CN101869706A - A compound astragalus polysaccharide and echinacea extract nanoemulsion adjuvant and its preparation method - Google Patents
A compound astragalus polysaccharide and echinacea extract nanoemulsion adjuvant and its preparation method Download PDFInfo
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Abstract
本发明公开了一种复方黄芪多糖和紫锥菊提取物纳米乳佐剂，该佐剂的粒径在1～100nm之间，由紫锥菊提取物、黄芪多糖、表面活性剂、助表面活性剂、油和蒸馏水组成。该产品外观呈透明淡黄色液体，粒径小于100nm；粘度较低，稳定性较好，配苗时无须特殊乳化，对设备要求低，产品质量容易控制。以卵清白蛋白OVA为模式抗原，证明本发明的复方黄芪多糖和紫锥菊提取物纳米乳佐剂在一定剂量上比使用铝佐剂能诱导较好的免疫反应，产生较高的抗体滴度，减少佐剂中油或者抗原的使用量，刺激Th1和Th2反应，增强机体的体液和细胞免疫作用，对机体具有良好的免疫保护作用。The invention discloses a compound astragalus polysaccharide and echinacea extract nanoemulsion adjuvant. The particle size of the adjuvant is between 1 and 100 nm, and the adjuvant is composed of echinacea extract, astragalus polysaccharide, surfactant, co-surfactant, oil and Distilled water composition. The appearance of this product is a transparent light yellow liquid, the particle size is less than 100nm; the viscosity is low, the stability is good, no special emulsification is required when seedlings are mixed, the requirements for equipment are low, and the product quality is easy to control. Taking ovalbumin OVA as a model antigen, it is proved that the compound astragalus polysaccharide of the present invention and the nanoemulsion adjuvant of Echinacea purpurea extract can induce a better immune response than aluminum adjuvant in a certain dose, produce higher antibody titers, reduce The amount of oil or antigen used in the adjuvant stimulates Th1 and Th2 responses, enhances the humoral and cellular immunity of the body, and has a good immune protection effect on the body.
Description
Technical field
The present invention relates to the vaccine adjuvant field, be specifically related to a kind of compound Radix Astragali polysaccharide and Echinacea extract Water-In-Oil nano emulsion adjuvant that can form compositions, have the immune effect of enhancement antigen, be used for the prevention and the treatment of Animal diseases with antigen.
Background technology
In recent years, along with the generation of increasing Animal diseases or infecting both domestic animals and human disease, also more and more higher to the demand and the requirement of production of vaccine.But actual often the discovery in the application of vaccine has many-sided shortcoming, variety of problems occurred: although 1. present commercialization inactivated vaccine and weak malicious Seedling all can alleviate clinical symptoms, can not stop the subinfection again of virus and the toxin expelling of sick pig; 2. the isolating viral pathogenicity in various places often also is different, even the repeatability of same virus neither be fine, and effective property, and often result of use is not good to cause the inactivated vaccine produced and attenuated vaccine; Though 3. inactivated vaccine safety, the protection that produces is not strong, often needs the immunity inoculation of repeated multiple times; 4. compare with inactivated vaccine, the inductive immunoreation persistent period of attenuated vaccine is longer, also more effective, the malicious problem but itself exist to loose, and have and return strong potential danger.
Therefore, the development of new vaccine adjuvant effectively improves the vaccine result of use, and the diffusing malicious problem when reducing the vaccine use will be the research emphasis of epidemic prevention.This project is intended by advanced nanotechnology, to prove that the herbal polysaccharide with good immunoenhancement result is applied in the blue-ear disease vaccine adjuvant, give full play to medicine sustained release that nanotechnology has, improve effect and the good immunoenhancement result of polysaccharide such as curative effect of medication, develop a kind of efficient, safe nano vaccine adjuvant, to solve the frequent problem that takes place of Animal diseases in the actual production.
For strengthening the immunogenicity of vaccine, add immunological adjuvant in the development of the viral vaccine of being everlasting, dna vaccination and polypeptide vaccine.Immunological adjuvant is a kind of immunomodulator, can mix prior to antigen or with antigen or inject simultaneously in the animal body, and the immunogenicity of energy non-specific ground enhancement antigen improves immune effect.Genus echinacea (Echinacea Moenck) plant is the wild flowers of a class Compositae that originate in America, also claims " SONGGUOJU ".Its Echinacea medicinal history can be traced back to North America Indians period.American Indian chews the back scum with the root of Echinacea and gulps down juice, is used for the treatment of cough and pharyngolaryngitis.Echinacea also can be treated tonsillitis, oral ulcer, acute abdominal pain, flu, septicemia, rabies, worm huge legendary turtle, snake venom, gonorrhea, burn, scald etc., and Echinacea always is regarded as SHENCAO in American Indian mind.The genus echinacea galenical can be used as immunological enhancement and regulator, and there have many dosage forms to be used in a large number to be clinical.And the Chinese medicine astragalus polysaccharide is indicating that as the effective antiviral substance of a class it will have important use in the vaccine adjuvant field, is paid attention to by more and more researchers.
Nano-emulsion is meant that the diameter of emulsion droplet is in a kind of special Emulsion of nanoscale.Generally speaking, thermodynamically stable, the isotropism that two kinds of non soluble liquids is formed under Action of Surfactant, appearance transparent or dispersion translucent, particle diameter 1-100nm are called nanoemulsions.Nanoemulsions is as the new drug carrier that has potentiality, and have the incomparable advantage of other drug carrier: physical stability is good; Can improve the dissolubility of insoluble drug; Can promote macromole water soluble drug absorption in vivo, improve bioavailability of medicament, or the like.The oil adjuvant is to use animal at present to use wider adjuvant in the inactivated vaccine, but this class adjuvant is generally water in oil emulsion, unstable, than thickness, need exert oneself during injection, and mineral oil is difficult for disperseing and metabolism in body, is easy to generate local side reactions such as erythema, pustule, influences the quality of animal shape and meat.And nano-emulsion Water-In-Oil adjuvant of the present invention replaces nonmetabolizable mineral oil with vegetable oil or animal oil, and sifting property surface of good activating agent adopts more advanced emulsifying process, and it is low to prepare viscosity, the novel adjuvant Seedling that poison is secondary little, easy to use.
Summary of the invention
At above-mentioned problems of the prior art and defective, the object of the present invention is to provide a kind of compound Radix Astragali polysaccharide and echinacea purpurea herb nanoemulsion adjuvant, this nano emulsion adjuvant production technology is simple, viscosity is suitable, good stability, the efficient and little Water-In-Oil adjuvant of side effect.
Realize that foregoing invention purpose technical scheme is, a kind of compound Radix Astragali polysaccharide and echinacea purpurea herb nanoemulsion adjuvant, the particle diameter of this nano emulsion adjuvant is between 1～100nm, be made up of following weight percentages: Echinacea extract 0.10%～2.00%, astragalus polysaccharides 0.10%～10.00%, surfactant 15.00%～60.00%, cosurfactant 1.00%～15.00%, oil 1.50%～25.00%, surplus are distilled water, and the gross weight of above-mentioned raw materials is 100%.
The optimum ratio of compound Radix Astragali polysaccharide of the present invention and echinacea purpurea herb nanoemulsion adjuvant is: Echinacea extract 0.50%～1.50%, astragalus polysaccharides 1.00%～5.00%, surfactant 25.00%～45.00%, cosurfactant 5.00%～10.00%, oil 5.00%～20.00%, surplus is a distilled water, and the gross weight of above-mentioned raw materials is 100%.
Surfactant of the present invention is the non-ionic surface active agent of HLB＞8, preferentially selects one or more the mixing in poly-sorbester p17, sorbester p18, tween 80 and the Tween-60 for use.
Cosurfactant of the present invention is one or more in ethanol, Macrogol 200, glycerol and the propylene glycol.
Oil of the present invention is one or more in Oleum Arachidis hypogaeae semen, olive oil, isopropyl myristate, vitamin E oil, liquid paraffin and the wheat germ oil.
Another object of the present invention provides the preparation method of above-mentioned compound Radix Astragali polysaccharide and Echinacea extract water-in-oil type nanoemulsion adjuvant, may further comprise the steps:
1) take by weighing astragalus polysaccharides, Echinacea extract, surfactant, cosurfactant, oil and distilled water, standby;
2) formation that at room temperature surfactant, cosurfactant and oil stirred, gained is oil phase;
3) under the room temperature, astragalus polysaccharides and Echinacea extract are dissolved in the distilled water, stirring is dissolved it fully, and gained is water;
4) under the room temperature, oil phase slowly is added drop-wise to aqueous phase, constantly stirs in the time of dropping, until forming transparent system, promptly.
Compared with prior art, compound Radix Astragali polysaccharide of the present invention and the following advantage of Echinacea extract water-in-oil type nanoemulsion adjuvant tool:
1) preparation method of the present invention simply, does not need special installation; Made compound nanometer emulsion adjuvant viscosity is low, convenient injection, deposits the maintenance clear state in 4～8 ℃ or room temperature, phenomenons such as medicine is separated out, layering can not occur.
2) raw material of the present invention easily obtains, and has better biocompatibility; Compound nanometer emulsion adjuvant has strengthened immunogenicity of antigens when identical antigen use amount, improved the immune effect of vaccine, has reduced aquaculture cost to a certain extent; Form by improved formulations, reduced the injection reaction of animal vaccine.
Need not special emulsifying when 3) compound nanometer emulsion adjuvant of the present invention is joined Seedling, only need simple the mixing, low for equipment requirements, simple to operate, product quality is controlled easily.
Description of drawings
Fig. 1 is the transmission electron microscope photo of nano emulsion adjuvant.
The specific embodiment
The physicochemical property of test example 1 nano emulsion adjuvant
1.1 outward appearance and pH value nano emulsion adjuvant of the present invention is a light yellow transparent liquid, good fluidity, pH are 6.71.
1.2 viscosity conveying end internal diameter is the 1.2mm glass pipette, draws the 1mL sample, room temperature condition record down flows out the 0.4mL needed time of sample (s), is its viscosity.Nano emulsion adjuvant viscosity of the present invention is 4.60s.
1.3 the morphologic observation nano emulsion adjuvant drops on the copper mesh that is covered with supporting film after suitably diluting with distilled water, leaves standstill 10min, drips Salkowski's solution negative staining 5min on copper mesh of 3% again, volatilizes naturally, nano-emulsion is spheroidal under the transmitted electron mirror, sees accompanying drawing 1.
1.4 particle diameter and distribution Zetasizer Nano ZS nano particle size instrument thereof record the particle size distribution of nano emulsion adjuvant.Its mean diameter is 45.2nm, and particle diameter accounts for 97.7% at the particle of 33.8～78.8nm, only accounts for 2.3% greater than 78.8nm or less than the particle of 33.8nm.The particle size distribution range of nano emulsion adjuvant of the present invention is narrow, and size ratio is more even.
1.5 study on the stability
Nano emulsion adjuvant still keeps the outward appearance of transparent homogeneous in the centrifugal 10min of 10000rpm speed, layering and medicine do not occur and phenomenon such as separates out.
Nano emulsion adjuvant is sub-packed in the cillin bottle, after packing, place, in the 15th day and sampling in the 30th day respectively under 4,25,37,40,60 ℃ and high light (4500 ± 500) the 1x condition, measure pH value, emulsion droplet particle diameter and medicament contg (astragalus polysaccharides) and wait each index, see Table 1.Nano emulsion adjuvant is placed 30d under 4,25,37,40 ℃ and illumination condition, liquid keeps the clear state, does not see layering, flocculation and breakdown of emulsion, and pH value, emulsion droplet particle diameter and medicament contg are not seen significant change.But under 60 ℃ of conditions, it is muddy that nano emulsion adjuvant becomes, and puts and recover the clear attitude under the room temperature again.Show that nano emulsion adjuvant of the present invention is comparatively stable room temperature or 4 ℃.
The stability of table 1 nano emulsion adjuvant
The immunological adjuvant effect test of test example 2 nano emulsion adjuvants
The ICR female mice is divided into 12 groups at random, 10 every group.The concrete normal saline group that is respectively, OVA antigen (100 μ g/ only) group, all the other each groups add aluminium glue (Alum 50 μ g/ are only) respectively on OVA antigen (100 μ g/ are only) basis, or by the astragalus polysaccharides (APS) of various dose and the nano emulsion adjuvant of Echinacea extract (EM) preparation.Little mouse's head is exempted from back 21d and is carried out secondary immunity, cervical region subcutaneous injection 0.4ml medicine, and two exempt from back 2 week blood sampling, and separation of serum, ELISA detect serum OVA inducing specific antibody horizontal, and experimental data is used
Expression, all data SAS software processes is checked the diversity between each group.And observation immunity front and back mice behavior and body weight change.
Respectively organize for the first time mice after the immunity and occur on a small quantity being afraid of cold outside stress and body weight descend to some extent, but very fast disappearance, the body weight trend that restore normal growth gradually; Two respectively organize the mice body weight after exempting from, and significant change does not take place, and mice is all no abnormal variation at aspects such as appetite and ability to acts.Each is organized the influence that OVA exempted from the mice serum specific antibody level and sees Table 2.
Table 2 each the group exempted from mice serum OVA inducing specific antibody horizontal influence (n=10,
)
Annotate:
aExpression is compared P＜0.05 with the OVA antigen group;
bExpression is compared P＜0.05 with the Alum+OVA antigen group.
As shown in Table 2, behind the mice secondary immunity, each test group serum OVA specific IgG of Alum+OVA antigen group and NE (APS+EM), IgG1 and IgG2a antibody horizontal all are significantly higher than OVA antigen control group (P＜0.05); NE (APS 100 μ g+EM 50 μ g)+OVA antigen group, NE (APS100 μ g+EM 100 μ g)+OVA antigen group, NE (APS 100 μ g+EM 200 μ g)+OVA antigen group, NE (APS 200 μ g+EM 50 μ g)+OVA antigen group, NE (APS 200 μ g+EM 100 μ g)+OVA antigen group, the IgG of NE (APS 400 μ g+EM 50 μ g)+OVA antigen group and NE (APS 400 μ g+EM 100 μ g)+each test group of OVA antigen group, IgG1 and IgG2a antibody horizontal and Alum+OVA antigen group be there was no significant difference on statistics, but the IgG of NE (APS 200 μ g+EM 200 μ g)+OVA antigen group and NE (APS 400 μ g+EM 200 μ g)+OVA antigen group, IgG1 and IgG2a antibody horizontal are significantly higher than Alum+OVA antigen group (P＜0.05); And IgG, IgG1 and IgG2a antibody horizontal there was no significant difference between each test group of NE (APS+EM), wherein (APS400 μ g+EM 200 μ g)+three kinds of antibody horizontals of OVA antigen group are the highest with NE again.
It is reported that astragalus polysaccharides and Echinacea extract all have effect (Matsuzaki etc., 2007 of regulating immunity of organism, enhancing body resistance; Predy etc., 2005; Canike etc., 2007; Biggs etc., 2007), its concrete manifestation can promote Th1 type and Th2 type antibody response.Th1 type immunne response mainly produces antibody such as IgG2a, and the Th2 type mainly produces antibody such as IgG1.This test finds that nano emulsion adjuvant produces specific antibody level when APS 200 μ g+EM, 200 μ g and APS 400 μ g+EM 200 μ g, obviously is better than aluminium glue.Therefore astragalus polysaccharides and echinacea purpurea herb nanoemulsion adjuvant can strengthen the immunocompetence of vaccine, promote Th1 and Th2 immunne response, reduce antigenic using dosage, thereby reach the immunogenicity of enhancement antigen, improve the vaccine result of use, and reduce the immunoreation in the vaccine use.
Further introduce product of the present invention and preparation method below by embodiment.
Embodiment 1
1) accurately take by weighing or measure following component, standby:
2) at room temperature Tween 80, glycerol, liquid paraffin and propylene glycol are stirred, form solution, gained is oil phase;
3) under the room temperature, astragalus polysaccharides and Echinacea extract are dissolved in the distilled water, stirring is dissolved it fully, and gained is water;
4) under the room temperature, oil phase slowly is added drop-wise to aqueous phase, constantly stirs in the time of dropping, until forming transparent system, promptly.
Embodiment 2
Embodiment 3
Embodiment 4
Embodiment 5
Embodiment 6
Embodiment 7
Embodiment 8
Embodiment 9
Embodiment 10
Claims (3)
1. compound Radix Astragali polysaccharide and echinacea purpurea herb nanoemulsion adjuvant is characterized in that the particle diameter of this nano emulsion adjuvant is made by following weight percentages between 1～100nm:
Echinacea extract 0.10%～2.00%
Astragalus polysaccharides 0.10%～10.00%
Surfactant 15.00%～60.00%
Cosurfactant 1.00%～15.00%
Oil 1.50%～25.00%
Surplus is a distilled water
The gross weight of above-mentioned raw materials is 100%;
Described surfactant is one or more in sorbester p17, sorbester p18, Tween 80, the polysorbate60;
Described cosurfactant is one or more in ethanol, Polyethylene Glycol, glycerol and the propylene glycol;
Described oil is one or more in Oleum Arachidis hypogaeae semen, olive oil, isopropyl myristate, vitamin E oil, liquid paraffin and the wheat germ oil.
2. compound Radix Astragali polysaccharide according to claim 1 and echinacea purpurea herb nanoemulsion adjuvant is characterized in that, are made by following weight percentages:
Echinacea extract 0.50%～1.50%, astragalus polysaccharides 1.00%～5.00%, surfactant 25.00%～45.00%, cosurfactant 5.00%～10.00%, oil 5.00%～20.00%, surplus is a distilled water, and the gross weight of above-mentioned raw materials is 100%.
3. the preparation method of described compound Radix Astragali polysaccharide of claim 1 and echinacea purpurea herb nanoemulsion adjuvant is characterized in that, comprises the following steps:
1) take by weighing astragalus polysaccharides, Echinacea extract, surfactant, cosurfactant, oil and distilled water, standby;
2) formation that at room temperature surfactant, cosurfactant and oil stirred, gained is oil phase;
3) under the room temperature, astragalus polysaccharides and Echinacea extract are dissolved in the distilled water, stirring is dissolved it fully, and gained is water;
4) under the room temperature, oil phase slowly is added drop-wise to aqueous phase, constantly stirs in the time of dropping,, promptly get compound Radix Astragali polysaccharide and echinacea purpurea herb nanoemulsion adjuvant until forming transparent system.
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| CN103083658A (en) * | 2011-10-27 | 2013-05-08 | 普莱柯生物工程股份有限公司 | Vaccine adjuvant composition for treatment or prevention of swine infectious diseases |
| CN103142702A (en) * | 2013-03-21 | 2013-06-12 | 河北科技师范学院 | Chinese herbal medicine injection liquid for treating porcine reproductive and respiratory syndrome and preparation method thereof |
| CN103610738A (en) * | 2013-12-10 | 2014-03-05 | 济南三峰生物工程有限责任公司 | Traditional Chinese medicine particle for preventing and treating avian influenza and improving chicken flock immunity and preparation method thereof |
| CN106924730A (en) * | 2017-02-14 | 2017-07-07 | 商丘美兰生物工程有限公司 | PRV Aloe Vera Gel nano emulsion adjuvant inactivated vaccine and preparation method thereof |
| CN108578688A (en) * | 2018-04-03 | 2018-09-28 | 武汉轻工大学 | The preparation method of multiple nano-emulsion vaccine adjuvant |
| CN113209287A (en) * | 2021-02-04 | 2021-08-06 | 中国农业科学院农业环境与可持续发展研究所 | Water-in-oil type nano vaccine adjuvant, preparation method and application thereof |
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| CN103083658A (en) * | 2011-10-27 | 2013-05-08 | 普莱柯生物工程股份有限公司 | Vaccine adjuvant composition for treatment or prevention of swine infectious diseases |
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| CN103142702A (en) * | 2013-03-21 | 2013-06-12 | 河北科技师范学院 | Chinese herbal medicine injection liquid for treating porcine reproductive and respiratory syndrome and preparation method thereof |
| CN103610738A (en) * | 2013-12-10 | 2014-03-05 | 济南三峰生物工程有限责任公司 | Traditional Chinese medicine particle for preventing and treating avian influenza and improving chicken flock immunity and preparation method thereof |
| CN106924730A (en) * | 2017-02-14 | 2017-07-07 | 商丘美兰生物工程有限公司 | PRV Aloe Vera Gel nano emulsion adjuvant inactivated vaccine and preparation method thereof |
| CN106924730B (en) * | 2017-02-14 | 2019-11-08 | 商丘美兰生物工程有限公司 | Porcine pseudorabies virus Aloe Vera Gel nano emulsion adjuvant inactivated vaccine and preparation method thereof |
| CN108578688A (en) * | 2018-04-03 | 2018-09-28 | 武汉轻工大学 | The preparation method of multiple nano-emulsion vaccine adjuvant |
| CN108578688B (en) * | 2018-04-03 | 2022-02-11 | 武汉轻工大学 | Preparation method of multiple nanoemulsion vaccine adjuvant |
| CN113209287A (en) * | 2021-02-04 | 2021-08-06 | 中国农业科学院农业环境与可持续发展研究所 | Water-in-oil type nano vaccine adjuvant, preparation method and application thereof |
| CN113209287B (en) * | 2021-02-04 | 2022-07-08 | 中国农业科学院农业环境与可持续发展研究所 | Water-in-oil type nano vaccine adjuvant, preparation method and application thereof |
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Application publication date: 20101027 |