CN109316489A - Application of the sulphation Echinacea polysaccharide as Immune Profile In Chicks reinforcing agent - Google Patents

Application of the sulphation Echinacea polysaccharide as Immune Profile In Chicks reinforcing agent Download PDF

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Publication number
CN109316489A
CN109316489A CN201811209345.2A CN201811209345A CN109316489A CN 109316489 A CN109316489 A CN 109316489A CN 201811209345 A CN201811209345 A CN 201811209345A CN 109316489 A CN109316489 A CN 109316489A
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sulphation
group
echinacea
polysaccharide
application
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刘翠
姚丽丽
谭盈
白霖
郭世宁
石达友
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South China Agricultural University
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South China Agricultural University
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K20/00Accessory food factors for animal feeding-stuffs
    • A23K20/10Organic substances
    • A23K20/163Sugars; Polysaccharides
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23KFODDER
    • A23K50/00Feeding-stuffs specially adapted for particular animals
    • A23K50/70Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75Feeding-stuffs specially adapted for particular animals for birds for poultry
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • A61P37/04Immunostimulants

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Husbandry (AREA)
  • Food Science & Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Birds (AREA)
  • Molecular Biology (AREA)
  • Dermatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Application the invention discloses sulphation Echinacea polysaccharide as Immune Profile In Chicks reinforcing agent;Preparing the application in chicken immunity enhancing drug or feed;As well as the application of cyclophosphamide antagonist.Sulphation Echinacea of the present invention is for the chick because of hypoimmunity after using cyclophosphamide to carry out modeling for immunosuppressor, chick peripheral blood lymphocytes and cytokines can be obviously increased, the immunity for promoting chick, shows that sulphation Echinacea polysaccharide can be used as Immune Profile In Chicks reinforcing agent and be applied.

Description

Application of the sulphation Echinacea polysaccharide as Immune Profile In Chicks reinforcing agent
Technical field
The present invention relates to veterinary vaccination preparation technique fields, more particularly, to sulphation Echinacea polysaccharide as chick The application of immunopotentiator.
Background technique
In recent years, the development of China's aviculture is going from strength to strength, but it is easy to appear during a large amount of cultivation big The disease of batch, for example, chicken is in the breeding process, the heat stress that summer high temperature easily causes causes its immune function to decline, draws Send out disease a series of;Especially chick, due to its own immunity natively deviation, during cultivation, the change of feed, The temperature and humidity of henhouse and the variation of external environment condition are easy to cause hypoimmunity, influence its speed of growth, and be easy hair Raw some inhibitive ability of immunity diseases, can make the immunity of chick worse, and disease incidence rises, and disease treatment is difficult, and make feedstuff-meat ratio, Feedstuff-egg ratio improves, and causes cultivation difficult, and aquaculture cost increases, and seriously affects the development of aviculture.
Currently, it is easily caused when mainly being puted prevention first inhibitive ability of immunity disease, but be immunized with immune both at home and abroad biggish Stress reaction can also excite the generation of disease if the immunity of body is lower sometimes.Herbal polysaccharide is because having improvement body to exempt from The extensive bioactivity such as epidemic disease function, antiviral, anti-oxidant, inhibition tumour growth, anti-aging, and have green natural, poison secondary Act on it is small, be not likely to produce the features such as drug resistance, by the extensive research of domestic and foreign scholars, become the hot spot of veterinary applications research.
A large number of studies show that many Chinese medicines and its effective component have certain immunological enhancement, but their work With weaker, especially Chinese medicine, due to complicated component, quality is difficult to control, and foreign countries are difficult to receive.Therefore, it is necessary to improve Chinese medicine The immunization of polysaccharide makes it suitable for promoting and applying on veterinary clinic.
Summary of the invention
It is an object of the invention to provide sulphation Echinacea polysaccharide as young with defect aiming at the shortcomings in the prior art The application of chicken immune reinforcing agent.The present invention by Echinacea polysaccharide carry out sulphation, sulphation Echinacea polysaccharide is prepared, And it is tested for the immunity improvement result of chick, the results showed that sulphation Echinacea polysaccharide can obviously increase chick peripheral blood Lymphocyte and cytokines promote the immunity of chick, and maintain each internal organs of chick in the general level of the health;It is indicated above Sulphation Echinacea polysaccharide is a kind of Immune Profile In Chicks reinforcing agent safely, effectively, quality controllable, is had in terms of veterinary clinic latent Application value.
Above-mentioned purpose of the invention is achieved by following scheme:
Application of the sulphation Echinacea polysaccharide as Immune Profile In Chicks reinforcing agent.It is made by immunosuppressor of cyclophosphamide Mould, for the chick of hypoimmunity after modeling, sulphation Echinacea polysaccharide can obviously increase chick peripheral blood lymphocytes and Cytokines promote the immunity of chick, show that sulphation Echinacea polysaccharide can be used as the progress of Immune Profile In Chicks reinforcing agent Using.
The present invention simultaneously also protection sulphation Echinacea polysaccharide prepare chicken immunity enhancing drug or feed in answer With.
Preferably, the sulphation Echinacea polysaccharide increases in chick peripheral blood lymphocytes drug or feed in preparation Using.
Preferably, the sulphation Echinacea polysaccharide increases in chick cytokines drug or feed in preparation Using.
Preferably, the cytokines are one of IL-2, IL-6 or IFN-γ or a variety of.
Sulphation Echinacea polysaccharide is also within the scope of the present invention as the application of cyclophosphamide antagonist.
Sulphation Echinacea polysaccharide of the present invention is prepared by following procedure:
S1. it is always more that Echinacea the extraction and purifying of Echinacea polysaccharide: is extracted from Echinacea complete stool using decoction alcohol precipitation method Sugar, then using the protein in sevage method Polysaccharide removing, the purifying of DEAE-52 cellulose column, freeze-drying obtains purifying purple cone Chrysanthemum polysaccharide;
S2. the vulcanization of Echinacea polysaccharide: sulphation modification is carried out to Echinacea polysaccharide using chlorosulfonic acid-pyridine method, by band There is the three-neck flask of stirrer and condensing unit to set in ice-water bath;The anhydrous pyridine 100mL of pre-cooling is first added, stirring is allowed to abundant It is cooling;16.7mL chlorosulfonic acid is added dropwise again, sees in flask a large amount of faint yellow solids occur, stops reaction, esterifying reagent system It is standby to complete;It weighs the purified Echinacea polysaccharide of 3g to set in conical flask, appropriate n,N-Dimethylformamide is added;By above-mentioned esterification Reagent is poured into conical flask, covers tightly bottle stopper;Oscillating reactions 3h conical flask being placed in 80 DEG C of water-baths;After reaction, It takes out conical flask to be cooled to room temperature, is neutralized to pH=7 with the NaOH solution of 5molL-1;3 are slowly added to when shaking conical flask The dehydrated alcohol of~4 times of volumes makes ethyl alcohol final concentration 80%, stands for 24 hours;Suction filtration takes precipitating, after adding suitable quantity of water dissolution precipitating, It is packed into bag filter, first uses tap water flowing water dialysis 48h, then for 24 hours with distilled water dialysis, merge dialyzate and be concentrated under reduced pressure, freeze and do It is dry, sulphation Echinacea polysaccharide is made.
With improvement phend-sulphuric acid and the acid barium chromate method measurement of ultrasound, polysaccharide matter in the Echinacea polysaccharide being prepared Measuring accounting is 30.00%, sulfate radical quality accounting 35.00%.
Compared with prior art, the invention has the following advantages:
Sulphation Echinacea of the present invention is for because of immunity after using cyclophosphamide to carry out modeling for immunosuppressor Low chick can obviously increase chick peripheral blood lymphocytes and cytokines, promote the immunity of chick, show Sulphation Echinacea polysaccharide can be used as Immune Profile In Chicks reinforcing agent and be applied.
Detailed description of the invention
Fig. 1 is influence of the EPP and SEPP to immunosupress chicken peripheral blood Serum IL-2.
Fig. 2 is influence of the EPP and SEPP to IL-6 content in immunosupress chicken peripheral blood serum.
Fig. 3 is influence of the EPP and SEPP to IFN-γ content in immunosupress chicken peripheral blood serum.
Specific embodiment
The present invention is made combined with specific embodiments below and further being elaborated, the embodiment is served only for explaining this Invention, is not intended to limit the scope of the present invention.Test method as used in the following examples is normal unless otherwise specified Rule method;Used material, reagent etc., unless otherwise specified, for the reagent and material commercially obtained.Embodiment 1
The preparation of sulphation Echinacea polysaccharide, detailed process is as follows:
(1) it is always more that Echinacea the extraction and purifying of Echinacea polysaccharide: is extracted from Echinacea complete stool using decoction alcohol precipitation method Sugar, 673g Echinacea complete stool, 80% ethyl alcohol soaked overnight, 70 DEG C of water-baths 2 times (each 1h) of reflux wrap up medicinal material with gauze, It is decocted repeatedly with water 2 times, intense fire is heated to boiling, and mild fire maintains its boiling, merges and decocts gained medical fluid twice, and concentration makes to give birth to Concentration reaches 1g/mL, 4500r/min, is centrifuged 5min, and removal of impurities takes supernatant alcohol precipitation 3 times repeatedly, be freeze-dried Echinacea is more Sugar-like product;Again using the protein in sevage method Polysaccharide removing, the purifying of DEAE-52 cellulose column, freeze-drying is purified Echinacea polysaccharide (EPP);
(2) vulcanization of Echinacea polysaccharide: sulphation modification is carried out to Echinacea polysaccharide using chlorosulfonic acid-pyridine method, and excellent Change modification process condition, sulphation Echinacea polysaccharide (SEPP) is made through dialysis purification, freeze-drying in the polysaccharide after modification.
With improvement phend-sulphuric acid and the acid barium chromate method measurement of ultrasound, in the sulphation Echinacea polysaccharide being prepared Polysaccharide quality accounting is 30.00%, sulfate radical quality accounting 35.00%.
2 application examples of embodiment
1, prepared by immunosuppression model
Modeling is carried out with cyclophosphamide immunosuppressor, cyclophosphamide immunosupress is mainly metabolized by cyclophosphamide and is produced Object phosphamide mustargen and methacrylaldehyde play a role.Its metabolite can generate covalent bond with DNA, DNA double chain is caused to be broken, Alkylation, protein and DNA interconnection, to interfere cell growth and breeding.The immune function of Immune Profile In Chicks system after modeling It can be reduced with activity, the inferior capabilities resisted the disease are easy the state by cause pathogeny imcrobe infection.
Specific modeling process is as follows: 320 plumage, 1 age in days Lingnan Yellow cock, after adaptive feeding 14d, is randomly divided into 8 Group, every group 40, respectively saline control group (NC), cyclophosphamide group (CY), SEPPH group, SEPPM group, SEPPL group, EPPH group, EPPM group, EPPL group, in addition to NC group, remaining 7 groups are respectively adopted intramuscular injection CY and carry out modeling, continuously inject 3d, Daily injection volume is 80mg/Kg, and the continuous 3d of NC group injects same amount of normal saline.
Wherein SEPPH group, SEPPM group and SEPPL group respectively represent administration sulphation Echinacea polysaccharide high dose group, in Dosage group and low dose group, dosage are respectively each stomach-filling 10mg/kg, 5mg/kg and 2.5mg/kg;EPPH group, EPPM group With EPPL group, high dose group, middle dose group and the low dosage of administration Echinacea polysaccharide (to carry out sulphation processing) are respectively represented Group, dosage are respectively each stomach-filling 10mg/kg, 5mg/kg and 2.5mg/kg.Echinacea polysaccharide used by the present embodiment It is that embodiment 1 is prepared with sulphation Echinacea polysaccharide.
Administration process: the continuous 3d of NC group injects same amount of normal saline, remaining 7 groups are respectively adopted continuous 3d intramuscular injection CY Modeling is carried out, starts to be administered for 24 hours after 3d, dosage is shown in Table 1.
The test of table 1 grouping and processing
The growing state of chicken group is observed after administration daily, the 7th, 14,21 and 28d record every group of meat after first administration respectively Chicken body weights, each group randomly select 5 chickens, weigh in, take liver,spleen,kidney, weigh and record weight, count and calculate Organ index.Statistical result is shown in Table 2.
Influence of the table 2SEPP and EPP to CY immunosupress chicken weight and organ index
Note: same column shoulder mark same letter indicates that difference is not significant (P > 0.05) without letter person, the different letter persons of shoulder mark It indicates significant difference (P < 0.05).
As shown in Table 2, chick weight is lower than NC group, and each SEPP, EPP administration group chick weight after CY modeling during test Be above CY group, wherein in first administration 7d, 21d, each SEPP, EPP administration group chick weight be all remarkably higher than CY group (P < 0.05), and not significant (P > 0.05) with NC group difference;Immune organs of chicken treated organ index result after CY modeling during comparative test It was found that liver index, spleen index, renal index are below NC group after administration 7d, wherein spleen index and renal index be substantially less than NC group (P < 0.05), each SEPP administration group after 7d is administered chick liver index, spleen index, renal index with NC group without significant difference (P > 0.05)。
2, sulphation Echinacea Polysaccharides in Chicken peripheral lymphocyte proliferation measures
The 7th, 14,21 and 28d after first administration, each group randomly select 5 sterile Culling heart bloods of chicken, with being prepared in advance Hank ' s liquid doubling dilution acquisition blood sample, the blood sample after doubling dilution is carefully slowly added in separation of lymphocytes with 1:1 Liquid upper layer, 2000r/min are centrifuged 10min, draw middle layer milky cloud cell, wash 2 times with Hank ' s, 1500r/ Min is centrifuged 10min, is added and contains 2% dual anti-and 10% fetal calf serum RPMI1640 culture solution.Lymph obtained above is thin The concentration of born of the same parents is adjusted to 2 × 106A/mL, is added 96 hole cell versions, and every 100 μ L of hole is placed in 37 DEG C, 5%CO2It is cultivated in incubator 30 μ L (5mg/mL) of MTT is added in 44h, every hole, continues to cultivate 4h, A is measured in microplate reader570Value.Testing result is shown in Table 3 institutes Show.
The influence that table 3 SEPP and EPP converts immunosupress chicken peripheral blood lymphocytes
Note: with table 2.
As shown in Table 3, CY group chick peripheral blood lymphocytes transformation is lower than NC group after modeling during test, and respectively gives Medicine treatment group can improve chick peripheral blood lymphocytes transformation after modeling;In first administration 7d, SEPPM and EPPM The peripheral blood lymphocytes A of group570Value is significantly higher than CY group (P < 0.05);When 14d, SEPPH group lymphocyte A570It is worth highest, The lymphocyte A of middle SEPPH, SEPPM group and EPP each group570Value is all remarkably higher than CY group (P < 0.05);When 21d, the leaching of SEPPH group Bar cell A570It is worth highest, wherein the lymphocyte A of SEPPH, SEPPM and EPP each group570Value is noticeably greater than CY group;When 28d, SEPPL group lymphocyte A570Value highest and it is significantly higher than CY group (P < 0.05).
3, sulphation Echinacea Polysaccharides in Chicken cytokines measure
The 7th, 14,21 and 28d after first administration randomly select 5 chicken Culling heart blood 3mL for every group respectively, analyse at room temperature Serum out is dispensed with 2mL centrifuge tube, is respectively used to ELISA method measurement Cytokine of Serum IL-2, IL-6 and IFN-γ.
Test result is as shown in Figures 1 to 3, and Fig. 1 to Fig. 3 is respectively IL-2, IL-6 and IFN-γ in chicken peripheral blood serum Content results.Letter meaning of the Fig. 1 into Fig. 3 is the same as consistent in table 2.
As can be known from Fig. 1, by comparing each group chicken peripheral blood Serum IL-2, chicken periphery after CY modeling during test Blood Serum IL-2 reduces, and chicken peripheral blood Serum IL-2 increases after drug treatment.When first administration 7d, NC group, SEPPH group, SEPPM group and EPPH group IL-2 content are above CY group;When 14d, SEPP each group and EPP each group IL-2 content are significant Higher than CY group (P < 0.05);When 21d, SEPPL group IL-2 content highest and it is significantly higher than NC group and CY group (P < 0.05);When 28d, SEPPL group IL-2 content highest.
As can be known from Fig. 2, compare IL-6 content in each group chicken peripheral blood serum, chicken peripheral blood blood after CY modeling during test IL-6 content reduces in clear, and IL-6 content increases in chicken peripheral blood serum after drug treatment.When first administration 7,14,28d, respectively SEPP group and EPP group IL-6 content increase, but not significant with CY group difference;When 21d, EPP each group, SEPPM group and SEPPL group are aobvious It writes and is higher than CY group and NC group (P < 0.05).
IFN-γ content in each group chicken peripheral blood serum is compared by Fig. 3 it is found that chicken peripheral blood blood after CY modeling during test IFN-γ content reduces in clear, and IFN-γ content increases in chicken peripheral blood serum after drug treatment.
In conclusion EPP and SEPP can significantly improve CY induction immunosupress chicken weight, liver index, spleen index and kidney Index, and peripheral lymphocyte proliferation is improved, in the infrared spectroscopy of SEPP, in 808.98cm-1There is strong flexible vibration in place Dynamic peak is C-O-SO3Related symmetry C-O-S absorption peak shows that the success SEPP effect of EPP sulphation modification is substantially better than EPP。
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention The limitation of shield range can also be made on the basis of above description and thinking for those of ordinary skill in the art Other various forms of variations or variation, there is no necessity and possibility to exhaust all the enbodiments.It is all of the invention Made any modifications, equivalent replacements, and improvements etc., should be included in the protection of the claims in the present invention within spirit and principle Within the scope of.

Claims (7)

1. application of the sulphation Echinacea polysaccharide as Immune Profile In Chicks reinforcing agent.
2. sulphation Echinacea polysaccharide is preparing the application in chicken immunity enhancing drug or feed.
3. applying according to claim 2, which is characterized in that the sulphation Echinacea polysaccharide increases chick periphery in preparation Application in blood lymphocyte drug or feed.
4. applying according to claim 2, which is characterized in that the sulphation Echinacea polysaccharide increases chick periphery in preparation Application in blood cytokine drug or feed.
5. applying according to claim 4, which is characterized in that the cytokines are IL-2, IL-6 or IFN-γ One of or it is a variety of.
6. application of the sulphation Echinacea polysaccharide as cyclophosphamide antagonist.
7. polysaccharide quality accounting is in the sulphation Echinacea polysaccharide according to claim 1 to 6 any applications 53.00%, sulfate radical quality accounting 34.48%.
CN201811209345.2A 2018-10-17 2018-10-17 Application of the sulphation Echinacea polysaccharide as Immune Profile In Chicks reinforcing agent Pending CN109316489A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113413455A (en) * 2021-06-28 2021-09-21 宁夏农林科学院动物科学研究所(宁夏草畜工程技术研究中心) Immunity enhancing composition and application thereof

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CN108641005A (en) * 2018-04-03 2018-10-12 华南农业大学 A kind of preparation method and applications of sulphation Echinacea polysaccharide

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CN108641005A (en) * 2018-04-03 2018-10-12 华南农业大学 A kind of preparation method and applications of sulphation Echinacea polysaccharide

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Publication number Priority date Publication date Assignee Title
CN113413455A (en) * 2021-06-28 2021-09-21 宁夏农林科学院动物科学研究所(宁夏草畜工程技术研究中心) Immunity enhancing composition and application thereof
CN113413455B (en) * 2021-06-28 2023-02-14 宁夏农林科学院动物科学研究所(宁夏草畜工程技术研究中心) Immunity enhancing composition and application thereof

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