improving treatment hepatic fibrosis clinical efficacy further." pharmacology " records, and the amygdaloside in Semen Persicae can produce a large amount of hydrocyanic acids after intestinal digestive enzyme decomposes, and occurs hydrocyanism.Although never there is patient clinically to occur poisoning event because taking supporting vital QI and dispersing blood stasis compound recipe, how to prevent potential serious toxic and side effects very necessary.Therefore, further secondary development supporting vital QI and dispersing blood stasis Chinese medicine compound, curative effect is improved again in 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 is to provide a kind of 5 component botanical drug compositions for the treatment of hepatic fibrosis and preparation method thereof, can significantly improve curative effect, reduces side effect, has good potential applicability in clinical practice.
A kind of 5 component botanical drug compositions for the treatment of hepatic fibrosis of the present invention, raw material is made up of Cordyceps mycelium, Semen Persicae and Herb Gynostemmae Pentaphylli.
The compatibility dosage of described 5 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, 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 and amygdaloside 1 ~ 2mg.
Preferably further to 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 and amygdaloside 1mg.
A kind of preparation method for the treatment of 5 component botanical drug compositions of hepatic fibrosis of the present invention, comprising:
(1) 5 components described in extraction: Cordyceps crude polysaccharides, Cordyceps fat-soluble ingredient, amygdaloside, gypenoside and Herb Gynostemmae Pentaphylli polysaccharides;
1. Cordyceps fat-soluble ingredient: get Cordyceps mycelium powder; put into the extraction kettle of supercritical extraction instrument after granulating with 20% ethanol water, extraction conditions: temperature is 45 DEG C, pressure is 30MPa; time is 2h; flow is 35kg/h, and separation condition is: first order separating pressure is 10MPa, and temperature is 50 DEG C; second level separating pressure is 6MPa; temperature is 40 DEG C, collects the grease being separated and parsing, obtains Cordyceps fat-soluble ingredient;
2. Cordyceps crude polysaccharides: the Cordyceps mycelium slag after supercritical extraction is boiled 3 hours in 10 times of NaOH aqueous solutions of pH=8, totally 2 times, after merging, centrifugally to remove slag; supernatant concentration, adding ethanol to final concentration is 75%, and refrigerator is placed; separate out polysaccharide, filter, clean dry;
3. amygdaloside: take Semen Persicae medical material 1kg, add 50% ethanol of 10 times amount, reflux 90min, filter, filtrate reduced in volume is to solid-liquid ratio 1:1, get the HZ-818 resin 250ml that pretreatment is good, wet method dress post, with deionized water rinsing extremely without alcohol taste, Semen Persicae is extracted concentrated solution with after 1BV/h flow velocity upper prop, first with 2BV deionized water rinsing, speed is 2BV/h, discard water wash liquid, again with 3BV50% ethanol elution amygdaloside, speed is 2BV/h, collect 50% ethanol elution, concentrating under reduced pressure, dry, obtain light yellow solid powder, after measured, the yield of Semen Armeniacae Amarum glycoside component is about 0.8%, wherein amygdaloside content is about 63%,
4. gypenoside: take Gynostemma pentaphyllum 1kg, add 60% ethanol of 20 times amount, reflux, extract, 2h, filter, filtrate reduced in volume is to extractum shape, add 30% ethanol to solid-liquid ratio 1:3, ultrasonic dissolution 15min, centrifugal, supernatant crosses macroporous adsorbent resin, get the HZ-816 resin 2L that pretreatment is good, wet method dress post, with deionized water rinsing extremely without alcohol taste, by Herb Gynostemmae Pentaphylli extract with 1/4BV/h flow velocity upper prop, rinse with 1/2BV/h flow velocity 2BV water, discard leacheate, again under identical flow velocity, with 2BV30% alcohol flushing, discard leacheate, last with 3BV95% ethanol elution gypenoside, collect 95% ethanol elution, concentrating under reduced pressure, dry, obtain faint yellow solid powder, after measured, the yield of gypenoside class component is about 1%, wherein Herb Gynostemmae Pentaphylli total glycosides content is about 40%,
5. Herb Gynostemmae Pentaphylli polysaccharides: will 4. in extraction process Herb Gynostemmae Pentaphylli medicinal residues naturally dry, take medicinal residues 500g, with 15 times amount deionized water reflux, extract, 1h, filter, filtrate reduced in volume, to solid-liquid ratio 1:1, adds the ethanol 900ml of 95%, to alcohol content about 70%, precipitate with ethanol spends the night, and filters, filter cake 95% ethanol twice, dries, and pulverizes, obtain 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 compatibility dosage, component stock solution is made.
Described component stock solution is applied to the preparation of the specific part slow release of intestinal, and consumption, according to age of route of administration, patient, body weight, the adjustment such as type and the order of severity of disease for the treatment of, can be used by one or many.
Described plant amedica compositions can be made into solid preparation, as tablet or capsule.
Described plant amedica compositions can be made into microcapsule.
Through experiment for many years and clinical research, the mechanism of action of FUZHENG HUAYU JIAONANG anti-hepatic fibrosis and material base are clearer and more definite.Amygdaloside in component, the Semen Persicaes such as the Cordyceps polysaccharide in research discovery Cordyceps mycelium and the salviol acid A in Radix Salviae Miltiorrhizae, B have remarkable effect of anti hepatic fibrosis.As Cordyceps polysaccharide can promote I, III Collagen Type VI degraded, obviously reduce the content of fibrosis rat liver tissue TGF-β 1 and TBRI thereof; Amygdaloside in Semen Persicae significantly can 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, suppress go down to posterity hepatic stellate cell (HSC) propagation and collagenation, suppress the signal transduction of TGF-β 1 in HSC born of the same parents, thus reduce the total amount of the HSC secretion TGF-B1 that goes down to posterity and suppress it to activate.And salviol acid A suppresses collage synthesis by significant anti peroxidation of lipid damaging action.Once Effect of Fuzheng Huayu Recipe in Treating CCl was used
4the in type rat liver fibrosis of induction, finds that each herbal medicine in Fuzheng Huayu Recipe mainly contains following characteristics: the 1) principal agent of collagen fiber degraded in anti-hepatic fibrosis, promotion liver in the Semen Persicae side of being; 2) Radix Salviae Miltiorrhizae is to improving liver function, and short albumin synthesis and reduction serum bilirubin level play a major role; 3) Cordyceps mycelium is the factor of influence reducing serum total bilirubin content; 4) Herb Gynostemmae Pentaphylli is that the party protects hepatocyte, reduces the principal agent of serum ALT activities.
The present invention picks in Fuzheng Huayu Recipe the Cordyceps with " righting " effect, has Semen Persicae that " blood circulation promoting and blood stasis dispelling " act on and has the 3 taste Chinese medicines such as Herb Gynostemmae Pentaphylli that " heat-clearing and toxic substances removing " act on.Therefrom extract and purification 5 kinds of active components: 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, screen, and analyze by NONMEN method these 5 kinds of extraction components, the optimum compatibility dosage of active component group filtering out Fuzheng Huayu Recipe is: Cordyceps crude polysaccharides (240mgkg
-1), Cordyceps fat-soluble ingredient (0.1mlkg
-1), Herb Gynostemmae Pentaphylli polysaccharides (7.3mgkg
-1), gypenoside (20mgkg
-1) and amygdaloside (1mgkg
-1).
beneficial effect
Plant amedica combination of components raw material of the present invention is made up of Cordyceps mycelium, Semen Persicae and Herb Gynostemmae Pentaphylli.There is through zoopery the antifibrosis therapy effect of anti-hepatic fibrosis and other organs, its effect be made up of Cordyceps mycelium, Radix Salviae Miltiorrhizae and Herb Gynostemmae Pentaphylli identical, the more former compound plant medicine of curative effect has the trend of potentiation, be conducive to Clinical pharmacokinetics and pharmacological research, have a good application prospect.
Accompanying drawing explanation
In the following drawings, A group is supporting vital QI and dispersing blood stasis former side's extractum group; C group is the present invention;
Fig. 1 is normal group, model group, A group and C group rat liver HE colored graph (× 200);
Fig. 2 is normal group, model group, A group and C group rat liver sirius red colored graph (× 100);
Fig. 3 is normal group, model group, A group and C group rat liver collagen deposition area change;
Fig. 4 is the changes of contents of normal group, model group, A group and C group liver tissues of rats Hyp;
Fig. 5 is that normal group, model group, A group and C group rat blood serum ALT are active;
Fig. 6 is that normal group, model group, A group and C group rat blood serum AST are active;
Fig. 7 is normal group, model group, A group and C group rat blood serum Alb content;
Fig. 8 is normal group, model group, A group and C group rat blood serum TBiL content.
Detailed description of the invention
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after the content of having read the present invention's instruction.
Embodiment 1
The anti-hepatic fibrosis curative effect of supporting vital QI and dispersing blood stasis compound recipe active component compound recipe:
1.1 animals:
Wistar male rat 59, SPF level, body weight 150 ± 10g, Chinese Academy of Sciences's Shanghai Experimental Animal Center provides, and credit number: SCXK (Shanghai): 2007-0005.All rat feedings are in Shanghai Univ. of Traditional Chinese Medicine Experimental Animal Center SPF laboratory, and full diet, freely drinks water.
1.2 main agents:
N-nitrosodimethylamine (dimethylnitrosamine, DMN), purchased 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, above all purchased from Solution on Chemical Reagents in Shanghai company of Chinese Medicine group.Neutral gum, lot number: 60090715, purchased from Shanghai Yi Yang Instrument Ltd..0.9% sodium chloride injection (Sodium Chloride Injection), lot number: 090903, purchased from Shuanghe Pharmaceutical Ind Co., Ltd., Anhui.Pentobarbital sodium (Pentobarbital Sodium, Lot.No.WS20090920, German subpackage.Hydroxyproline (hydroxyproline, Hyp) standard substance, analytical pure, Sigma-Aldrich Chemie GmbH, Germany product.Perchloric acid (Perchloric acid), analytical pure, lot number: 20070807 purchased from Tao Pu chemical plant, Shanghai.Serum liver functional reagent box, comprise glutamate pyruvate transaminase (alanine aminotransamine, ALT), glutamic oxaloacetic transaminase, GOT (aspartate aminotransferase, AST), albumin (albumin, Alb), total bilirubin (total bilirubin, T.Bil), all Bioengineering Research Institute is built up purchased from Nanjing.
1.3 medicines:
Cordyceps crude polysaccharides, Cordyceps fat-soluble ingredient, amygdaloside, gypenoside, the same Part I 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%.During application, dilution is the solution gavage liquid of 94.5mg/ml.
1.4 key instruments:
FIM20 ice machine, Dutch Phillips product.The biochemical special refrigerator of upper Pedicellus et Pericarpium Trapae, purchased from upper marine Pedicellus et Pericarpium Trapae company.Cryogenic refrigerator (-70 DEG C), Forma scientific Products.Desk centrifuge (microfuge lite), Beckman Products.AA-200 analytical balance, Denver instrument company of inverted microscope (37 × B) U.S. product.Pure water system (HPLC/UP), Labconco Products.CQX25-12 ultrasonic cleaner, Shanghai must ultrasonic company limited product.Rotary microtome (RM2035), HI1220 bake sheet machine, HI1210 thermostat water bath, and LEICA ASP300 automatic dehydrator, LEICA EG1160 paraffin wax embedding, all purchased from German Leica company.Instant camera, Polariod Products.XW-80A type vortex mixer, its woods Bel instrument manufacturing company limited of Haimen, Jiangsu.ML-902 time constant-temperature magnetic stirring apparatus, Shanghai Pujiang analytical tool factory produces.PH meter (
), Beckmanwc Products.1000 μ l, 200 μ l, 20 μ l sample injectors, French Gilson company manufactures.The multi-functional microplate reader of M5, Molecular Devices company of the U.S. produces.Precellys24 homogenizer, French Bertin company produces.
2.1 model preparations
Wistar male rat 59, after conforming 3 days, is divided into normal group (10) and modeling group (49) at random by the method for table of random number.Modeling group 0.5%DMN presses 2mlkg
-1body weight lumbar injection, injects 2/3 of full dosage first, every day 1 time, continuously rest 4d after injection 3d, totally 3 weeks, within first 3 days of the 4th week, injects 0.5%DMN full dose, 2/3 amount and 1/2 amount respectively.Normal rats gives the sodium chloride injection of identical time and position lumbar injection equivalent.
2.2 administration
Modeling the 4th week, get 3 rats at random and put to death observation hepatic lesions situation, after confirming have hepatic fibrosis to change, the method for remaining 46 rat table of random numbers is divided into 1 model group (18) and 2 treatment groups (A, C), A is supporting vital QI and dispersing blood stasis former side's extractum group; C is the present invention, often organizes 14.C treatment group component aqueous solution prepares dosage according to table 1, with 5mlkg
-1body weight gavage.Wherein Cordyceps fat-soluble ingredient is due to can not be soluble in water very well, and every 100g rat body weight expense is very little, so press 0.5mlkg with after edible oil dilution
-1the other gavage of body weight.A group administration supporting vital QI and dispersing blood stasis extractum, dosage 5mlkg
-1body weight, normal group and model group isodose drinking water and edible oil gavage, totally 4 weeks every day 1 time.
Table 1 Chinese drug-treated group grouping gavage component extract compatibility dosage
2.3 sample reception
Experiment terminates rear Rat Fast and can't help water, takes rat body weight, with 3% pentobarbital sodium 2mlkg after 24h
-1the dosage lumbar injection of body weight, postanesthetic rat opens abdominal cavity completely, if any ascites, takes ascites weight after blotting liquid to claim overweight dry cotton ball.Then take a blood sample through postcava with 10ml syringe, leave standstill after 2 hours at 4 DEG C, 3000rpm, 15min are centrifugal, get supernatant and are sub-packed in 1.5ml centrifuge tube ,-70 DEG C of cryopreservation.Animal is got after blood puts to death, and takes off liver and spleen claims its weight record, then gets about 1.0cm × 0.8cm × 0.3cm size hepatic tissue 2 pieces, fixing in 10% neutral buffered formalin.
2.4 Serum bichemisbry detect
ALT activity adopts reitman-frankel method; AST activity adopts reitman-frankel method; Alb content adopts Bromocresol green; TBiL content adopts sodium benzoate-caffeine colorimetry.
2.5 hepatic tissue hydroxyproline (Hydroxyproline, Hyp) assays
JamallShi method is adopted to measure
2.6 liver histologicals are observed
2.6.1 dyeing
The hepatic tissue that formaldehyde is fixing, dehydration of alcohol step by step after 24h, dimethylbenzene is transparent, 56 DEG C of paraffin embedding, 4 μm of thick sections, for HE, sirius red dyeing, om observation.
2.6.2 hepatic tissue hemorrhagic necrosis grading standard
The hepatic injury of the rat model of DMN induction, based on hemorrhagic necrosis.Make standard by oneself, under light microscopic (200 ×), observe classification.(see 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 hepatic fibrosis degree.
Table 3 hepatic tissue Collagen fiber deposition degree by stages
2.6.4 hepatic tissue Collagen fiber deposition area image procossing semi-quantitative analysis
Often open sirius red staining section and get corner and central authorities' totally 5 position pictures taken respectively, adopt image-pro plus6.1 computed in software to go out every pictures collagen deposition area, average.
2.7 statistical method
SPSS18.0 software statistics system is adopted to carry out statistical disposition.Measurement data with
represent, adopt ANOVA program to carry out one factor analysis of variance, carry out homogeneity test of variance, and compare between two by LSD program; Ranked data Ridit method is checked.(with α=0.05 for notable level carries out statistical test).
3 results
3.1 rat ordinary circumstances
Each group of rat modeling situation: modeling at the end of 4 weeks without rats death.At the end of experiment in 8 weeks, normal rats is without death, dead 3 of model group, dead 2 of A group, dead 2 of C group, and time dead, the rat mental status is very poor, and weight loss is obvious, and feed reduces, and may be that liver failure is dead.8 weeks experiment at the end of find that model group 11 rats have ascites, AS occurrence be the AS occurrence of 68.8%, C group minimum be 33.3%.(see table 4)
A situation arises for table 4 each group rats death and ascites
3.2 respectively organize rat body weight, liver body ratio, spleen body comparatively frequently
Compare with normal group, model group rats body weight significantly reduces, and spleen is heavy obviously to be increased, and spleen body is than increasing; Compare with model group, each group rat body weight all has reply, and wherein the amplitude of A group is comparatively large, has significant difference.All the other each index changes no significant difference between A, C group and model group.(see table 5)
The comparison of rats'liver weight, spleen weight, liver body ratio, spleen body ratio respectively organized by table 5
| Grouping | N | Body weight (g) | Liver heavy (g) | Spleen heavy (g) | Liver body ratio | Spleen body ratio |
| Normal group | 10 | 324±23.3 | 8.95±1.02 | 0.71±0.074 | 0.028±0.0019 | 0.0022±0.0003 |
| Model group | 16 | 256±43.9 ＃＃ | 7.54±1.96 | 1.44±0.263 ＃ | 0.029±0.0040 | 0.0059±0.0017 ＃ |
| A group | 12 | 290±36.9 * | 8.56±2.01 | 1.34±0.278 | 0.029±0.0043 | 0.0047±0.0011 |
| C group | 12 | 276±31.9 | 8.23±2.07 | 1.41±0.219 | 0.029±0.0046 | 0.0052±0.0011 |
Compare with normal group:
#p < 0.05,
##p < 0.01; Compare with model group:
*p < 0.05
3.3 rat liver pathological observations
3.3.1 gross examination of skeletal muscle
Normal rats liver color is scarlet, and matter is tender soft, smooth surface, clear-cut margin; Spleen color is dark red, and matter is medium.Model group rats Liver on Judge Hardness is comparatively hard, and color is dark red, and edge is blunt, rough surface, and liver volume obviously reduces compared with normal group, and spleen obviously increases.Treat each group of liver size compared with model group, color and luster and quality to be all improved, spleen has reducing in various degree.
3.3.2 hepatic tissue HE dyes and observes
Normal rats lobules of liver clear in structure, hepatocyte is radially arranged to surrounding along liver rope by central vein, and without degeneration necrosis, sinus hepaticus has no narrow and expansion.Compared with normal group, model group normal configuration is destroyed, and portal area spoke gathers; Hepatocyte arrangement disorder, although hepatocellular degeneration is not serious, has 5 examples to have obvious hemorrhagic necrosis to accompany inflammatory cell infiltration, has the 2 routine gallbladders that obviously become silted up in addition; Sinus hepaticus is narrow.Above-mentioned various pathological change also has existence in each treatment group, but has alleviating in various degree compared with model group.Compare between each treatment group, A group sinus hepaticus is narrow, hepatocellular degeneration degree of necrosis is lighter.(see Fig. 1, table 6)
Table 6 is group liver tissues of rats hemorrhagic necrosis grading respectively
3.3.3 the dyeing of hepatic tissue sirius red is observed
Observed result shows, and normal rats liver only has a small amount of Collagen fiber deposition around portal area and central vein.Model group rats portal area expands Liver Collagen fiber laydown, and fibrous septum is obviously formed, and part holds formation pseudolobuli.Compared with model group, each treatment group collagen fiber hyperplasia, deposition degree obviously alleviate, and wherein obvious with C group, rare complete fibrous septum, some intervals become elongated, slim.(see figure 2)
3.4 respectively organize rat liver collagen deposition semi-quantitative analysis
Each group of rat liver fibrosis by standard sxemiquantitative classification, and carries out analysis display with Ridit, and compared with normal group, model group significance (P < 0.01), respectively organizes no significant difference.In treatment group, compared with model group, only C group significance (P < 0.01).(see table 7)
Table 7 respectively group rat liver collagen deposition sxemiquantitative Ridit is analyzed
Compare with normal group:
##p < 0.01; Compare with model group:
*p < 0.01.
3.5 rat liver collagen deposition area image procossing
Model group rats collagen deposition area has obvious increase (P < 0.01) compared with normal group, and C group has significant minimizing, has statistical significance (P < 0.01).(see table 8, Fig. 3)
Table 8 component formula is on the impact of rat liver collagen deposition
Compare with normal group:
##p < 0.01; Compare with model group:
*p < 0.01
3.6 liver tissues of rats hydroxyproline content changes
Compared with same period normal group, the Hyp content of model group rats hepatic tissue obviously increases (P < 0.01); Compared with model group, the Hyp content of A group liver tissues of rats obviously reduces (P < 0.01); C 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 formula is on the impact 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 indicators
3.7.1 different component formula is on the impact of rats with liver cirrhosis Serum ALT, AST activity
Compared with same period normal group, model group rats serum ALT activities significantly raises, (P < 0.01); Each group of Drug therapy compared with model group, A group rat blood serum ALT is active significantly reduces (P < 0.05, P < 0.01).(see table 10, Fig. 5).Compared with same period normal group, model group rats serum AST is active significantly to be raised, (P < 0.05); Each treatment group and the same period model group compare the impact of rat blood serum AST activity without remarkable change.(see table 10, Fig. 6)
Table 10 component formula is on the impact of Liver Function ALT, AST
Compare with normal group:
#p < 0.05;
##p < 0.01; Compare with model group:
*p < 0.05.
3.7.2 different component formula is on the impact of rats with liver cirrhosis serum Alb, TBiL activity
Compared with same period normal group, model group rats serum Alb is active significantly reduces (P < 0.01); Give each group of Drug therapy with the improvement had compared with model group the same period in various degree, wherein A group has remarkable statistical significance (P < 0.01), and C group rat blood serum Alb is active significantly raises (P < 0.05).Compared with normal group, model group rats serum T BiL is active significantly to be raised, (P < 0.05); Give each group of Drug therapy with the improvement had compared with model group the same period in various degree, wherein A group, C group rat blood serum TBiL activity all significantly reduce (P < 0.01).(see table 11, Fig. 7,8).
Table 11 component formula is on the impact of 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.
Claims (7)
1. treat 5 component botanical drug compositions of hepatic fibrosis, be 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, amygdaloside 0.1 ~ 4mg; Wherein, the extracting method of each component is as follows:
1. Cordyceps fat-soluble ingredient: get Cordyceps mycelium powder, put into the extraction kettle of supercritical extraction instrument after granulating with 20% ethanol water, extraction conditions: temperature is 45 DEG C, pressure is 30MPa, time is 2h, flow is 35kg/h, and separation condition is: first order separating pressure is 10MPa, and temperature is 50 DEG C, second level separating pressure is 6MPa, temperature is 40 DEG C, collects the grease being separated and parsing, obtains Cordyceps fat-soluble ingredient;
2. Cordyceps crude polysaccharides: the Cordyceps mycelium slag after supercritical extraction is boiled 3 hours in 10 times of NaOH aqueous solutions of pH=8, totally 2 times, after merging, centrifugally to remove slag, supernatant concentration, adding ethanol to final concentration is 75%, and refrigerator is placed, separate out polysaccharide, filter, clean dry;
3. amygdaloside: take Semen Persicae medical material 1kg, add 50% ethanol of 10 times amount, reflux 90min, filter, filtrate reduced in volume is to solid-liquid ratio 1:1, get the HZ-818 resin 250ml that pretreatment is good, wet method dress post, with deionized water rinsing extremely without alcohol taste, Semen Persicae is extracted concentrated solution with after 1BV/h flow velocity upper prop, first with 2BV deionized water rinsing, speed is 2BV/h, discard water wash liquid, again with 3BV50% ethanol elution amygdaloside, speed is 2BV/h, collect 50% ethanol elution, concentrating under reduced pressure, dry, obtain light yellow solid powder, after measured, the yield of Semen Armeniacae Amarum glycoside component is about 0.8%, wherein amygdaloside content is about 63%,
4. gypenoside: take Gynostemma pentaphyllum 1kg, add 60% ethanol of 20 times amount, reflux, extract, 2h, filter, filtrate reduced in volume is to extractum shape, add 30% ethanol to solid-liquid ratio 1:3, ultrasonic dissolution 15min, centrifugal, supernatant crosses macroporous adsorbent resin, get the HZ-816 resin 2L that pretreatment is good, wet method dress post, with deionized water rinsing extremely without alcohol taste, by Herb Gynostemmae Pentaphylli extract with 1/4BV/h flow velocity upper prop, rinse with 1/2BV/h flow velocity 2BV water, discard leacheate, again under identical flow velocity, with 2BV 30% alcohol flushing, discard leacheate, last with 3BV 95% ethanol elution gypenoside, collect 95% ethanol elution, concentrating under reduced pressure, dry, obtain faint yellow solid powder, after measured, the yield of gypenoside class component is about 1%, wherein Herb Gynostemmae Pentaphylli total glycosides content is 40%,
5. Herb Gynostemmae Pentaphylli polysaccharides: will 4. in extraction process Herb Gynostemmae Pentaphylli medicinal residues naturally dry, take medicinal residues 500g, with 15 times amount deionized water reflux, extract, 1h, filter, filtrate reduced in volume, to solid-liquid ratio 1:1, adds the ethanol 900ml of 95%, to alcohol content about 70%, precipitate with ethanol spends the night, and filters, filter cake 95% ethanol twice, dries, and pulverizes, obtain 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. a kind of 5 component botanical drug compositions for the treatment of hepatic fibrosis according to claim 1, it is characterized in that: by per kilogram of body weight, described in be respectively Cordyceps crude polysaccharides 230 ~ 250mg, Cordyceps fat-soluble ingredient 0.1 ~ 0.2ml, gypenoside 10 ~ 30mg, Herb Gynostemmae Pentaphylli polysaccharides 7 ~ 8mg and amygdaloside 1 ~ 2mg.
3. a kind of 5 component botanical drug compositions for the treatment of hepatic fibrosis according to claim 1, it is characterized in that: by per kilogram of body weight, described in be respectively Cordyceps crude polysaccharides 240mg, Cordyceps fat-soluble ingredient 0.1ml, gypenoside 20mg, Herb Gynostemmae Pentaphylli polysaccharides 7.3mg and amygdaloside 1mg.
4. 5 component botanical drug compositions according to claim 1, is characterized in that: the preparation making the specific part being applied to intestinal.
5. 5 component botanical drug compositions according to claim 5, is characterized in that: described preparation is tablet or capsule.
6. 5 component botanical drug compositions according to claim 6, is characterized in that: microcapsule made by described capsule.
7. a preparation method for 5 component botanical drug compositions for the treatment of hepatic fibrosis as claimed in claim 1, comprising:
(1) described 5 components are extracted: Cordyceps crude polysaccharides, Cordyceps fat-soluble ingredient, amygdaloside, gypenoside and Herb Gynostemmae Pentaphylli polysaccharides;
1. Cordyceps fat-soluble ingredient: get Cordyceps mycelium powder, put into the extraction kettle of supercritical extraction instrument after granulating with 20% ethanol water, extraction conditions: temperature is 45 DEG C, pressure is 30MPa, time is 2h, flow is 35kg/h, and separation condition is: first order separating pressure is 10MPa, and temperature is 50 DEG C, second level separating pressure is 6MPa, temperature is 40 DEG C, collects the grease being separated and parsing, obtains Cordyceps fat-soluble ingredient;
2. Cordyceps crude polysaccharides: the Cordyceps mycelium slag after supercritical extraction is boiled 3 hours in 10 times of NaOH aqueous solutions of pH=8, totally 2 times, after merging, centrifugally to remove slag, supernatant concentration, adding ethanol to final concentration is 75%, and refrigerator is placed, separate out polysaccharide, filter, clean dry;
3. amygdaloside: take Semen Persicae medical material 1kg, add 50% ethanol of 10 times amount, reflux 90min, filter, filtrate reduced in volume is to solid-liquid ratio 1:1, get the HZ-818 resin 250ml that pretreatment is good, wet method dress post, with deionized water rinsing extremely without alcohol taste, Semen Persicae is extracted concentrated solution with after 1BV/h flow velocity upper prop, first with 2BV deionized water rinsing, speed is 2BV/h, discard water wash liquid, again with 3BV50% ethanol elution amygdaloside, speed is 2BV/h, collect 50% ethanol elution, concentrating under reduced pressure, dry, obtain light yellow solid powder, after measured, the yield of Semen Armeniacae Amarum glycoside component is about 0.8%, wherein amygdaloside content is about 63%,
4. gypenoside: take Gynostemma pentaphyllum 1kg, add 60% ethanol of 20 times amount, reflux, extract, 2h, filter, filtrate reduced in volume is to extractum shape, add 30% ethanol to solid-liquid ratio 1:3, ultrasonic dissolution 15min, centrifugal, supernatant crosses macroporous adsorbent resin, get the HZ-816 resin 2L that pretreatment is good, wet method dress post, with deionized water rinsing extremely without alcohol taste, by Herb Gynostemmae Pentaphylli extract with 1/4BV/h flow velocity upper prop, rinse with 1/2BV/h flow velocity 2BV water, discard leacheate, again under identical flow velocity, with 2BV 30% alcohol flushing, discard leacheate, last with 3BV 95% ethanol elution gypenoside, collect 95% ethanol elution, concentrating under reduced pressure, dry, obtain faint yellow solid powder, after measured, the yield of gypenoside class component is about 1%, wherein Herb Gynostemmae Pentaphylli total glycosides content is 40%,
5. Herb Gynostemmae Pentaphylli polysaccharides: will 4. in extraction process Herb Gynostemmae Pentaphylli medicinal residues naturally dry, take medicinal residues 500g, with 15 times amount deionized water reflux, extract, 1h, filter, filtrate reduced in volume, to solid-liquid ratio 1:1, adds the ethanol 900ml of 95%, to alcohol content about 70%, precipitate with ethanol spends the night, and filters, filter cake 95% ethanol twice, dries, and pulverizes, obtain 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 compatibility dosage, component stock solution is made.
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