CN103356717B - Rhizoma Dryopteris Crassirhizomatis extract and its use in preparation of viral disease controlling medicines - Google Patents
Rhizoma Dryopteris Crassirhizomatis extract and its use in preparation of viral disease controlling medicines Download PDFInfo
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Abstract
The invention provides a Rhizoma Dryopteris Crassirhizomatis extract and its use in the preparation of viral disease controlling medicines. The Rhizoma Dryopteris Crassirhizomatis extract is an extract obtained through ethyl acetate extraction-ethanol refluxing extraction, can be added to drinking water or feeds to improve the humoral immunity and cellular immunity functions of animals, can control highly pathogenic avian influenza, newcastle disease virus, infectious bursal disease viruses and weaned pig diarrhea, and can also control the occurrence and epidemic of immunosuppressive diseases. The Rhizoma Dryopteris Crassirhizomatis extract has the advantages of easily available raw material, low cost, and small side effects, and is suitable for controlling the epidemic diseases of animals having impaired immunities after failed immunities.
Description
Technical field
The present invention relates to one and there is raising poultry cellular immunization and humoral immunization, play control animal viral disease, the preparation method and application of the Rhizoma Dryopteris Crassirhizomatis extract of the disease caused by pig infectious diarrhea poison, bird flu, Avian pneumo-encephalitis virus, infectious bursa of Fabricius virus, belong to field of veterinary.
Background technology
Since entering 21 century, animal viral disease is as the outburst in the world of bird flu, newcastle, not only have a strong impact on and hinder the sound development of animal husbandry, even cause its propagation in the mankind, cause a series of Public Health Emergencies, cause great harm to people ' s health, social stability.Therefore, research and initiative biological veterinary new product, the great demand of country ensureing China's animal husbandry development to become, safeguard people ' s health and life security, maintain social stability and promote economic development.
The dry rhizome that Rhizoma Dryopteris Crassirhizomatis (RhizomaDryopteridisCrassirhizomatis) is Dryopteridaceae plant dryopteris crassirhizoma (DryopteriscrassirhizomaNakai) and petiole residual base, mainly be distributed in the China such as Heilungkiang, Jilin, Liaoning, Shandong, Hebei and Inner Mongolia northern, at present without GAP planting base.Mainly contain multiple phloroglucinol compounds in Rhizoma Dryopteris Crassirhizomatis rhizome and petiole residual base, aerial parts contains terpenoid etc.Wherein phloroglucinol compounds (dryocrassin ABBA, Filixic Acids ABA and albaspidin AA etc.) has antioxidation, anthelmintic, malaria, antitumor, antibacterial (staphylococcus aureus, micrococcus catarrhalis, diphtheroid etc.) and antiviral (influenza virus, bird flu virus, adenovirus etc.) activity.
Rhizoma Dryopteris Crassirhizomatis is gone through an edition pharmacopeia by China and is recorded, the bibliographical information of domestic existing many influenza A, hepatitis and the acquired immune deficiency syndrome (AIDS) etc. that cause as bacterial pneumonia, upper respiratory tract infection and viral infection about Rhizoma Dryopteris Crassirhizomatis prescription therapeutic multi-infection disease and patent.As: (1) treatment influenza virus: compound rhizome of Cyrtomium aspirin tablet patent is mainly used in treatment influenza (application number CN03108749.3, publication number is CN1466981).Vinegar beverage with rhizoma polystichi isatis root has the grippal effect for the treatment of (application number CN200510015819.6, publication number is CN1954731) and the application (application number: CN200910082162.3, publication number be CN10862391A) of Rhizoma Osmundae compositions in preparation treatment human influenza medicine.Chinese medicine composition flavour of a drug such as (composition) Fructus Forsythiae, Flos Lonicerae, Herba Ephedrae, Semen Armeniacae Amarums has kill virus, brings down a fever, antiinflammatory, and experiment confirms there is clear and definite anti-influenza A H 1 N 1 virus (application number CN200910075211.0, publication number is CN101991694A).(2) atypical pneumonia virus: the vitro Drug anti-SARS virus drug efficacy study formed with Chinese prescription (Fructus Forsythiae, Flos Lonicerae, Radix Isatidis, Semen Armeniacae Amarum, Mentholum, Herba Houttuyniae, Radix Et Rhizoma Rhei, Herba Pogostemonis, Rhizoma Dryopteris Crassirhizomatis, Radix Rhodiolae, Herba Ephedrae, Radix Glycyrrhizae and Gypsum Fibrosum) shows compositions to the suppression medium effective concentration (IC50) of SARS virus for 3.63mg/ml, therapeutic index is 40.33, illustrate that antiviral compound substrate concentration of the present invention is greater than 0.09mg/ml and obviously can suppresses SARS virus (number of patent application: CN03143211.5, application publication number: CN1483463).(3) prevent and treat mumps virus: number of patent application CN200810225995.6, publication number is CN101732468A; (4) bronchitis is prevented and treated: be made up of Fructus Forsythiae, Flos Lonicerae, Radix Isatidis, Radix Et Rhizoma Rhei, Herba Pogostemonis, Rhizoma Dryopteris Crassirhizomatis, Radix Rhodiolae etc., can effectively antibacterial, antiinflammatory, cough-relieving, reduce phlegm, immunity moderation, clinical experiment confirms all there is significant curative effect (number of patent application CN200810104909.6, publication number CN101564458) to acute/chronic bronchitis.
Patent in livestock and poultry concentrates on: (1) avian influenza prevention: the prescription medicine be made up of Fructus Forsythiae, Flos Lonicerae, Radix Isatidis, Rhizoma Dryopteris Crassirhizomatis, the Radix Astragali etc. is to poultry avian influenza prevention Be very effective, use safety, convenience (application number CN200510011432.3, publication number is CN1682843).A kind of Chinese medicine extract for birds flu-preventing of disclosure of the invention and preparation method thereof in addition.This Chinese medicine extract contains Flos Lonicerae, Fructus Forsythiae, Radix Scutellariae, Radix Isatidis, Rhizoma Dryopteris Crassirhizomatis, Herba Houttuyniae, the Radix Astragali, Radix Bupleuri, for control bird flu, there is drug effect feature remarkable and safe and convenient to use (application number is CN200710123069.3, and publication number is CN101332255).In addition.Japan Patent is reported by Herba Houttuyniae, Caulis Lonicerae, Radix Isatidis, Rhizoma Dryopteris Crassirhizomatis, the Radix Angelicae Dahuricae, Rhizoma Paridis, Rhizoma Belamcandae, make tablet, capsule, granule, injection and oral liquid have anti-avian influenza effect (preparation of Chinese medicine ingredients for preventing and treating bird flu and application, application number: JP2011111841A, the patent No.: JP2011207895 (A)).
(2) fish streptococcicosis is prevented and treated: Korean Patent report utilizes Radix Sophorae Flavescentis, Thymi Serpylli Herba extract to mix according to 1:0.2-1:5 weight ratio, Herba thymi vulgaris mixes according to 1:0.2-1:5 with Rhizoma Dryopteris Crassirhizomatis extract, effectively can prevent and treat fish streptococcicosis (application number KR20070134699A, the patent No.: KR20090066949 (A)).
The extract biological activity aspect of Rhizoma Dryopteris Crassirhizomatis, report is had to may be used for control virus abroad: can effectively suppress hiv protease active as Korean Patent report utilizes Rhizoma Dryopteris Crassirhizomatis extract to prepare medicine and functional food, for preventing and treating acquired immune deficiency syndrome (AIDS) (application number: KR20080074370A, the patent No.: KR20100012927 (A)).Canadian Patent report utilizes Rhizoma Dryopteris Crassirhizomatis extract to have, and anti-A type infects, effect (CA2271622) of hepatitis C and acquired immune deficiency syndrome (AIDS).Rhizoma Osmundae extract has the effect of influenza and bird flu virus, and provides application High Performance Liquid Chromatography/Mass Spectrometry on line analytical processing mensuration Rhizoma Dryopteris Crassirhizomatis extract finger printing.The finger printing of disclosure of the invention and technology thereof can be used for the Variety identification of Rhizoma Dryopteris Crassirhizomatis and the quality monitoring (publication number is CN1846717) of Rhizoma Osmundae extract.
In a word, the patent of the independent medication of domestic, external Rhizoma Dryopteris Crassirhizomatis is mainly used in preventing and treating the report of psoriasis effect, antitumor, anticancer, the aspect such as anti-AIDS, hepatitis virus malaria, mostly wherein said Rhizoma Dryopteris Crassirhizomatis extract is to be obtained by ethanol or methanol extraction, is showed no by ethyl acetate lixiviate-alcohol reflux preparation technology.Meanwhile, utilize extract for preventing and treating avian influenza, newcastle, infectious bursa of Fabricius, renal type infectious bronchitis and promotion or strengthen immunity of livestock and also have no report.In view of the animal viral disease generation of current China and epidemic rate have exceeded vaccine development speed, China lacks the special antiviral drugs of poultry.In addition, 2005 and the Ministry of Agriculture of China in 2012 issue the documents and forbid that antiviral drugs such as amantadine, rimantadine, acyclovir, morpholine (two) guanidine, ribavirin etc. the mankind use are applied in livestock medicine, abuse in strict monitoring veterinary drug, to ensure livestock food safety.Therefore, Rhizoma Dryopteris Crassirhizomatis extract is developed to be suitable for poultry antiviral special-purpose medicaments imperative.
Summary of the invention
Technical problem to be solved by this invention is to provide the herbal medicine of a kind of new raising immunity of livestock, effectively treatment livestock and poultry viral disease.
In order to solve the problem, the present inventor is by a large amount of laboratorys and clinical verification, find that Rhizoma Dryopteris Crassirhizomatis extract obtains extract by ethyl acetate lixiviate-alcohol reflux, there is extractum yield high, the effect that antiviral activity is strong, effective control diarrhea of weaned piglets, bird flu, newcastle, fabricius bursa, infectious bronchitis virus, thus the survival rate effectively improving poultry.
The present invention evaluates the antiviral efficacy of Rhizoma Dryopteris Crassirhizomatis extract by Experimental infection in body, finds that extract has the effect of the following livestock and poultry virus infection of control, as newcastle (F4) and bursal disease virus (LX).In vivo test shows: continuous 5 days oral after, Rhizoma Dryopteris Crassirhizomatis extract can resist the attack of newcastle, infectious bursa of Fabricius, infectious bronchitis virus.For the porcine epizootic diarrhea continuous oral 14 days of clinical onset, can the survival rate of available protecting piglet.
Accordingly, the invention provides the application of Rhizoma Dryopteris Crassirhizomatis extract in preparation raising immune drug or viral diseases medicine.
Described virosis especially comprises high pathogenic avian influenza, Avian pneumo-encephalitis virus, bursal disease virus, renal type infectious bronchitis virus and control immunosuppressive disease, as avian visceral lymphomatosis, mycotoxin disease, pig breeding-breath syndrome virus (pig blue-ear disease), porcine circovirus 2 type (Porcine circovirus desease), porcine pseudorabies and swine eperythrozoonosis.
The present invention also provides a kind of medicine improving fowl poultry immune ability, anti-livestock and poultry viruses disease, and it contains Rhizoma Dryopteris Crassirhizomatis extract.Described medicine can further include pharmaceutic adjuvant, includes but not limited to one or more in starch, dextrin, lactose, stevioside, sucrose, glycyrrhizin, high fructose syrup, sucralose, xylitol, Sorbitol, glucose, pregelatinized Starch and/or carboxymethyl starch sodium.Described medicine can be tablet, granule, soluble powder, oral liquid, injection or lyophilized injectable powder.
Rhizoma Dryopteris Crassirhizomatis extract of the present invention prepares in accordance with the following methods: pulverized by raw material, sieve, and sieved thing adds ethyl acetate and soaks, lixiviate, merge extractive liquid, and filter, reclaim under reduced pressure ethyl acetate, obtains brown extractum; Residue medicinal residues alcohol reflux, collects backflow, reclaims ethanol, concentrated acquisition extractum; The extractum that 2 times are collected is mixed, concentrates and obtain cream liquid, dry.
In said method, preferably, the method for ethyl acetate lixiviate is the ethyl acetate by ethyl acetate and medical material envelope-bulk to weight ratio 3-10 times amount, lixiviate 1-4 time at 4-30 DEG C, each 1-24h; The method of alcohol reflux is the 60%-95% ethanol with ethanol and medical material envelope-bulk to weight ratio 3-10 times amount, reflux, extract, 1-4 time, each 1-4h.More preferably, after being pulverized by Rhizoma Dryopteris Crassirhizomatis, 20 mesh sieves are crossed, the ethyl acetate that sieved thing adds ethyl acetate and medical material envelope-bulk to weight ratio 5-8 times amount lixiviate 2-3 time at 25 DEG C, each 2-8 hour, collects lixiviating solution and merges filtration, collect filtrate, reclaim ethyl acetate, obtain Rhizoma Dryopteris Crassirhizomatis crude extract 1; Residue medicinal residues add the alcohol reflux of 70% of ethanol and medical material envelope-bulk to weight ratio 5-8 times amount, reflux temperature 85 DEG C, extract 2-3 time, each 2-3 hour, collect filtrate, reclaim ethanol, obtain Rhizoma Dryopteris Crassirhizomatis crude extract 2, Rhizoma Dryopteris Crassirhizomatis extract 1 and 2 is mixed, concentrated, obtain extractum, obtain Rhizoma Dryopteris Crassirhizomatis extract.
The effective dose that described Rhizoma Dryopteris Crassirhizomatis extract improves immunity of livestock in preparation is 5-100mg/kg, and preferred dose is 25-50mg/kg; The effective dose for the treatment of animal viral disease is 10-200mg/kg, and preferred dose scope is 50-100mg/kg.
Compared with the Rhizoma Dryopteris Crassirhizomatis extract that Rhizoma Dryopteris Crassirhizomatis extract prepared by said method and existing method obtain, mainly have the advantage of the following aspects: the content of effective ingredient is higher, contained impurity is lower; The yield of product significantly improves, and has good prevention effect for animal viral disease.
Described administering mode is spice, drinking water administration or oral administration.
Poultry of the present invention mainly refer to meat pigeon, broiler, meat pigeon, laying hen and pig.
Rhizoma Dryopteris Crassirhizomatis extract of the present invention is the extract obtained with ethyl acetate lixiviate-alcohol reflux, poultry humoral immunization and cellular immune function is improved by drinking water or adding in feedstuff, play control bird flu, Avian pneumo-encephalitis virus, infectious bursa of Fabricius and diarrhea of weaned piglets, effectiveness is more than 85%.In addition, its raw material of the present invention is easy to get and cost compare is low, and side effect is little, is applicable to the animal epidemic control of immunologic function of livestock and birds lowly or after immuning failure, realizes healthy aquaculture.
Detailed description of the invention
Below by way of detailed description of the invention, the present invention is further described.Here want to be pointed out that, detailed description of the invention is below only for illustration of the present invention, those skilled in the art are under the prerequisite understanding the present invention's spirit and essence, the content that can disclose according to the present invention to be modified to the present invention in conjunction with prior art or is retouched, and these technical schemes all belong within scope of the present invention.
The effect of Rhizoma Dryopteris Crassirhizomatis extract prepared by the embodiment of the present invention 1 is better, but embodiment 2 ~ 5 is prepared Rhizoma Dryopteris Crassirhizomatis extract and also possessed relevant drug effect.
The preparation of embodiment 1 Rhizoma Dryopteris Crassirhizomatis extract
Cross 60 mesh sieves after being pulverized by Rhizoma Dryopteris Crassirhizomatis 100g, sieved thing adds 8 times amount (ml/g) ethyl acetate, 25 DEG C of lixiviate 5h, extracts 3 times continuously.Then merge extractive liquid, filter, concentrating under reduced pressure, obtains Rhizoma Dryopteris Crassirhizomatis extract extractum 1.
Residue Rhizoma Dryopteris Crassirhizomatis medicinal residues are added the ethanol of 8 times amount (ml/g) 70%, 85 DEG C of backflow 2h, extract 2 times continuously.Then merge extractive liquid, filters, and reclaim ethanol, concentrating under reduced pressure, obtains Rhizoma Dryopteris Crassirhizomatis extract extractum 2.
Rhizoma Dryopteris Crassirhizomatis extractum 1 and extractum 2 are mixed, obtains Rhizoma Dryopteris Crassirhizomatis effective active composition.
The preparation of embodiment 2 Rhizoma Dryopteris Crassirhizomatis extract
Cross 60 mesh sieves after being pulverized by Rhizoma Dryopteris Crassirhizomatis 100g, sieved thing adds 10 times amount (ml/g) ethyl acetate, 25 DEG C of lixiviate 20h, extracts 1 time continuously.Then merge extractive liquid, filter, concentrating under reduced pressure, obtains Rhizoma Dryopteris Crassirhizomatis extract extractum 1.
Residue Rhizoma Dryopteris Crassirhizomatis medicinal residues are added the ethanol of 5 times amount (ml/g) 75%, reflux, extract, 3h, extracts 4 times continuously.Then merge extractive liquid, filters, and reclaim ethanol, concentrating under reduced pressure, obtains Rhizoma Dryopteris Crassirhizomatis extract extractum 2.
Rhizoma Dryopteris Crassirhizomatis extractum 1 and extractum 2 are mixed, obtains Rhizoma Dryopteris Crassirhizomatis effective active composition.
The preparation of embodiment 3 Rhizoma Dryopteris Crassirhizomatis extract
Cross 20 mesh sieves after being pulverized by Rhizoma Dryopteris Crassirhizomatis 100g, sieved thing adds 3 times amount (ml/g) ethyl acetate, 15 DEG C of lixiviate 1h, extracts 3 times continuously.Then merge extractive liquid, filter, concentrating under reduced pressure, obtains Rhizoma Dryopteris Crassirhizomatis extract extractum 1.
Residue Rhizoma Dryopteris Crassirhizomatis medicinal residues are added the ethanol of 3 times amount (ml/g) 80%, reflux, extract, 1h, extracts 3 times continuously.Then merge extractive liquid, filters, and reclaim ethanol, concentrating under reduced pressure, obtains Rhizoma Dryopteris Crassirhizomatis extract extractum 2.
Rhizoma Dryopteris Crassirhizomatis extractum 1 and extractum 2 are mixed, obtains Rhizoma Dryopteris Crassirhizomatis effective active composition.
The preparation of embodiment 4 Rhizoma Dryopteris Crassirhizomatis extract
Cross 60 mesh sieves after being pulverized by Rhizoma Dryopteris Crassirhizomatis 100g, sieved thing adds 5 times amount (ml/g) ethyl acetate, 4 DEG C of lixiviate 24h, extracts 4 times continuously.Then merge extractive liquid, filter, concentrating under reduced pressure, obtains Rhizoma Dryopteris Crassirhizomatis extract extractum 1.
Residue Rhizoma Dryopteris Crassirhizomatis medicinal residues are added the ethanol of 6 times amount (ml/g) 60%, reflux, extract, 4h, extracts 1 time continuously.Then merge extractive liquid, filters, and reclaim ethanol, concentrating under reduced pressure, obtains Rhizoma Dryopteris Crassirhizomatis extract extractum 2.
Rhizoma Dryopteris Crassirhizomatis extractum 1 and extractum 2 are mixed, obtains Rhizoma Dryopteris Crassirhizomatis effective active composition.
The preparation of embodiment 5 Rhizoma Dryopteris Crassirhizomatis extract
Cross 60 mesh sieves after being pulverized by Rhizoma Dryopteris Crassirhizomatis 100g, sieved thing adds 6 times amount (ml/g) ethyl acetate, 4 DEG C of lixiviate 8h, extracts 3 times continuously.Then merge extractive liquid, filter, concentrating under reduced pressure, obtains Rhizoma Dryopteris Crassirhizomatis extract extractum 1.
Residue Rhizoma Dryopteris Crassirhizomatis medicinal residues are added the ethanol of 6 times amount (ml/g) 70%, reflux, extract, 2h, extracts 2 times continuously.Then merge extractive liquid, filters, and reclaim ethanol, concentrating under reduced pressure, obtains Rhizoma Dryopteris Crassirhizomatis extract extractum 2.
Rhizoma Dryopteris Crassirhizomatis extractum 1 and extractum 2 are mixed, obtains Rhizoma Dryopteris Crassirhizomatis effective active composition.
The preparation of embodiment 6 Rhizoma Dryopteris Crassirhizomatis granule
The Rhizoma Dryopteris Crassirhizomatis extract (embodiment 1) of 100g is used 500ml n-hexane dissolution, adds sucrose 100g, cyclodextrin 200g, conventionally make granule 400g.
The preparation of embodiment 7 Rhizoma Dryopteris Crassirhizomatis soluble powder
The Rhizoma Dryopteris Crassirhizomatis extract (embodiment 1) of 100g is used 500ml acetic acid ethyl dissolution, is prepared into Rhizoma Dryopteris Crassirhizomatis solution; Cyclodextrin solution is prepared into the beta cyclodextrin 200g 1000ml that adds water; 2 kinds of solution are positioned in homogenizer, stir 7 minutes with 4000rpm/min, 50 DEG C of oven dry, pulverize, cross 20 mesh sieves, be prepared into soluble powder 300g.
The preparation of embodiment 8 Rhizoma Dryopteris Crassirhizomatis oral liquid
By the Rhizoma Dryopteris Crassirhizomatis extract 300mlN of 100g, dinethylformamide dissolves, and is prepared into Rhizoma Dryopteris Crassirhizomatis solution, then adds aquesterilisa to 1000ml, obtains the oral liquid of 10%.
The optimization of experimental example 1 Rhizoma Dryopteris Crassirhizomatis extraction process
Test objective: with extractum yield and active constituent content and animal model curative effect for investigating object, relatively this extraction process and the difference in the past reporting extraction process
1 material
1.1 Rhizoma Dryopteris Crassirhizomatiss: purchased from Anguo City, Hebei province Chang Da prepared slices of Chinese crude drugs company limited.Appraisal basis Primary Reference " Chinese veterinary pharmacopoeia " 2010 editions two and " Beijing's prepared slices of Chinese crude drugs standard ".
95% ethanol, ethyl acetate: analytical pure, purchased from chemical reagents corporation of Beijing.
The blue belit in firm jail: analytical pure, purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
Dryocrassine ABBA standard substance: purchased from Ze Lang bio tech ltd, Nanjing, content is greater than 98%.
1.2 laboratory animals: 28 age in days SPF chickens 64, male, purchased from Beijing laboratory animal company limited of dimension tonneau China.
1.3 strains: artificial challenge's strain IBDVLX strain (Academy of Agricultural Sciences of Beijing animal and veterinary institute), with tissue homogenate every chickling eye dripping, collunarium 0.2mL altogether.Start medication after 24h, use 5 days continuously.
2 methods
The preparation method of 2.1 samples
Sample 1 ~ 5: the method by embodiment 1 ~ 5 is obtained Rhizoma Dryopteris Crassirhizomatis extract.Get extractum 0.1g respectively, add 5ml dissolve with ethanol, precision measures subsequent filtrate 1mL in 100mL measuring bottle, is settled to scale, shakes up, to obtain final product with 95% ethanol dilution.Shake up, as need testing solution.
Sample 6: get after Rhizoma Dryopteris Crassirhizomatis medicinal material coarse powder 1000kg pulverizes and cross 60 mesh sieves, add 95% ethanol 100ml, backflow 1h, is cooled to room temperature, extractum weight of weighing.Get 0.1g extractum and add 5ml dissolve with ethanol, precision measures subsequent filtrate 1mL in 100mL measuring bottle, is settled to scale, shakes up, to obtain final product with 95% ethanol dilution.Shake up, as need testing solution (Gao Zengping, Su Yulei, Beijing University of Chinese Medicine's journal, 2009,32:259-262).
Sample 7: get after Rhizoma Dryopteris Crassirhizomatis medicinal material coarse powder 1000kg pulverizes and cross 60 mesh sieves, add 95% ethanol 100ml, merceration 2h, supersound extraction 40min, filter, reclaim ethanol, measure the weight of extractum.Then, take 0.1g extractum, add 5ml dissolve with ethanol, get subsequent filtrate 1mL in 100mL measuring bottle, be settled to scale with 95% ethanol dilution, shake up, to obtain final product.Shake up, as need testing solution (Gao Zengping, Su Yulei, Beijing University of Chinese Medicine's journal, 2009,32:259-262).
2.2 sample tests methods
The foundation of total phloroglucinol standard curve: get Dryocrassine ABBA standard substance is solvent with chloroform, is mixed with the Dryocrassine ABBA solution of concentration 1mg/ml, as standard solution.Precision takes Dryocrassine ABBA standard substance 5.375mg and is placed in 5ml volumetric flask, add chloroform to dissolve and standardize solution, precision measures 50 μ l, 75 μ l, 100 μ l, 125 μ l, 150 μ l, 175 μ l, 200 μ l put in 2ml volumetric flask, chloroform standardize solution, obtained concentration is respectively 25 μ g/ml, 37.5 μ g/ml, 50 μ g/ml, 62.5 μ g/ml, 75 μ g/ml, 87.5 μ g/ml, the solution of 100 μ g/ml, solution is transferred to 10ml scale in vitro, add the blue belit developer in the firm jail of 5ml0.5%, lucifuge reaction 20min, absorbance is measured in 476nm place.With absorbance (OD) be abscissa X, concentration is vertical coordinate Y, the standard curve of obtained total phloroglucinol.
Sample total phloroglucinol assay: get respectively above-mentioned 7 kinds of sample liquids 2ml supernatant prepared to 10ml scale in vitro, add the blue developer in the firm jail of 5ml0.5%, lucifuge reaction 20min, measure the absorbance of each sample respectively at 476nm place.
2.3 test groupings: be divided into Rhizoma Dryopteris Crassirhizomatis extract 7 dosage test groups, counteracting toxic substances matched group is set simultaneously, often organize 8 SPF chickens, with the administration of oral administration gavage mode, every day 2 times.
2.4 counteracting toxic substances methods: the fabricius bursa tissue getting the ill SPF chicken of artificial challenge's infectious bursa of Fabricius virus, grind with the glass grinding device of sterilizing, 1 fabricius bursa adds 1ml sterile saline, make tissue homogenate, multigelation three times, centrifuging and taking supernatant, supernatant normal saline carries out 1:10 dilution, add gentamycin according to 4000 units/ml in diluent, act on after 30 minutes and using.Except blank group, other respectively organizes equal counteracting toxic substances, and every chicken carries out eye dripping, collunarium amounts to 0.2ml.Counteracting toxic substances successive administration 5 days after 12 hours, oral administration gavage administration.
2.5 evaluation indexes: with the efficiency of the yield of extractum, total phloroglucinol Content evaluation 7 kinds of extracting method.After artificial bursal disease virus counteracting toxic substances, observe it and survive quantity, evaluate its 7 kinds of extracting method protection features with survival rate.
3 results
3.1 different extraction processes are on the impact of Rhizoma Dryopteris Crassirhizomatis extract yield
With extractum productive rate and total phloroglucinol content for inspection target, 7 kinds of technique extractum yields are respectively 14.5%, 12.0%, 11.3%, 9.8%, 12.6%, 8.4% and 6.2%; Total phloroglucinol content is respectively 182.5,154.4,122.8,98.5,145.3,68.3 and 45.4mg/g, and as can be seen here, ethyl acetate lixiviate-alcohol reflux technique is totally better than ethanol refluxing process and ethanol immersion.Meanwhile, during ethyl acetate lixiviate, room temperature (25 DEG C) extracting effect is better than 4 DEG C that report in method 3.Secondly, alcohol reflux concentration 70% is better than 95% concentration in method 2.Therefore, ethyl acetate lixiviate-alcohol reflux is selected to be Optimization Technology.
The comparison of table 1 Different Extraction Method medicine yield
The Rhizoma Dryopteris Crassirhizomatis extract that 3.2 different extraction processes obtain is on the impact of artificial challenge's fabricius bursa model
After attacking with bursal disease virus; the sample prepared of oral distinct methods respectively; its protective rate is respectively 100.0%, 87.5%, 75.0%, 75.0%, 87.5%, 62.5% and 62.5%; from overall protective rate; sample 1-5 protective rate significance is higher than sample 6 and sample 7, and sample 1 protective rate pole significance is higher than sample 6-7.The extract property evident in efficacy that this prompting adopts ethyl acetate lixiviate-ethanol refluxing process to obtain is higher than independent alcohol steep, separately alcohol reflux thing.
The different extraction process of table 2 is to SPF chicken bursa therapeutic effect
| Grouping | Chicken quantity (only) | Dosage (mg/kg) | Survival rate (%) |
| Sample 1(method 1) | 8 | 100 | 100.0 |
| Sample 2(method 2) | 8 | 100 | 87.5 |
| Sample 3(method 3) | 8 | 100 | 75.0 |
| Sample 4(method 4) | 8 | 100 | 75.0 |
| Sample 5 (method 5) | 8 | 100 | 87.5 |
| Sample 6(method 6) | 8 | 100 | 62.5 |
| Sample 7(method 7) | 8 | 100 | 62.5 |
| Virus control group | 8 | - | 40.0 |
3 conclusions:
This test confirms that ethyl acetate lixiviate-alcohol reflux technique can improve Rhizoma Dryopteris Crassirhizomatis extract and total phloroglucinol content, obtains extract for treating chicken bursa effect simultaneously and is better than traditional ethanol circumfluence method and ethanol immersion.
The test of experimental example 2 Rhizoma Dryopteris Crassirhizomatis treatment artificial challenge SPF avian influenza
Test objective: determine whether Rhizoma Dryopteris Crassirhizomatis extract has treatment high pathogenic avian influenza effect
1 materials and methods
1.1 laboratory animals: 6 week age SPF chicken 60, purchased from Beijing dimension tonneau China laboratory animal company limited.Raise in negative pressure isolator.
1.2 medicines: Rhizoma Dryopteris Crassirhizomatis solubilized powder is prepared according to method described in embodiment 7; Matched group medicine Buddha's warrior attendant gastral cavity amine (HohnsonMattheyCompany, the U.S., Lot10118375, analytical pure is 99%).
1.3 test groupings: dosage group, Rhizoma Osmundae low dose group, astragalus polysaccharides group, infection matched group and blank group in Rhizoma Osmundae high dose group, Rhizoma Osmundae, amount to 6 groups, often organize 10 chickens.
Table 3 tests grouping and process
| Grouping | Quantity (only) | Administration concentration | Route of administration | Administration number of times |
| High dose group | 10 | 100mg/kg | Oral | 2/ day |
| Middle dosage group | 10 | 50mg/kg | Oral | 2/ day |
| Low dose group | 10 | 25mg/kg | Oral | 2/ day |
| Amantadine matched group | 10 | 50mg/kg | Oral | 2/ day |
| Virus control group | 10 | Ethyl acetate+water | Oral | 2/ day |
| Blank group | 10 | Ethyl acetate+water | Oral | 2/ day |
1.4 strains: H 5 N 1 avian influenza HB-bp Strain (A/Chicken/Hebei/102/2005), by viral dilution to 10
-4, nasal cavity gives 0.2ml, and after counteracting toxic substances 12 hours start administration, and continuous use uses 5 days, every day 2 times.
1.5 evaluation indexes:
Survival rate and weight gain index: Continuous Observation 10 days, every day viewing test chicken spirit, appetite, active situation, makes a record.On-test and at the end of, test chicken is weighed.Calculate body weight increase rate, survival rate and mean survival time.
2 results
2.1 Rhizoma Dryopteris Crassirhizomatis extracts are on the impact of SPF chicken body weight after counteracting toxic substances
After being infected by collunarium, after artificial challenge's bird flu virus, high dose group, middle dosage group and low dose group body weight increase rate are respectively 80.0%, 76.8% and 80.4%, and amantadine matched group is 65.2%.High, the middle dosage group of statistical analysis Rhizoma Dryopteris Crassirhizomatis and low dose group the relative weight gain difference on effect are not remarkable, but significance is higher than amantadine group (P<0.05).
After table 4 counteracting toxic substances, Rhizoma Dryopteris Crassirhizomatis affects result to SPF chicken rate of body weight gain
Note: show significant difference P<0.05 with a-b, b-c in string; A-c shows difference extremely significantly P<0.01.
After 2.2 counteracting toxic substances, Rhizoma Dryopteris Crassirhizomatis extract is on SPF chicken survival rate and time-to-live impact
After bird flu virus counteracting toxic substances, Rhizoma Osmundae high dose group, middle dosage group, low dose group survival rate are respectively 80.0%, 70.0% and 70.0%, and amantadine matched group is 40.0%.On the time-to-live, Rhizoma Dryopteris Crassirhizomatis three dosage group mean survival times are 8.2 days, 7.5 days and 7.5 days, and amantadine matched group is 3.8 days.
Table 5 Rhizoma Dryopteris Crassirhizomatis extract is on artificial challenge SPF adult livability and the impact of time-to-live
| Grouping | Amount of survival (only) | Survival rate (%) | Mean survival time |
| High dose group | 8 | 80.0 a | 8.2 a |
| Middle dosage group | 7 | 70.0 a | 7.5 a |
| Low dose group | 7 | 70.0 a | 7.5 a |
| Amantadine matched group | 4 | 40.0 b | 3.8 b |
| Virus control group | 2 | 20.0 c | 2.5 c |
| Blank group | 10 | 100.0 | 10.0 |
Note: show significant difference P<0.05 with a-b, b-c in string; A-c shows difference extremely significantly P<0.01.
3 conclusions
After this result of the test bird flu virus counteracting toxic substances, Rhizoma Dryopteris Crassirhizomatis is with the oral SPF chicken of 25mg/kg, and continuous oral 5 days, can treat the attack that bird flu causes, and survival rate is to 70%, and significance is higher than amantadine matched group.
The therapeutic test of experimental example 3 Rhizoma Dryopteris Crassirhizomatis treatment artificial challenge SPF newcastle disease
Test objective: determine whether Rhizoma Dryopteris Crassirhizomatis extract has treatment newcastle disease effect
1 materials and methods
1.1 laboratory animals: 5 week age SPF chicken 85, purchased from Jinan Si Pafasi poultry company limited.Raise in negative pressure isolator.
1.2 medicines: Rhizoma Dryopteris Crassirhizomatis solubilized powder is prepared according to method described in embodiment 7; Astragalus polysaccharides powder, purchased from Beijing Centre Biology Co., Ltd., lot number 20100605.
1.3 test groupings: dosage group, Rhizoma Osmundae low dose group, astragalus polysaccharides group, infection matched group and blank group in Rhizoma Osmundae high dose group, Rhizoma Osmundae.
Table 6 tests grouping and process
| Grouping | Quantity (only) | Administration concentration | Route of administration | Administration number of times |
| High dose group | 10 | 100mg/kg | Oral | 2/ day |
| Middle dosage group | 10 | 50mg/kg | Oral | 2/ day |
| Low dose group | 10 | 25mg/kg | Oral | 2/ day |
| Astragalus polysaccharides matched group | 10 | 50mg/kg | Oral | 2/ day |
| Virus control group | 10 | 0 | - | - |
| Blank group | 10 | 0 | - | - |
1.4 strains and counteracting toxic substances Avian pneumo-encephalitis virus (Beijing Strain F4), purchased from China Veterinary Drugs Supervisory Inst..By viral dilution to 10
-5, nasal cavity gives 0.1ml, and after counteracting toxic substances 12 hours start administration, and continuous use uses 5 days.
1.5 evaluation indexes:
1.5.1 survival rate and weight gain index: duration of test, every day viewing test chicken spirit, appetite, active situation, makes a record.On-test and at the end of, test chicken is weighed.Calculate body weight increase rate, survival rate.
1.5.2 virus isolated rate: after counteracting toxic substances the 10th day, the chicken of survival implements euthanasia, asepticly takes chicken liver, lungs, and inoculation 9-10 age in days SPF Embryo Gallus domesticus, measures its Avian pneumo-encephalitis virus content.
2 results
After the oral SPF chicken of 2.1 Rhizoma Dryopteris Crassirhizomatis extract, body weight is affected
After being infected by collunarium, after Newcastle Disease Virus F4 strain, high dose group, middle dosage group and low dose group body weight increase rate are respectively 88.2%, 91.4% and 80.4%, and astragalus polysaccharides matched group is 65.9%.Statistical analysis testing drug low dose group the relative weight gain Be very effective is lower than high dose group and middle dosage group, and significance is higher than astragalus polysaccharides matched group.
The oral rear impact on SPF chicken rate of body weight gain of table 7 Rhizoma Dryopteris Crassirhizomatis
Note: show significant difference P<0.05 with a-b, b-c in string; A-c shows difference extremely significantly P<0.01.
2.2 Rhizoma Dryopteris Crassirhizomatis extracts are on SPF chicken survival rate and the impact of time-to-live
After the strong poison of artificial challenge, testing drug high dose group, middle dosage group, low dose group survival rate are respectively 80.0%, 80.0%, 60.0%, and astragalus polysaccharides matched group is 40.0%.On the time-to-live, Rhizoma Dryopteris Crassirhizomatis three dosage group mean survival times are 7.2 days, 8.5 days and 6.0 days, and astragalus polysaccharides matched group is 6.3 days.
Table 8 Rhizoma Dryopteris Crassirhizomatis extract is on artificial challenge SPF adult livability and the impact of time-to-live
| Grouping | Amount of survival (only) | Survival rate (%) | Mean survival time |
| High dose group | 8 | 80.0 a | 7.2 a |
| Middle dosage group | 8 | 80.0 a | 8.5 b |
| Low dose group | 6 | 60.0 b | 6.0 a |
| Astragalus polysaccharides matched group | 4 | 40.0 c | 4.5 c |
| Virus control group | 4 | 20.0 | 3.5 |
| Blank group | 10 | 100.0 | 10.0 |
Note: show significant difference P<0.05 with a-b, b-c in string; A-c shows difference extremely significantly P<0.01.
3 conclusions:
After this result of the test display newcastle counteracting toxic substances, Rhizoma Dryopteris Crassirhizomatis is with the oral SPF chicken of 50mg/kg, and continuous oral 5 days, the survival rate of chicken is to 80%, and significance is higher than astragalus polysaccharides group.
Experimental example 4 Rhizoma Dryopteris Crassirhizomatis extract is to the therapeutic effect of infectious bursal disease
Test objective: determine whether Rhizoma Dryopteris Crassirhizomatis extract has the curative effect for the treatment of infections chicken cloacal bursa virus
1 materials and methods
1.1 laboratory animals: 26 age in days SPF chickens, male, purchased from Beijing laboratory animal company limited of dimension tonneau China.
1.2 strains: artificial challenge's strain IBDVLX strain (Academy of Agricultural Sciences of Beijing animal and veterinary institute), tissue homogenate every chickling eye dripping, collunarium be 0.2mL altogether.Start medication after 24h, use 5 days continuously.
1.3 medicines: astragalus polysaccharides powder, purchased from Beijing Centre Biology Co., Ltd., lot number 20100605; Rhizoma Dryopteris Crassirhizomatis extract oral liquid prepared by embodiment 8.
1.4 test groupings: be divided into Rhizoma Dryopteris Crassirhizomatis extract 3 dosage test groups (150mg/kg body weight, 100mg/kg body weight, 50mg/kg body weight), positive controls administration astragalus polysaccharides 100mg/kg body weight; Counteracting toxic substances matched group, normal healthy controls group are set simultaneously, often organize 10 SPF chickens, with the administration of oral administration gavage mode.
1.5 counteracting toxic substances methods
Get the fabricius bursa tissue of the ill SPF chicken of artificial challenge's infectious bursa of Fabricius virus, grind with the glass grinding device of sterilizing, 1 fabricius bursa adds 1ml sterile saline, make tissue homogenate, multigelation three times, centrifuging and taking supernatant, supernatant normal saline carries out 1:10 dilution, add gentamycin according to 4000 units/ml in diluent, act on after 30 minutes and using.Except blank group, other respectively organizes equal counteracting toxic substances, and every chicken carries out eye dripping, collunarium amounts to 0.2ml.Counteracting toxic substances successive administration 5 days after 12 hours, oral administration gavage administration.
1.6 observation index
Body weight increase rate=the relative weight gain/normal healthy controls weight gain × 100%; Immune Organs Index=immune organ weight in wet base/body weight × 100%; Survival rate=survival number of elements/experiment quantity × 100%.
2 results
2.1 Rhizoma Dryopteris Crassirhizomatiss are on the impact of artificial challenge chicken body weight
After artificial challenge's virus, high dose group, middle dosage group, low dose group body weight increase rate are respectively 96.3%, 67.9% and 62.8%, and astragalus polysaccharides matched group is 43.2%.The result of the test display weightening finish effect of Rhizoma Osmundae extract and dosage present amount-result relation, and high dose group extremely significance higher than astragalus polysaccharides matched group.
Table 9 various dose is on the impact of body weight
Note: with a-b, b-cP<0.05, a-cP<0.01 in string
2.2 impacts on the survival rate of chicken bursa artificial challenge
As can be seen from Table 10, bracken high dose group, middle dosage group, low dose group are respectively 90.0%, 90% and 70%, and astragalus polysaccharides matched group and infection matched group are respectively 50% and 40%.Result display high dose and middle dosage group difference are not remarkable, but middle dosage significance is higher than low dose group (P<0.05), and pole significance is higher than astragalus polysaccharides matched group (P<0.01).
Table 10 Rhizoma Dryopteris Crassirhizomatis extract is on the impact of survival rate
| Group | Number of elements | Survival number of elements | Survival rate |
| 150mg/kg | 10 | 9 | 90.0 a |
| 100mg/kg | 10 | 9 | 90.0 a |
| 50mg/kg | 10 | 7 | 70.0 b |
| Astragalus polysaccharides 100mg/kg | 10 | 5 | 50.0 c |
| Infect matched group | 10 | 4 | 40.0 |
| Normal healthy controls group | 10 | 10 | 100.0 |
2.3 impacts on the immune organ after chicken bursa artificial challenge
Table 11 result display Rhizoma Dryopteris Crassirhizomatis high, medium and low dosage group fabricius bursa average index is respectively 1.58,1.56 with 1.35, astragalus polysaccharides is 0.95, through statistical analysis Rhizoma Dryopteris Crassirhizomatis high dose group, middle dosage group significance higher than astragalus polysaccharides group (P<0.01), and diversity is remarkable.
Immune Organs Index after table 11 Rhizoma Dryopteris Crassirhizomatis extract treatment fabricius bursa artificial challenge
3 conclusions
Rhizoma Dryopteris Crassirhizomatis extract is oral with 100mg/kg, and the infection effectively can treated infectious bursa of Fabricius virus and cause for continuous 5 days, effect is better than the astragalus polysaccharides of 100mg/kg.
Experimental example 5 Rhizoma Dryopteris Crassirhizomatis extract poultry immunity strengthens function test
Test objective: determine whether Rhizoma Dryopteris Crassirhizomatis extract has and strengthen chicken humoral immunization and Study On Cellular Immune
1 materials and methods
1.1 medicines and vaccine
Cyclophosphamide (Cy): purchased from Tianjin Jinshi Pharmaceutical Co., Ltd., lot number 20111002, accurate word H12021006, the 50mg/ sheet of traditional Chinese medicines.
Levamisole (LH): purchased from Shandong Kernel and Hall Pharmaceutical Industry Co., Ltd., lot number: 110102, accurate word H37020819, the 25mg/ sheet of traditional Chinese medicines; .
Rhizoma Dryopteris Crassirhizomatis soluble powder: embodiment 7.
Newcastle disease vaccine: CS2 strain is purchased from Qingdao company limited of foreign country.
T cell subclass antibodies: Mouseanti-chickenCD3, Mouseanti-chickenCD4, Mouse anti-chickenCD8(SouthernBiotech, USA): Bo Leide bio tech ltd, Beijing
1.2 animals: 21 age in days SPF chickens, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
2 methods
2.1 test groupings
90 21 age in days SPF chickens are divided into 5 groups at random, often organize 18.Blank group, cyclophosphamide group, levamisole group, Rhizoma Dryopteris Crassirhizomatis group, Rhizoma Dryopteris Crassirhizomatis+cyclophosphamide group respectively.
2.2 immunity and administering modes
21 Japanese instar chicklings all carry out Newcastle disease attenuated vaccine CS2 strain immunity, and immunization ways is eye dripping, collunarium, every 0.2mL.The administering mode of medicine mainly based on oral, once a day, successive administration 2 weeks, drug withdrawal 1 week.Blank group oral normal saline, Rhizoma Dryopteris Crassirhizomatis+cyclophosphamide group only presses prescribed dose oral a kind of medicine every day.Grouping and administrations refer to table 12.Administration first day takes eye dripping collunarium mode immunity newcastle disease vaccine CS2 strain.
2.3 Testing index
Body weight change situation: record initial average weight, records low 1,2,3 week average weight situation of change respectively.
Antibody horizontal detects: administration first day takes eye dripping collunarium mode immunity newcastle disease vaccine CS2 strain, respectively at the 7th, 14,21 day wing venous collection serum, by newcastle epidemic disease antibody level in blood clotting Inhibition test examination volume.Immune Organs Index: respectively at after administration the 7th, 14 day, and after drug withdrawal 1 week, often organize random selecting 3 chickens and dissect, weigh its spleen, fabricius bursa, thymus organ weight, calculate shoot formation, shoot formation=organ weight/body weight * 100%(mg/g).
The detection of T lymphocyte and subgroup: after 1 week, detect peripheral blood T lymphocyte content with drug withdrawal in 14 days respectively at administration.
Table 12 divides into groups and administrations
| Grouping | Dosage | Administration number of times | Test number of elements |
| Rhizoma Dryopteris Crassirhizomatis group | 100mg/kg | 2 days/time | 18 |
| Levamisole group | 10mg/kg | 2 days/time | 18 |
| Cyclophosphamide group | 50mg/kg | 2 days/time | 18 |
| Rhizoma Osmundae+cyclophosphamide group | 100mg/kg+50mg/kg | 1 day/time, the two interval uses | 18 |
| Normal saline group | —— | —— | 18 |
2.4 data analysiss and process
Test data EXCEL2003 and SPSS13.0 statistical software carry out date processing and one-dimensional variance analysis, and experimental data represents with (X ± SD).Significant difference is P<0.05, and difference is extremely significantly P<0.01.
3 results
3.1 Rhizoma Dryopteris Crassirhizomatiss are on the impact of SPF chicken body weight
Table 13 shows SPF chicken body weight change situation, and under the prerequisite of original body mass there was no significant difference, administration is after 2 weeks, and cyclophosphamide group significance is lower than all the other each group, and display immunosuppression model is successfully prepared.After 3rd week, Rhizoma Dryopteris Crassirhizomatis group body weight significance higher than levamisole group, cycli phosphate amide group and normal saline group, but not remarkable with Rhizoma Osmundae+cyclophosphamide group difference.This prompting Rhizoma Osmundae can block the inhibitory action of cyclophosphamide.
Table 13 medicine is on the impact of SPF chicken body weight
| ? | Rhizoma Osmundae group | Levamisole group | Cyclophosphamide | Rhizoma Osmundae+cyclophosphamide group | Normal saline group |
| Original body mass | 153.36±17.45 | 152.09±17.49 | 152.18±17.35 | 151.55±17.32 | 154.09±16.67 |
| First week | 258.10±22.17 a | 243.58±28.42 a | 206.64±22.58 c | 233.27±15.14 b | 240.09±30.44 a |
| Second week | 295.88±29.75 a | 280.00±30.26 a | 241.62±48.11 b | 279.25±39.10 a | 282.44±41.68 a |
| 3rd week | 423.01±51.46 a | 359.17±50.89 b | 334.33±13.49 c | 385.4±47.91 a | 365.50±61.21 b |
Note: show significant difference P<0.05 with a-b, b-c in a line; A-c shows difference extremely significantly P<0.01.
3.2 medicines are on the impact of SPF Immune Organs of Chicken index
The results are shown in Table shown in 14, after the 2nd week, its bursal index of cyclophosphamide processed group, the equal significance of thymus index reduce, and this illustrates that immunosuppression model is set up.Compared with levamisole group, after Rhizoma Dryopteris Crassirhizomatis soluble powder is oral can significance improve fabricius bursa, spleen shoot formation.Along with the prolongation of administration time, Rhizoma Osmundae+cyclophosphamide group is compared with levamisole group, normal saline group, and Immune Organs Index difference is not remarkable, and this prompting Rhizoma Osmundae can block the inhibitory action of cyclophosphamide.
Table 14 medicine is on the impact of SPF Immune Organs of Chicken index
Note: show significant difference P<0.05 with a-b, b-c in a line; A-c shows difference extremely significantly P<0.01.
3.3 medicines are on the impact of newcastle epidemic disease antibody level
Table 15 show orally separately to pass through, antibody IgY against chicken Newcastle Disease continuous rise after levamisole and Rhizoma Osmundae+cyclophosphamide, 21 days antibody horizontals start to decline.14th day Rhizoma Dryopteris Crassirhizomatis group, levamisole hydrochloride group antibody horizontal are higher than normal saline group and cyclophosphamide group, but difference is not significantly (P<0.05).After 14 days, 21 days, Rhizoma Osmundae group is higher than Rhizoma Osmundae+cyclophosphamide group, and difference is not remarkable.But significance is higher than cyclophosphamide group and saline control group.
The oral rear impact on newcastle epidemic disease antibody level of table 15 Rhizoma Osmundae
| Project | 7 days HI value (log 2） | 14 days HI value (log 2） | 21 days HI value (log 2） |
| Rhizoma Osmundae group | 7.6 a | 9.0 a | 8.5 a |
| Levamisole group | 7.1 a | 8.5 a | 6.5 b |
| Rhizoma Osmundae+cyclophosphamide group | 7.0 a | 8.0 a | 7.8 a |
| Cyclophosphamide group | 6.0 a | 6.2 b | 5.0 c |
| Normal saline | 6.3 a | 6.8 b | 6.6 b |
The impact of 3.4 medicine human peripheral blood T lymphocyte content
The impact of table 16 medicine human peripheral blood T lymphocyte and lymphocyte subgroup
| Group | Rhizoma Osmundae group | Levamisole group | Cyclophosphamide group | Rhizoma Osmundae+cyclophosphamide group | Normal saline group |
| 14 days CD3 values | 69.49±5.08 a | 43.85±8.93 b | 29.7±10.97 c | 49.80±4.87 b | 50.25±6.20 b |
| 21 days CD3 values | 82.81±1.33 a | 40.5±5.60 b | 25.15±22.08 c | 42.12±13.34 b | 52.2±11.18 b |
| 14 days CD4/CD8 | 1.52±0.07 a | 1.32±0.04 b | 0.95±0.39 c | 1.27±0.04 b | 1.28±0.12 b |
| 21 days CD4/CD8 | 1.45±0.03 a | 1.03±0.06 b | 0.77±0.18 c | 1.08±0.13 b | 1.04±0.27 b |
Table 16 shows Rhizoma Osmundae individually dosed group of T cell CD3 molecule subgroup, and in administration, after 14 days and 21 days, quantity significance is higher than levamisole group and Rhizoma Osmundae+cyclophosphamide group (P<0.05), and pole significance is higher than cyclophosphamide group (P<0.01).Rhizoma Dryopteris Crassirhizomatis group CD4+/CD8+ ratio significance is higher than levamisole group and Rhizoma Osmundae+cyclophosphamide group (P<0.05), and pole significance is higher than cyclophosphamide group (P<0.01).This prompting Rhizoma Dryopteris Crassirhizomatis extract effectively can improve the T cell immunity of poultry, is conducive to the removing (as virus, born of the same parents' endophyte and parasite) of pathogenic microorganism in cells.
4 conclusions
Rhizoma Dryopteris Crassirhizomatis extract effectively can increase the Immune Organs Index (fabricius bursa, thymus) of chicken and improve the overall immune organ function of poultry.T lymphocyte quantity can be improved after continuing medication, promote the differentiation of t lymphocyte subset group, inducing cellular immune, thus play Promote immunity effect.
The test of experimental example 6 Rhizoma Dryopteris Crassirhizomatis extract regulation and control pigling immunity
Test objective: whether checking Rhizoma Dryopteris Crassirhizomatis extract by oral ablactational baby pig, can promote the immunity of piglet and the generation of resist the disease and popular.
1 material
1.1 medicine
Astragalus polysaccharides (45% content): purchased from Hebei Ruigao Animal Pharmaceutical Co., Ltd., lot number 2011100701.
Rhizoma Osmundae extract: every gram of granule contains crude drug 1 gram, effluent north health company limited Medium scale (by embodiment 6) all over the world.
1.2 animal
40 ages in days are selected in this test, and Du × length × large ablactational baby pig of the health that nest is other, parity is close, average weight is 12.61 ± 0.23kg about, amounts to 100.
1.3 test kit
Porcine Interferon-gamma test kit, porcine interleukin-4 test kit: purchased from Invitrogen company.Hog cholera antibody test kit, PorCheck CSFV Ab 2.0antibody test kit, is produced by PRIONICS company.
2 methods
2.1 groupings and daily ration composition
According to test pig body weight and sex, by 100 ablactational baby pig, be divided into 5 processed group, often organize 20 piglets, test totally 14 days full phase.Concrete grouping situation is as follows: Rhizoma Osmundae 100mg/kg, 50mg/kg and 25mg/kg, astragalus polysaccharides group 100mg/kg and blank daily ration group.Each processed group does not all add any other medicines, and all daily rations are all fed with mash form.Feeding and management is carried out according to the administrative standard of modern farming.
2.2 evaluation index
Within experimental period, carry out evaluating its function with piglet sickness rate, Scours index, growth performance, antibody horizontal and T cell subgroup and cytokine.Sickness rate (%)=morbidity head number/test piglet number × 100; Diarrhea rate (%)=diarrhoea head number × Diarrhoea days/(test piglet number × test natural law) × 100
Antibody horizontal: test first 7 days, carry out hog cholera immune to piglet, tests and carries out foot and mouth disease immunity on the 1st day.Carry out vena cava anterior blood sampling to ablactational baby pig when testing the 15th day, often repeat 4, separation of serum, subpackage is preserved and is detected.Test kit is adopted to detect the antibody horizontal of its swine fever virus.
Cell subsets ratio: after feeding continuously 15 days, often repeat 4 and gather 2ml peripheral blood with Sterile vacuum anticoagulant blood-collecting pipe, 4 DEG C of preservations, carry out Flow cytometry T cell subset proportions in 12 hours.
Cytokine levels: feed continuously after 15 days, often repeats 4 to ablactational baby pig and carries out vena cava anterior blood sampling, separation of serum, and subpackage is preserved, and adopts test kit to detect interleukin-4 content and gamma interferon content.
3 results and analysis
3.1 Rhizoma Dryopteris Crassirhizomatis piglet clinical onset rate impacts
After table 17 shows oral Rhizoma Dryopteris Crassirhizomatis, 25mg/kg group piglet diarrhea rate reaches 2%, but 50mg/kg and 100mg/kg dosage group significance reduces piglet sickness rate and diarrhoea quantity.Blank group sickness rate reaches 10%, and diarrhea rate reaches 8%.
Table 17 Rhizoma Dryopteris Crassirhizomatis is on the impact of ablactational baby pig survival rate and diarrhea rate
3.2 Rhizoma Dryopteris Crassirhizomatiss are on the impact of piglet hog cholera antibody level
After table 18 shows oral 100,50 and 25mg/kg Rhizoma Dryopteris Crassirhizomatis, the hog cholera antibody level of piglet is significantly higher than blank group (P < 0.05), and various dose group Rhizoma Dryopteris Crassirhizomatis is difference remarkable (P > 0.05) compared with astragalus polysaccharides matched group.
Table 18 Rhizoma Dryopteris Crassirhizomatis affects result to hog cholera antibody level
| Grouping | Quantity | Hog cholera antibody level |
| Rhizoma Dryopteris Crassirhizomatis 100mg/kg | 10 | 62.10±12.15 a |
| Rhizoma Dryopteris Crassirhizomatis 50mg/kg | 10 | 58.12±2.50 a |
| Rhizoma Dryopteris Crassirhizomatis 25mg/kg | 10 | 53.10±10.24 a |
| Astragalus polysaccharides group 100mg/kg | 10 | 60.22±19.13 a |
| Blank group | 10 | 37.50±10.20 b |
The impact of 3.2 Rhizoma Dryopteris Crassirhizomatis human peripheral blood T cell subgroup ratios
After table 19 result shows oral 100,50 and 25mg/kg Rhizoma Dryopteris Crassirhizomatis, in peripheral blood, CD4+/CD8+ ratio is all significantly higher than astragalus polysaccharides matched group and blank group, and diversity significantly (P < 0.05).Astragalus polysaccharides group is compared with blank group, and difference is not remarkable.In addition, after oral Rhizoma Dryopteris Crassirhizomatis, in peripheral blood, IL-4 and IFN-γ content presents increase along with the increase of drug dose, shows docs-effect pattern.100,50mg/kg two group difference is not remarkable, but significance is higher than low dose group (P < 0.05), and pole significance is higher than astragalus polysaccharides group and blank group (P < 0.01).
The impact of table 19 Rhizoma Dryopteris Crassirhizomatis human peripheral blood T cell subgroup and cytokine
4 conclusions
This experimental result display Rhizoma Dryopteris Crassirhizomatis continuous oral 14 days, 50mg/kg and 100mg/kg dosage group can effectively reduce Linchuan sickness rate, diarrhea rate, improve piglet hog cholera antibody level, increase T cell subgroup ratio and improve IL-4 and IFN-γ in cytokine, thus improving the immunologic function of piglet.
Claims (7)
1. Rhizoma Dryopteris Crassirhizomatis extract, it obtains by the following method:
20 mesh sieves are crossed, the ethyl acetate that sieved thing adds ethyl acetate and medical material envelope-bulk to weight ratio 5-8 times amount lixiviate 2-3 time at 25 DEG C, each 2-8 hour after being pulverized by Rhizoma Dryopteris Crassirhizomatis, collect lixiviating solution and merge filtration, collect filtrate, reclaim ethyl acetate, obtain Rhizoma Dryopteris Crassirhizomatis crude extract 1; Residue medicinal residues add the alcohol reflux of 70% of ethanol and medical material envelope-bulk to weight ratio 5-8 times amount, reflux temperature 85 DEG C, extract 2-3 time, each 2-3 hour, collect filtrate, reclaim ethanol, obtain Rhizoma Dryopteris Crassirhizomatis crude extract 2, Rhizoma Dryopteris Crassirhizomatis extract 1 and 2 is mixed, concentrated, obtain extractum, obtain Rhizoma Dryopteris Crassirhizomatis extract.。
2. Rhizoma Dryopteris Crassirhizomatis extract according to claim 1 improves the application in immunity of livestock medicine or viral diseases medicine in preparation.
3. application according to claim 2, is characterized in that, described raising immunity of livestock refers to and strengthens cellular immunization and humoral immunization; Virosis is bird flu virus, Avian pneumo-encephalitis virus, bursal disease virus, virosis caused by renal type infectious bronchitis virus.
4. improve a medicine for fowl poultry immune ability or viral diseases, it contains Rhizoma Dryopteris Crassirhizomatis extract according to claim 1.
5. medicine according to claim 4, it also comprises one or more in starch, dextrin, lactose, stevioside, sucrose, glycyrrhizin, high fructose syrup, sucralose, xylitol, Sorbitol, glucose, pregelatinized Starch and/or carboxymethyl starch sodium.
6. the medicine according to claim 4 or 5, it is tablet, granule, soluble powder, oral liquid, injection or lyophilized injectable powder.
7. a preparation method for Rhizoma Dryopteris Crassirhizomatis extract, it comprises step:
20 mesh sieves are crossed, the ethyl acetate that sieved thing adds ethyl acetate and medical material envelope-bulk to weight ratio 5-8 times amount lixiviate 2-3 time at 25 DEG C, each 2-8 hour after being pulverized by Rhizoma Dryopteris Crassirhizomatis, collect lixiviating solution and merge filtration, collect filtrate, reclaim ethyl acetate, obtain Rhizoma Dryopteris Crassirhizomatis crude extract 1; Residue medicinal residues add the alcohol reflux of 70% of ethanol and medical material envelope-bulk to weight ratio 5-8 times amount, reflux temperature 85 DEG C, extract 2-3 time, each 2-3 hour, collect filtrate, reclaim ethanol, obtain Rhizoma Dryopteris Crassirhizomatis crude extract 2, Rhizoma Dryopteris Crassirhizomatis extract 1 and 2 is mixed, concentrated, obtain extractum, obtain Rhizoma Dryopteris Crassirhizomatis extract.
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| CN102813713A (en) * | 2012-08-01 | 2012-12-12 | 中国农业大学 | Rhizoma dryopteris crassirhizomae and fructus crataegi composition, preparation method and application of composition |
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| 液相色谱_串联质谱法分析绵马贯众抗禽流感病毒活性部位的化学成分;陈建新等;《质谱学报》;20060730;第27卷;118-120 * |
| 绵马贯众抗禽流感病毒活性部位的高效液相色谱_质谱法分析;陈建新等;《分析测试学报》;20080630;第27卷(第6期);623-626 * |
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Effective date of registration: 20180209 Address after: 068253 new industry demonstration area of Luanping County, Chengde City, Hebei Patentee after: Chengde Purun Biopharmaceutical Co., Ltd. Address before: 100193 Beijing city Haidian District Old Summer Palace Road 2 West Campus of China Agricultural University animal medicine science building 307 Patentee before: China Agricultural University |