CN103800320B - Application of medicinal composition containing dehydroandrographolide succinate and pharmaceutically-acceptable salt thereof to preparation of veterinary immunopotentiator - Google Patents
Application of medicinal composition containing dehydroandrographolide succinate and pharmaceutically-acceptable salt thereof to preparation of veterinary immunopotentiator Download PDFInfo
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Abstract
The invention provides an application of a medicinal composition containing dehydroandrographolide succinate and a pharmaceutically-acceptable salt thereof to preparation of a veterinary immunopotentiator. The dehydroandrographolide succinate is 14-desoxy-11,12-didehydroandrographolide-3,19-dihemisuccinate. The medicinal composition comprises 0.1-99 percent of dehydroandrographolide succinate and the salt thereof and the balance of carrier. By adopting the medicinal composition, the animal cell and humoral immune functions can be enhanced, the effect of regulating animal organism immunity is achieved, and especially, remarkable effects are achieved on the aspects of improvement on immune antibodies of animal vaccines and prevention of animal diseases.
Description
Technical field
The present invention relates to a kind of application of andrographolide in terms of enhancing human body immunity power, can be used to prepare immunostimulant for animals
Agent.
Background technology
Andrographolide is potassium sodium dehydroandroandrograsuccinate succinate, is from Jing esters after effective component extracting in Chinese medicine Herba Andrographis
Change, be dehydrated, be prepared from into salt, its chemical name 14- deshydroxy -11, dehydrogenation andrographolide -3 of 12- bis-, 19- disuccinic acids half
Ester potassium/sodium salt.Andrographolide has heat-clearing and toxic substances removing and antivirus action, be clinically mainly used in treat by virus cause it is viral
Pneumonia and upper respiratory tract infection, the medicine that market gets the Green Light 188 mainly includes crude drug and injection, Clinical practice
Dosage form has two kinds of injection and injection powder pin, can be used for intramuscular injection and intravenous injection.
Potassium sodium dehydroandroan drographolide succinate injection has anti-inflammation, and heat-clearing and toxic substances removing promotes adrenal cortex function and sedation, to virus
Property pneumonia, viral upper respiratory tract infection and acute bacillary dysentery etc. have good therapeutic effect.(the andrographolide treatment virus such as Lin Na
Property upper respiratory tract infection effect observation, Youjiang River name race medical college journal, the 6th phase in 2006,933-935) report, andrographolide pair
The infantile acute upper respiratory infection that influenza virus, respiratory syncytial viruss, adenoviruss, Coxsackie viruss cause has treatment to make
With effect is better than ribavirin.(andrographolide assistant controls the clinical observation of mycoplasma pneumonia in children, publication modern medicine to Zhang Yong etc.
Health, the 15th phases of volume 21 in 2005,1970-1971) report, on the basis of conventional macrolide class antibiotic therapy, plus scorching amber
Peaceful treatment mycoplasma pneumonia in children has good efficacy, and effect is better than conventional therapy and ribavirin injection.
The patent of Publication No. CN101049312A discloses one kind by andrographolide, cephalosporins and pharmaceutically acceptable auxiliaries
Composition antiviral, the pharmaceutical composition of antibacterial.
The patent of Publication No. CN 101531649A discloses one kind by 14- deshydroxy -11, in the dehydrogenation Herba Andrographis of 12- bis-
Ester -3, the salt that the esters of 19- disuccinic acids half are constituted with adamantane.
The patent of Publication No. CN 101073625A disclose it is a kind of containing general flavone from blackberry lily and andrographolide or its
The pharmaceutical composition of derivant, with infection and antiinflammatory action.
The patent of Publication No. CN 1939380A discloses a kind of medicine for infection, antiviral and antipyretic-antalgic
Compositions, by 14- deshydroxy -11, dehydrogenation andrographolide -3 of 12- bis-, 19- disuccinic acid half ester salt and group made by Flos Lonicerae
Compound.
The patent of Publication No. CN 1554337A discloses a kind of pharmaceutical composition with antibiosis and antiviral functions, by
Baicalin and water miscible 14- deshydroxy -11, dehydrogenation andrographolide -3 of 12- bis-, 19- disuccinic acid half ester salt compares 1/ with weight
0.2～5 is effective medicinal ingredient and acceptable auxiliary adding ingredient composition in medicine.
The patent of Publication No. CN 1939420A discloses a kind of new antibacterial and antiviral drug compositionss and its preparation side
Method, it by 14- deshydroxy -11, dehydrogenation andrographolide -3 of 12- bis-, 19- disuccinic acids half ester or its pharmaceutically acceptable salt with
Fructus Forsythiae or Fructus Forsythiae extract are made.
However, there are problems that in above-mentioned patent following two：
1. antibacterials are combined into composition compound preparation with other antiviral drugs, be unfavorable for quality control.
2. effect of the andrographolide in prescription is antiviral.
To so far, the andrographolide used in veterinary clinic there is no document announcement, also improve immunity without using andrographolide
Application report in terms of power.Because animals and human beingses have the difference of essence, especially poultry exists poor with mammalian metabolism
It is different, and the non-clinical pharmacological research with regard to andrographolide shows：Andrographolide has antipyretic, antiinflammatory, tranquilizing soporific and killing in vitro
The effect of virus, has no report and shows that it has the function of enhancing human body immunity power.
The content of the invention
Inventor is found surprisingly that in test, andrographolide while antiviral, also with enhancing human body immunity power
Function.Zooblast and humoral immune function can be improved, with the effect for adjusting animal body immunity, especially animal is being improved
The immune antibody and prevention Animal diseases aspect of vaccine has beat all effect.
The invention provides a kind of pharmaceutical composition containing andrographolide and its pharmaceutically acceptable salt prepare it is for animals
Purposes in terms of immunostimulant, to improve premunition of the animal body to immune effect of vaccine and body to pathogen, wherein, it is scorching
Amber is rather 14- deshydroxy -11, dehydrogenation andrographolide -3 of 12- bis-, 19- disuccinic acid half esters, scorching amber in described pharmaceutical composition
Peaceful and its salt content is 0.1-99%, and remaining is carrier.
Preferably, the pharmaceutically acceptable salt includes potassium salt, sodium salt, k-na salt, magnesium salt, calcium salt, zinc salt, preferred sodium
Salt, k-na salt, potassium salt.
Preferably, the dosage form of the immunostimulant for animals includes peroral dosage form and injection type, and the peroral dosage form is
Pre-mixing agent, the injection type includes injection, injection powder pin.
Preferably, the carrier includes coating materials, plasticizer, wetting agent, the filler in pre-mixing agent, in injection type
PH regulators.
The coating materials include cellulose acetate-phthalate, hydroxypropylmethyl cellulose phthalate, poly- third
One or two in olefin(e) acid resin, polyvinyl acetate phthalic acid ester, succinic acid hydroxypropyl methylcellulose.
The plasticizer is one or two in phthalic acid ester, Oleum Ricini, Polyethylene Glycol.
The wetting agent includes water and ethanol.
The filler includes glucose, Lactose, starch, dextrin, Sodium Chloride, sodium thiosulfate.
Described PH regulators are in citric acid, cysteine, potassium dihydrogen phosphate, sodium dihydrogen phosphate, hydrochloric acid, sodium hydroxide
One or more.
Preferably, described pharmaceutical composition is applied to livestock or poultry；The domestic animal be pig, cattle, sheep, dog, cat, the family
Fowl is chicken, duck, goose；More preferably pig and chicken.
It is highly preferred that described pharmaceutical composition is used to strengthen the immunity of ND Vaccine and swine Fever Vaccine, and it is used for
The prevention and treatment of chicken bursal disease and porcine contagious pleuropneumonia.
It is highly preferred that in terms of andrographolide, consumption of the described pharmaceutical composition in animal is：Gastrointestinal administration, pig, cattle,
Sheep, 1.6-10mg/kg body weight, poultry 3-20mg/kg body weight, dog 2-12mg/kg, cat 3-18mg/kg；It is administered as 1 day 1 time, even
Use 3-5 days；
Injection is used, pig, cattle, sheep, 0.5-3mg/kg, poultry 1-6mg/kg, dog 0.6-4mg/kg, cat 0.9-5mg/kg；
It is administered as 1 day 1 time, is used in conjunction 3-5 days.
Various pharmaceutical preparatioies containing andrographolide and its immunostimulant for animals of salt of the present invention adopt existing system
Conventional method production in medicine field.
Pharmaceutical preparation containing andrographolide and its immunostimulant for animals of salt of the present invention, its prescription and preparation side
Method is：
1st, the pre-mixing agent of andrographolide and its salt：
Andrographolide 0.1-50% (w/w)
Coating materials 0.1-50% (w/w)
Filler 0-99.8% (w/w)
Wherein carrier includes coating materials, filler, specifically refer to coating materials required for powder coating technology, plasticizer,
Wetting agent, coating materials and plasticizer distinguish preferred hydroxypropylmethyl cellulose phthalate-Oleum Ricini, polyacrylic acid tree
Ester-Polyethylene Glycol.
(1) coating material, plasticizer are weighed in proportion, and by the 10-100 extraordinarily wetting agent of solid matter total amount, mixing is equal
It is even.
(2) principal agent andrographolide and its salt are accurately weighed.
(3) principal agent and coating solution are added into fluidized bed, is coated using spray method.
(4) it is dried, obtains final product andrographolide coating powder.
(5) carrier is proportionally added into full dose, obtain final product.
2nd, the injection of andrographolide and its salt：
Andrographolide 0.5-50% (w/w)
PH regulator 0.01-1% (w/w)
Inject water to 100% (w/w)
Wherein PH regulators refer to the acid or alkaline matter for maintaining injection pH value and adding, including citric acid, half Guang
Propylhomoserin, potassium dihydrogen phosphate, sodium dihydrogen phosphate, hydrochloric acid, sodium hydroxide, optimization citric acid, cysteine, sodium hydroxide, most preferably
Sodium hydroxide..
(1) PH regulators are weighed by weight proportion, plus water for injection 80ml, stirring and dissolving.
(2) solution is adjusted to pH value 6-8, the medicinal charcoal stirring of (w/v) is inhaled plus 0.07% with 1mol/L sodium hydroxide solutions
Attached 20min, filtering decarbonization.
(3) add 0.1-50g andrographolide stirring and dissolving, adjusted to corresponding pH value to 6-7 with 1mol/L sodium hydroxide solutions,
100ml is injected water to, is filtered, in being sub-packed in 5ml ampoule bottles, every 5ml, sealing, 115 DEG C of sterilizing 30min obtain final product this
The bright described injection containing andrographolide.
3rd, the injectable powder of andrographolide and its salt：
Andrographolide 90-99% (w/w)
PH regulator 1-10% (w/w)
Wherein PH regulators refer to the acid or alkaline matter for maintaining injection pH value and adding, including citric acid, half Guang
Propylhomoserin, potassium dihydrogen phosphate, sodium dihydrogen phosphate, hydrochloric acid, sodium hydroxide, optimization citric acid, cysteine, sodium hydroxide, most preferably
Sodium hydroxide..
(1) PH regulators are weighed by weight proportion, plus water for injection 80ml, stirring and dissolving.
(2) solution is adjusted to pH value 6-8, the medicinal charcoal stirring of (w/v) is inhaled plus 0.07% with 1mol/L sodium hydroxide solutions
Attached 20min, filtering decarbonization.
(3) add 0.1-50g andrographolide stirring and dissolving, adjusted to corresponding pH value to 6-8 with 1mol/L sodium hydroxide solutions,
100ml is injected water to, is filtered, in being sub-packed in 5ml ampoule bottles, lyophilization is obtained final product.
Pharmaceutical composition containing andrographolide and its pharmaceutically acceptable salt of the present invention, can not only improve animal
Cell and humoral immune function, also with the overall effect for strengthening animal body immunity, are especially improving exempting from for animal vaccine
Epidemic disease antibody and prevention Animal diseases aspect have beat all effect.
Specific embodiment
The present invention is further described with reference to specific embodiment, advantages of the present invention and feature will be with description more
For clear.But these embodiments are only exemplary, do not constitute any restriction to the scope of the present invention.Those skilled in the art
It should be understood that can carry out to the details of technical solution of the present invention and form without departing from the spirit and scope of the invention
Modification is replaced, but these modifications and replacement are each fallen within protection scope of the present invention.
The andrographolide used in the embodiment of the present invention and its salt adopt 14- deshydroxy -11, dehydrogenation andrographolide -3 of 12- bis-,
19- disuccinic acid half ester k-na salts, purchased from Sichuan Guanghan City Wei Kang plants company limited, lot number 100712, content is 94%.
The type fluid beds of Mini Glatt 3, German Glatt companies.
Prepare the preparation of the andrographolide pre-mixing agent of embodiment 1,5%
(1) ethanol for weigh hydroxypropylmethyl cellulose phthalate 100g, Oleum Ricini 70g in proportion, Jia 40%
2000ml is dissolved, mix homogeneously.
(2) principal agent 14- deshydroxy -11, dehydrogenation andrographolide -3 of 12- bis-, 19- disuccinic acid half ester potassium sodium are accurately weighed
106g。
(3) by principal agent and coating solution (for purely being included) plus fluidized bed, it is coated using spray method.
(4) it is dried, obtains final product andrographolide pre-mixing agent 192g, plus starch is obtained final product to 2000g, subpackage.
Prepare the preparation of the andrographolide powder of embodiment 2,10%
(1) the ethanol 5000ml for weigh polyacrylic acid resin 280g, Polyethylene Glycol 220g in proportion, Jia 60% carries out molten
Solution, mix homogeneously.
(2) principal agent 14- deshydroxy -11, dehydrogenation andrographolide -3 of 12- bis-, 19- disuccinic acid half ester potassium sodium are accurately weighed
319g。
(3) principal agent and coating solution are added into fluidized bed, is coated using spray method.
(4) it is dried, obtains final product andrographolide pre-mixing agent 573g, plus glucose is obtained final product to 3000g, subpackage.
Prepare the preparation of the ' Tanhuning ' injection of embodiment 3,1%
A, citric acid 2.7g is weighed, L-cysteine hydrochloride 0.5g, Sodium Chloride 2.5g, plus water for injection 400ml, stirring
Dissolving.
B, solution is adjusted with 1mol/L sodium hydroxide solutions to pH value 6-7, plus 0.07% (w/v) medicinal charcoal stirring and adsorbing
20min, filtering decarbonization.
C, plus 14- deshydroxy -11, dehydrogenation andrographolide -3 of 12- bis-, 19- disuccinic acid half ester potassium sodium 5.3g stirring and dissolving,
Adjusted to corresponding pH value to 6-7 with 1mol/L sodium hydroxide solutions, inject water to 500ml, filtered, be sub-packed in 5ml peaces
In small jar bottle, every 5ml, sterilizing obtains final product the injection containing andrographolide.
Prepare the preparation of the ' Tanhuning ' injection of embodiment 4,20%
A, weigh potassium dihydrogen phosphate 8.7g, sodium dihydrogen phosphate 7.5g, plus water for injection 400ml, stirring and dissolving.
B, solution is adjusted with 1mol/L sodium hydroxide solutions to pH value 6-7, plus 0.07% (w/v) medicinal charcoal stirring and adsorbing
20min, filtering decarbonization.
C, plus 14- deshydroxy -11, dehydrogenation andrographolide -3 of 12- bis-, 19- disuccinic acid half ester potassium sodium 106.4g stirrings are molten
Solution, is adjusted to corresponding pH value to 6-8 with 1mol/L sodium hydroxide solutions, injects water to 500ml, is filtered, and is sub-packed in 5ml
In ampoule bottle, every 5ml, lyophilization, sealing, 115 DEG C of sterilizing 30min obtain final product the injection containing andrographolide of the present invention
Liquid.
Prepare the preparation of the Andrographolide in Andrographolide for Injection injectable powder of embodiment 5,10%
A, citric acid 25g is weighed, L- cysteine hydrochlorate 5g, plus water for injection 400ml, stirring and dissolving.
B, solution is adjusted with 1mol/L sodium hydroxide solutions to pH value 6-7, plus 0.07% (w/v) medicinal charcoal stirring and adsorbing
20min, filtering decarbonization.
C, plus 14- deshydroxy -11, dehydrogenation andrographolide -3 of 12- bis-, 19- disuccinic acid half ester potassium sodium 53.2g stirrings are molten
Solution, is adjusted to corresponding pH value to 6-7 with 1mol/L sodium hydroxide solutions, injects water to 500ml.
D, membrane filtration, in being sub-packed in 5ml ampoule bottles, every 5ml, lyophilization, sealing obtains final product note of the present invention
Penetrate with the pin of powder containing andrographolide.
Prepare embodiment 6, the preparation of Andrographolide in Andrographolide for Injection injectable powder
A, citric acid 0.8g is weighed, L- cysteine hydrochlorate 0.2g, plus water for injection 400ml, stirring and dissolving.
B, solution is adjusted with 1mol/L sodium hydroxide solutions to pH value 6-7, plus 0.07% (w/v) medicinal charcoal stirring and adsorbing
20min, filtering decarbonization.
C, plus 14- deshydroxy -11, dehydrogenation andrographolide -3 of 12- bis-, 19- disuccinic acid half ester potassium sodium 526.6g stirrings are molten
Solution, is adjusted to corresponding pH value to 6-7 with 1mol/L sodium hydroxide solutions, injects water to 5000ml.
D, membrane filtration, in being sub-packed in 5ml ampoule bottles, every 5ml, lyophilization.The medicine of embodiment 7, andrographolide preparation
Effect test
First, impact of the andrographolide to animal cell immunity function
Medicine：Injection liquid samples prepared by the embodiment of the present invention 5.
Reagent：RPMI-1640 culture fluid：GIBCO Products；LPS、ConA、MTT：Sigma companies；DMSO：Tianjin
De En chemical reagent company limited, lot number：X1110414；D-Hanks liquid is self-control (preparation method：Sequentially add 0.4g
KCl, 0.06g KH2PO4, 8.0g NaCl, 0.35g NaHCO3, 0.132gNa2HPO4·12H2O, 1.0g D-Glucose, 1ml
Phenol red (0.1%) is settled to 1000ml into 800ml distilled water after dissolving, autoclaving, 10 pounds of 10min are obtained final product.).It is used
Reagent source is as follows：NaHCO3, Beijing Chemical Plant, lot number：20070718；NaCl, Tianjin Yong great chemical reagent company limited,
Lot number：20100105；Na2HPO412H2O, Chengdu Ke Long chemical reagents factory, lot number：20091031；KCl2, Tianjin is triumphant
Logical chemical reagent company limited, lot number：20081209；KH2PO4, Chengdu Ke Long chemical reagents factory, lot number：20100125；Portugal
Grape sugar, Shantou Xilong Chemical Factory Co., Ltd, lot number：080328.
The dilution of medicinal liquid：Take aseptic cillin bottle 10, per bottle of addition RPMI-1640 complete culture solution 1ml, the 1st bottle of addition
Injection 1ml prepared by the embodiment of the present invention 5, mix homogeneously, doubling dilution is standby to the 10th bottle (being diluted to 1: 1024).
1. impact of the monomer andrographolide of the present invention to chicken splenic lymphocytes in-vitro multiplication
(1) preparation of chicken spleen lymphocyte suspension
7 Japanese instar chickling 3 is taken, cervical region sacrificed by exsanguination, medicining liquid dipping sterilization 30min, aseptic taking-up spleen is placed in plate
It is interior.With 10mL syringes draw 5mL D-Hanks liquid rinse repeatedly 5 times, by the splenocyte suspension after flushing collect it is aseptic from
In heart pipe, 1500r/min is centrifuged 5min, abandons supernatant, and ((pH is 7.4 Tris- to add 10mL erythrocyte cracked liquids
NH4C1), 5min, 1500r/min are stood, 5min is centrifuged, washed repeatedly 2 times with D-Hanks liquid, use calf serum containing 100ug/ml
RPMI-1640 complete culture solutions it is resuspended, Jing trypan blues count, it was demonstrated that viable count ＞ 95%, adjustment cell concentration be 1.0 ×
l07The splenocyte suspension of/mL, it is standby.
(2) lymphocyte transformation test
Lymphocyte transformation test is carried out using mtt assay, lymphocyte suspension obtained above is added into 96 hole cell trainings
In foster plate, per the μ L of hole 90, the μ L of medicinal liquid 100 of doubling dilution are then added per hole, be eventually adding mitogen (10 μ g/ml's
ConA or LPS), if ConA and LPS control wells, cell control well and culture fluid control wells.By culture fluid be put into 37 DEG C, saturation it is wet
Degree, the CO that volume fraction is 5%248h is cultivated in incubator, culture terminates front 4h, and the μ of MTT solution 10 of 5mg/ml is added per hole
L, continues to cultivate to terminating, and gently suctions out culture fluid supernatant, and 150 μ L dimethyl sulfoxide, micro oscillator concussion are added per hole
10min, the absorbance of microplate reader determinator 570nm, the statistical analysis softwares of SPSS 11.5 are analyzed.
(3) result of the test see the table below.
The impact of ' Tanhuning ' injection prepared by the present invention of table 1 to chicken splenic lymphocytes proliferation activityn
=4)
Note：In same row, mark " a " is represented compared with cell controls group, significant difference (p ＜ 0.05), and " A " represents difference pole
Significantly (p ＜ 0.01).Mark " b " represents that compared with having mitogen matched group significant difference (p ＜ 0.05), " B " represents that difference extremely shows
Write (p ＜ 0.01).
As it can be seen from table 1 the present invention prepare ' Tanhuning ' injection carry out 32-128 times dilute after can cooperate with LPS
Stimulate the propagation (p ＜ 0.05) of chicken splenic lymphocytes, the stimulation is most strong ((p ＜ 0.01)) in 1: 64 times of dilution, with
ConA is without synergism；
2. impact of the monomer andrographolide of the present invention to pig blood Lymphocyte Proliferation in Vitro
It is aseptic to take anticoagulation 1-2ml, addition to fill in the centrifuge tube of lymphocyte separation medium, 2000r/min centrifugations
15min, separates lymphocyte.Washed repeatedly 2 times with D-Hanks liquid, with the resuspended lymphocyte of culture fluid, Jing Trypan Blues are counted
Viable count ＞ 95%, adjustment cell concentration is 1 × 107/ ml, with MTT stainings (operational approach is with 1) is measured,
OD570The absorbance of each group cell is determined at nm, is analyzed with SPSS statistical softwares, the results are shown in Table 2.
The impact of ' Tanhuning ' injection prepared by the present invention of table 2 to pig blood lymphocyte proliferation activityn
=4)
Note：In same row, mark " a " is represented compared with cell controls group, significant difference (p ＜ 0.05), and " A " represents difference
Extremely significantly (p ＜ 0.01).Mark " b " represents that compared with mitogen matched group significant difference (p ＜ 0.05), " B " represents that difference extremely shows
Write (p ＜ 0.01).
From table 2 it can be seen that the present invention prepare ' Tanhuning ' injection carry out 32-128 times dilute after can with ConA,
LPS collaboration stimulate pig blood lymphocyte in-vitro multiplication (p ＜ 0.05), and when diluting for 32-64 times with ConA synergism
It is most strong ((p ＜ 0.01)), it is most strong ((p ＜ 0.01)) with LPS synergism in 64 times of dilutions.
Above result of the test shows that andrographolide preparation prepared by the present invention has under finite concentration stimulates lymphocyte to increase
The effect grown, and with the function of enhancing animal cell immunity.
2nd, impact of the monomer andrographolide of the present invention to animal nonspecific immunity
Test medicine：Andrographolide preparation prepared by the embodiment of the present invention 2.Control medicine：Astragalus polysaccharidess powder, 1g astragalus polysaccharidess
Powder 450mg containing astragalus polysaccharidess, lot number：1106151, Ai Disen (Beijing) Bioisystech Co., Ltd produce.
Healthy kunming mice 60, male and female half and half, body weight 18-22g is randomly divided into 3 groups of A, B, C, 20 per group, its
Middle A groups are andrographolide group, and B groups are astragalus polysaccharidess matched group, and C groups are blank control group.Andrographolide is made into according to dosage
The solution of 30mg/ml, astragalus polysaccharidess powder is made into the solution that concentration is 15mg/ml, is administered using gavage mode, given low
0.2ml/20g, once a day, successive administration 5 days.
1. impact of the andrographolide of the present invention to mice reticuloendothelial system phagocytic function
The 1h after last dose, per group of random 10 mices of picking, the prepared Chinese ink of 4 times of dilutions of tail vein injection saline,
Dosage be 0.05ml/10g body weight, timing immediately.5min (the t after injection1)、10min(t2) (advance with 20 μ l capillary tubies
Moistened with heparin) blood is taken in eye rear vein beard, it is added to 2ml 1mg.ml-1Na2CO3In shake up, use spectrophotometric determination
Absorbance at 600nm.Finally white mice is put to death, its body weight and liver, spleen weight are weighed respectively, and index (K) is cleaned up in calculating
With phagocytic index (α).Computing formula is as follows：
Clean up index K=(lgA1-lgA2)/(t2-t1)
Wherein, A1、A2Represent that 5min, 10min collect absorbance of the blood sample at 600nm respectively.
WithTo represent the K values and α values of test each group, t inspections, the significance of comparing difference between group are carried out.Experiment
The results are shown in Table 3.
The impact of andrographolide preparation prepared by the present invention of table 3 to mice Carbon clearance rateN=10)
Note：Each test group compares with blank control group, and " * " represents significant difference (p ＜ 0.05), and " * * " represents that difference extremely shows
Write.
Result of the test shows, the andrographolide preparation 30mg/ml successive administrations of the present invention 5 days, and mice cleans up index, phagocytosis
Index compared with blank control group, significant difference (p ＜ 0.05), better than astragalus polysaccharidess matched group, A groups are better than compared with B groups
B groups, but difference is not significantly (p ＞ 0.05).Illustrate that andrographolide can strengthen the phagocytic function of reticuloendothelial system, improve body to different
The Scavenging activity of thing, can enhancing body non-specific immunity.
2. impact of the monomer andrographolide of the present invention to mice reticuloendothelial system phagocytic function
The preparation of 20% chicken erythrocyte suspension：Fresh cock blood 5ml is taken, with brine 3 times, is often inferior to
2000r.min-1Centrifugation 10min, removes supernatant, and with normal saline the chicken erythrocyte suspension of 20 % (v/v) is made into.
Test method：With 24h after last 1 time administration, mice 10 is taken per group, the chicken of every mouse peritoneal injection 20% is red
Cell physiological salt aqueous suspension 1mL, cervical dislocation puts to death mice after 30min, and is faced upward position and be fixed on Mus plate, and center is cut off
Abdominal skin, trans-abdominal chamber saline injection 2mL rotates Mus plate 1min, suctions out abdominal cavity washing liquid 1mL, and average mark is dripped in microscope slide
On, it is put in the enamel box for being lined with wet gauze, in 37 DEG C of incubation 30min.Incubate complete, put in normal saline and rinse, remove not
Paster cell, dries, Wright's stain dyeing, microscope oil Microscopic observation, counts the phagocytic percentage of peritoneal macrophage.Per piece
200 are calculated as follows phagocytic percentage and phagocytic index.
Phagocytic percentage (%)=(number of macrophages of the phagocyte number/counting of phagocytosis chicken red blood cell) × 100%
The number of macrophages of the chicken red blood cell sum of phagocytic index=swallowed/count
Result of the test：It is shown in Table 4
Impact of the andrographolide preparation of the present invention of table 4 to mouse peritoneal phagocyte phagocytic functionN=10)
Note：In with data line, indicate identical shoulder mark and represent that difference is not notable.
Result of the test shows, the andrographolide successive administration of 30mg/ml 5 days, and Turnover of Mouse Peritoneal Macrophages is gulped down to chicken red blood cell
Rate is bitten compared with blank control group, significant difference (p ＜ 0.05), better than astragalus polysaccharidess matched group, difference not significantly (p ＞
0.05).Illustrate that andrographolide can strengthen the phagocytic function of Turnover of Mouse Peritoneal Macrophages, so as to the nonspecific immunity of enhancing body
Power.
3rd, impact of the monomer andrographolide of the present invention to animal body fluid immunity
1. impact of the monomer andrographolide of the present invention to laying hen vaccine immunity
Test medicine：Andrographolide preparation prepared by the embodiment of the present invention 2.Control medicine：Astragalus polysaccharidess powder, 1g astragalus polysaccharidess
Powder 450mg containing astragalus polysaccharidess, lot number：1106151, Ai Disen (Beijing) Bioisystech Co., Ltd produce.
1 age in days Avain chicken 150, normally raises to 7 ages in days, random 5 groups, 30 per group.From the beginning of the 8th day
Spice is administered, and A groups, B groups, C groups give the andrographolide preparation of the preparation of the embodiment of the present invention 2, and spice dosage is respectively per kg feedstuffs
0.15g, 0.5g, 1g (are converted to andrographolide active component consumption 3mg/kg.d-1Body weight, 10mg/kg.d-1Body weight 20mg/kg.d-
Body weight)；D groups give astragalus polysaccharidess powder, and per kg feedstuff 0.4g, E groups are matched group, any medicine are not given, by the dosage regimen of table 5
It is administered, successive administration 5 days.Newcastle disease IV vaccine collunarium, eye dripping carry out immunity during 14 age in days, while cervical region subcutaneous injection
Newcastle inactivated vaccine 0.2ml, 14d, 21d, the 28d after immunity 10 chickens of per group of extraction, gather anticoagulation, and HI tests are surveyed
Determine antibody level of serum, serum antibody measurement result is shown in Table 4.
Table 5 is tested concrete packet and is processed
The impact (log2) of andrographolide preparation prepared by table 6, the present invention to antibody IgY against chicken Newcastle Disease levelN=
10)
Note：In same column data, shoulder mark same letter person represents that difference is not notable.
As a result show, after giving the andrographolide preparation and astragalus polysaccharidess of the present invention preparation of various dose, newcastle disease vaccine
14d, 21d, 28d antibody horizontal is above blank control group after immunity, and significant difference (P ＜ 0.05) illustrates of the present invention
Andrographolide can promote the generation of antibody IgY against chicken Newcastle Disease.
2. impact of the Chinese medicine monomer andrographolide of the present invention to pig production performance and vaccine immunity
Test medicine：Andrographolide preparation prepared by the embodiment of the present invention 3.Control medicine：Astragalin injection, specification
10ml：0.1g, lot number：20101203, Shandong Bai Te Bioceuticals Inc..
21 age in days piglets 50, are randomly divided into 5 groups, 10 per group, after observation is without exception test are proceeded by.A groups, B
Andrographolide preparation prepared by group, the C groups injection embodiment of the present invention 4, dosage is respectively and (is converted to raw material by 0.5ml/10kg body weight
Consumption 0.5mg/kg body weight), 1.5ml/10kg body weight (being converted to raw material dosage 1.5mg/kg body weight), 3ml/10kg body weight (folding
It is counted as raw material dosage 3mg/kg body weight)；D groups inject astragalin injection, and 1ml/10kg, E group is made for injection 1ml normal saline
For control, successive administration 3 days, concrete dosage regimen refers to table 6.Each group animal carries out hog cholera lapinised virus vaccine and exempts from 25 ages in days
Epidemic disease is inoculated with, intramuscular injection, per 3 parts of pig.14d, 28d after immunity, each group randomly selects 8, and vena cava anterior blood sampling 1ml divides
From serum, the blocking rate that ELISA detection kit (U.S. IDEXX productions) determines hog cholera antibody is blocked using serum, illustratively
Book is operated, and determines hog cholera antibody potency in each serum.
Table 6, the concrete packet of test and process
Antibody horizontal (%) after table 7, the immunity of each group swinery swine Fever VaccineN=10)
Note：In same column data, indicate identical shoulder mark and represent that difference is not notable.
As can be seen from Table 7, andrographolide preparation prepared by the embodiment of the present invention, immunity are injected before piglet immunological swine Fever Vaccine
Afterwards hog cholera antibody level is higher than blank control group in 14d, 28d blood, and significant difference illustrates the andrographolide preparation of present invention preparation
The generation of hog cholera antibody after vaccine immunity can be stimulated, compared with blank control group, hence it is evident that improve the level of hog cholera antibody after immunity.
4th, impact of the monomer andrographolide of the present invention to animal disease resistant ability
1. impact of the monomer andrographolide of the present invention to artificial challenge's infectious bursal disease
Test medicine：Andrographolide preparation prepared by the embodiment of the present invention 2.Control medicine：Astragalus polysaccharidess powder, 1g astragalus polysaccharidess
Powder 450mg containing astragalus polysaccharidess, lot number：1106151, Ai Disen (Beijing) Bioisystech Co., Ltd produce.
Virus：Infections chicken cloacal bursa virus standard strain SNJ93 strains, purchased from China Veterinery Drug Inspection Office, malicious valency：2
×105.0Provirus are carried out 1000 times of dilutions by/0.1ml before use.
15 180 chickens of age in days SPF chickens are randomly divided into 6 groups, 30 per group, respectively 1,2,3 groups of andrographolide, astragalus polysaccharidess
Powder matched group, counteracting toxic substances matched group.From the beginning of 16 ages in days, each group is administered by the scheme of table 8, and 1,2,3 groups of andrographolide uses the present invention
Andrographolide preparation spice prepared by embodiment 2, spice dosage is respectively and (is converted to andrographolide per kg feedstuff 0.15g, 0.5g, 1g
Active component consumption 3mg/kg.d-1Body weight, 10mg/kg.d-1Body weight, 20mg/kg.d-Body weight)；D groups give astragalus polysaccharidess powder, often
Kg feedstuff 0.4g, each group successive administration 5 days.E counteracting toxic substances matched groups, do not give any medicine.F groups are blank control group, are not carried out
Any process.During 21 age in days, A, B, C, D, E group is carried out with the infections chicken cloacal bursa virus eye dripping of 1000 times of dilutions, collunarium
Counteracting toxic substances, every chicken 0.1ml.After counteracting toxic substances, daily the mental status, clinical symptoms, morbidity, the death condition of observation chicken, calculates morbidity
Rate, mortality rate compares each group difference with X 2 test.
Criterion：During test, occur fluffy spiritual depressed, feather, dysentery, tremble, loss of appetite, stay it is vertical, to thorn
The symptoms such as sharp habituation, it is judged to morbidity.There is above disease symptom dead, fabricius bursa enlargement occurs in dissection, inside there is inflammatory
Secretions or bleeding, it is judged to death.
The sickness rate of each group chicken group, mortality rate are shown in Table 9
Table 8 is tested concrete packet and is processed
Table 9, the morbidity of test chicken and death condition
Note：In same row, compared with blank control group, significant difference (P≤0.05), " * * " is represented and blank shoulder table " * "
Matched group is compared, significant difference (P≤0.01).
From the results shown in Table 9, spice give various dose the present invention prepare andrographolide preparation and the Radix Astragali it is many
After Icing Sugar 5 days, chicken group strengthens the resistance of artificial challenge's infections chicken cloacal bursa virus, and M ＆ M substantially drops
It is low.Wherein, compared with counteracting toxic substances matched group, sickness rate, mortality rate are significantly lower than counteracting toxic substances matched group, significant difference to 2,3 groups of andrographolide
(P≤0.05).Illustrate to be used in conjunction using 3-20mg/kg andrographolide and can improve within 5 days resistance of the chicken to infectious bursal disease.
2. impact of the andrographolide of the present invention to artificial challenge's porcine contagious pleuropneumonia
Test medicine：Andrographolide preparation prepared by the embodiment of the present invention 3.Control medicine：Astragalin injection, specification
10ml：0.1g, lot number：20101203, Shandong Bai Te Bioceuticals Inc..
Strain：Actinobacillus pleuropneumoniae 259, purchased from China Veterinery Drug Inspection Office.Infectious bacteria is in containing 10% chocolate
On agar, 5%CO2, under the conditions of 37 DEG C culture 24h, swept away with sterile saline, count, be made into sterile saline
1.75×109Cfu/ml concentration, it is standby.
60 age in days length are white, DABAI, 72 pigs of Duroc three way cross (detection serum antibody is feminine gender), be randomly divided into 6
Group, 12 per group.Andrographolide preparation prepared by A groups, B groups, the C groups injection embodiment of the present invention 4, dosage is respectively and presses 0.5ml/
10kg body weight (being converted to raw material dosage 0.5mg/kg body weight), 1.5ml/10kg body weight (are converted to raw material dosage 1.5mg/kg body
Again), 3ml/10kg body weight (being converted to raw material dosage 3mg/kg body weight)；D groups injection astragalin injection 1ml/10kg, E groups,
F groups inject 1ml normal saline as control, successive administration 5 days, and concrete dosage regimen refers to table 10.When 65 age in days, each group is moved
With Actinobacillus pleuropneumoniae, (concentration is 2.5 × 10 to thing9Cfu/ml) intranasal vaccination, per pig 1ml.Early daily after inoculation, evening
It is each once to observe pig breathing, appetite and the mental status, and rectal temperature is determined, Continuous Observation 8 days.The morbidity of record each group pig
Number and death toll, and pathology cut open inspection is carried out to dead pig.
Criterion：During observation, there is spiritual slight depressed, body temperature rising, cough, rapid breathing, difficulty, dog sitting posture
Breathing, it is judged to morbidity.There is the death of above symptom, take its liver, spleen and apply APP agar plate culture medium, 37 DEG C are cultivated 18h,
There is bacterial growth, smear, Gram’s staining, microscopy sees that shaft-like, the filmanetous colony red colonies that are dispersed in are judged to sense under microscope
Dye Actinobacillus pleuropneumoniae is dead.
According to morbidity and dead number of animals, incidence and mortality is calculated, the difference between each group is compared in X 2 test.
Concrete outcome is shown in Table 11.
Table 10, the concrete packet of test and process
Table 11, the condition of morbidity death statistics of each group animal
As can be seen from Table 11, inject 5 days after the andrographolide preparation and astragalin injection of embodiment of the present invention preparation,
Swinery strengthens the resistance of the contagious pleuropneumonia of artificial challenge, and M ＆ M is substantially reduced.Wherein, andrographolide
1st, 2,3 compared with counteracting toxic substances matched group, and counteracting toxic substances sequela rate reduces respectively 33.3%, 41.7%, 58.3%, and mortality rate drops respectively
It is low by 16.7%, 25.0%, 41.7%.Illustrate that andrographolide of the present invention can improve pig and Actinobacillus pleuropneumoniae is supported
Drag.
Presently preferred embodiments of the present invention is the foregoing is only, not to limit the present invention, all essences in the present invention
Within god and principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.
Claims (6)
1. use of the pharmaceutical composition containing andrographolide and its pharmaceutically acceptable salt in terms of poultry immunity reinforcing agent is prepared
On the way, wherein, andrographolide be 14- deshydroxy -11, dehydrogenation andrographolide -3 of 12- bis-, 19- disuccinic acid half esters, the drug regimen
Andrographolide and its salt content are 0.1-99% in thing, and remaining is carrier；Wherein, the poultry immunity reinforcing agent is prevention animal disease
The poultry immunity reinforcing agent of disease, the Animal diseases are infectious bursal disease.
2. purposes according to claim 1, wherein, the pharmaceutically acceptable salt include potassium salt, sodium salt, k-na salt,
Magnesium salt, calcium salt, zinc salt.
3. purposes according to claim 1, wherein, the pharmaceutically acceptable salt includes sodium salt, k-na salt, potassium salt.
4. purposes according to claim 1, wherein, the dosage form of the immunostimulant for animals includes peroral dosage form and injection
Dosage form, the peroral dosage form is pre-mixing agent, and the injection type includes injection, injection powder pin.
5. purposes according to claim 1, wherein, the carrier includes coating materials, plasticizer, the moistening in pre-mixing agent
Agent, filler, the PH regulators in injection type；The coating materials include cellulose acetate-phthalate, hydroxypropyl methyl
In cellulose phthalate, polyacrylic resin, polyvinyl acetate phthalic acid ester, succinic acid hydroxypropyl methylcellulose
One or two；The plasticizer be phthalic acid ester, Oleum Ricini, Polyethylene Glycol in one or two, the wetting agent
Including water and ethanol, the filler includes glucose, Lactose, starch, dextrin, Sodium Chloride, sodium thiosulfate, and described PH is adjusted
Section agent is one or more in citric acid, cysteine, potassium dihydrogen phosphate, sodium dihydrogen phosphate, hydrochloric acid, sodium hydroxide.
6. purposes according to claim 1, wherein, in terms of andrographolide, consumption of the described pharmaceutical composition in animal is：
Gastrointestinal administration, poultry 3-20mg/kg body weight is administered as 1 day 1 time, is used in conjunction 3-5 days；
Injection is used, and poultry 1-6mg/kg is administered as 1 day 1 time, is used in conjunction 3-5 days.
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| CN201210462098.3A CN103800320B (en) | 2012-11-15 | 2012-11-15 | Application of medicinal composition containing dehydroandrographolide succinate and pharmaceutically-acceptable salt thereof to preparation of veterinary immunopotentiator |
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