CN108641005A - A kind of preparation method and applications of sulphation Echinacea polysaccharide - Google Patents
A kind of preparation method and applications of sulphation Echinacea polysaccharide Download PDFInfo
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Abstract
The invention discloses a kind of preparation method and applications of sulphation Echinacea polysaccharide.The preparation method includes the following steps：S1. chlorosulfonic acid pyridine esterifying reagent is prepared；S2. the uniform solution of Echinacea polysaccharide is prepared using the Echinacea polysaccharide of purifying；S3. esterifying reagent is added in the uniform solution of Echinacea polysaccharide, confined reaction 3h is stirred at 80 DEG C；S4. after the reaction was complete, it is 7 to be cooled to room temperature and adjust pH, and a concentration of 80% ethyl alcohol of 3 ~ 4 times of volumes is added, staticly settles for 24 hours；S5. it takes precipitation to be dissolved in water to dialyse 3 days, concentrate dialysate, freeze-drying obtains sulphation Echinacea polysaccharide.The Echinacea polysaccharide of purifying is carried out the sulphation Echinacea polysaccharide of sulphation modification adaptive immune enhancing by the present invention; preparation process is simple; definite ingredients; quality controllable, drug effect is reliable, is suitable for large-scale production; there is significant immunoregulation effect to the Dendritic Cells of chicken; it can be widely applied in the immunoregulation medicament of birds and beasts, it is safe to use, it has no toxic side effect.
Description
Technical field
The invention belongs to Chinese medical extract research field, relate generally to a kind of sulphation Echinacea polysaccharide preparation method and
It is applied.
Background technology
The state of immunity of organisms has decisive role, Dendritic Cells conduct to the premunition and productivity of animal body
Full-time antigen presenting cell in animal body, its state and quantity play vital work to immune response in animal body
With.Chinese herbal medicine has unique superiority, specificity or can non-specifically enhance the immunity of body, for many years more
Sugar, which is studied, to be had the function of enhancing body's immunity to displaying herbal polysaccharide, and has that Chinese herbal medicine is safe and nontoxic, stable etc. has
Point does not have toxic side effect to the normal cell of body, is good Organism immunoregulation agent.
Echinacea（Echinacea purpurea）EP at home and abroad forms research hot many years, especially in North America
And Europe, they obtain with Echinacea Related product treatment toothache, skin disease, epilepsy, cancer, viral and bacteriosis
Preferable effect.It is a large amount of research shows that herbal polysaccharide has the effects that antiviral, antibacterial, antitumor.Echinacea polysaccharide has
There is the characteristic of general herbal polysaccharide, and clinically have practical application, but at present the research of Echinacea polysaccharide is collected more
In its extracting method and with other substances compounding additive agent field, for Echinacea polysaccharide Immune-enhancing effect performance boost itself
Lack research, obtained immunoregulation medicament activity is low, cannot effectively adjust animal body immunocompetence and resist the disease
The problem of ability, the efficient simple immunoregulation medicament of shortage.
Therefore it provides it is a kind of safe and effective, there is the immunological regulation herbal polysaccharide of application prospect to have on veterinary clinic
Very important meaning.
Invention content
It is an object of the invention to overcome deficiency in the prior art, a kind of preparation side of sulphation Echinacea polysaccharide is provided
Method, sulphation Echinacea polysaccharide not only possesses the characteristic of general polysaccharides, safe and nontoxic, stable, and sulphation modification can be into one
Step improves the immunological enhancement of herbal polysaccharide, and drug effect is relatively reliable.
Another object of the present invention is to provide a kind of sulphation Echinacea polysaccharide.
Another object of the present invention is to provide a kind of sulphation Echinacea polysaccharide in preparing chicken immune and adjusting drug
Using.
The above-mentioned purpose of the present invention is achieved by following technical solution：
A kind of preparation method of sulphation Echinacea polysaccharide, includes the following steps：
S1. chlorosulfonic acid-pyridine esterifying reagent is prepared；
S2. the uniform solution of Echinacea polysaccharide is prepared using the Echinacea polysaccharide of purifying；
S3. esterifying reagent is added in the uniform solution of Echinacea polysaccharide, 2.5 ~ 3.5h of confined reaction is stirred at 75 ~ 85 DEG C,
After the reaction was complete, it is cooled to room temperature and adjusts pH to neutrality, alcohol precipitation；
S4. it takes and precipitates the dialysis that is dissolved in water in S3, concentrate dialysate, freeze-drying obtains sulphation Echinacea polysaccharide.
The sulphation Echinacea polysaccharide component that the preparation method of the present invention is prepared is clear, contains Echinacea polysaccharide and sulphur
Acid group, wherein Echinacea polysaccharide have the characteristic of general polysaccharides, do not have toxic side effect, sulphation modification to the normal cell of body
The immunological enhancement of herbal polysaccharide can be further increased, it is quality controllable.Inventor stumbles in the preparation side of the present invention
The higher sulfate radical content of Echinacea polysaccharide sulfate radical obtained under method, has the function of good Organism immunoregulation
Wherein it is preferred to which the uniform solution of Echinacea polysaccharide described in S2, which is Echinacea polysaccharide, is dissolved in N, N- dimethyl formyls
The solution formed in amine.
It is highly preferred that a concentration of 9.5 ~ 10.5mg/mL of the uniform solution of Echinacea polysaccharide described in S2.
Preferably, the preparation process of chlorosulfonic acid-pyridine esterifying reagent is：Anhydrous pyridine is fully cooled at 0 DEG C, dropwise
Chlorosulfonic acid is added, drop finishes and forms faint yellow solid, obtains esterifying reagent, and the wherein volume ratio of anhydrous pyridine and chlorosulfonic acid is 100:
16.7。
Preferably, the preparation method of the Echinacea polysaccharide wherein purified described in S2 is：It is thick that water decoction-alcohol sedimentation prepares Echinacea
Polysaccharide, then the Echinacea polysaccharide purified is prepared by Sevage method removing proteins.
Wherein water decoction-alcohol sedimentation, which prepares Echinacea Thick many candies and operates, is：By complete stool Echinacea with 80% ethyl alcohol soaked overnight, 70
DEG C water-bath flows back 2h, is decocted with water, filters out the dregs of a decoction, filtrate is concentrated, and crude drug concentration is made to reach 1g/mL, pure in centrifuging and taking
Heavy, freeze-drying obtains Echinacea Thick many candies sample；
The concrete operations of Echinacea polysaccharide that Sevage method removing proteins prepare purifying are：By Echinacea Thick many candies and water with 1g：
The ratio of 100mL dissolves, addition Sevage reagents, and the volume ratio of Sevage reagents and Echinacea Thick many candies aqueous solution is 16:30,
15min is shaken with the rotating speed of 2500rpm/min, supernatant liquor is taken, ethyl alcohol is added after concentration, alcohol content is made to reach 70%, stands 24
Hour, take precipitation to be freeze-dried the Echinacea polysaccharide purified.
Echinacea polysaccharide is extracted using complete stool Echinacea, purifying, and raw material availability is high.
The sulphation Echinacea polysaccharide being prepared by the above method is also within protection scope of the present invention.
Preferably, it is 0.27 ~ 0.32 mg/mL, sulfuric acid to have the polyoses content for the Echinacea polysaccharide that the above method is prepared
Radical content is 0.25 ~ 0.3 mg/mL.The Echinacea polysaccharide of the present invention is extracted from Echinacea plant, safety and environmental protection, document
Research shows that sugared content and sulfate radical content in sulfuric acid Echinacea polysaccharide and immunoregulation effect have it is close contact, it is dense
Degree directly affects immunoregulation effect.
Application of the above-mentioned sulphation Echinacea polysaccharide in preparing chicken immune and adjusting drug.The sulphation Echinacea of the present invention
The Dendritic Cells of Polysaccharides in Chicken has significant immunoregulation effect, fills up the research blank of domestic and international veterinary drug Echinacea, can
The epidemic disease for being widely used in his birds such as chicken and beasts is adjusted in drug.
Apply the sulphation when preparing chicken immune and adjusting drug, wherein drug use purple above-mentioned sulphation Echinacea polysaccharide
Bore the use a concentration of 2 of chrysanthemum polysaccharide-7~2-9mg/mL。
Preferably, in the application sulphation Echinacea polysaccharide use a concentration of 2-8mg/mL。
The present invention has the following technical effects compared with prior art：
The present invention provides a kind of preparation methods of sulphation Echinacea polysaccharide, and the Echinacea polysaccharide of purifying, which is carried out sulphation, to be repaiied
The sulphation Echinacea polysaccharide of adaptive immune enhancing is adornd, preparation process is simple, definite ingredients, quality controllable, and drug effect is reliable, is suitable for
Large-scale production.The Dendritic Cells for the sulphation Echinacea Polysaccharides in Chicken that the present invention is prepared has significant immunological regulation
Effect, the epidemic disease that can be widely applied to birds and beasts is adjusted in drug, safe to use, is had no toxic side effect, and has wide disease
Prevention and control application market.
Description of the drawings
Fig. 1 is sulphation Echinacea polysaccharide prepared by embodiment 1（SEPP）Infrared spectrum.
Specific implementation mode
The present invention is further illustrated With reference to embodiment, but embodiment the present invention is not done it is any
The restriction of form.In order to further verify beneficial effects of the present invention.
The sulphation Echinacea polysaccharide of the present invention（SEEP）Polyoses content and sulfate radical content detection method it is as follows：
Polyoses content detects
A. the drafting of standard curve：5% phenol test fluid is configured to distilled water；With standard glucose（（D+）Anhydrous grape
Sugar）For standard items, weighs in 105 DEG C of dryings to standard glucose 100mg to the 100mL volumetric flasks of constant weight, water is added to be settled to
1mg/mL storing solutions are made in 100mL graduation marks, and drawing 10mL with liquid-transfering gun is settled to 100mL, obtains the glucose of 0.1mg/mL
Solution takes 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL to be added in test tube respectively, respectively adds water to 2mL, and 5% phenol is then added
1mL the and 5mL concentrated sulfuric acids, shaking shake up, and are placed at room temperature for 20min, then measure its light absorption value in 492nm, are pressed with the ultra-pure water of 2mL
Same operation is used as blank, and using the light absorption value of the corresponding concentration measured as Y-axis, it is bent to obtain corresponding standard for a concentration of X-axis
Line regression equation Y=9.8707x+0.0172, R >=0.999（n=3）, wherein X is concentration, and unit mg/mL, Y are light absorption value.
B. the measurement of sample size：5mgSEEP accurately is weighed, is dissolved in respectively in the super ultra-pure waters of 5mL, is made into 1mg/mL samples
Product solution, takes 200ul to add water to 2mL, and 5% phenol 1.0mL and concentrated sulfuric acid 5mL is then added, shakes up, and is so that blank reagent is added
Reference is measured according to aforesaid operationsA 490 Value calculates the content of polysaccharide in sample according to standard curve.
The measurement of sulfate radical content
A. the configuration of acid barium chromate solution:Precision weighs 1.93g potassium chromates and is dissolved in 1mL dilute hydrochloric acid with 2.4288g barium chlorides
In solution, it is settled to 100mL.
b. 1:The configuration of 1 ammonium hydroxide:20mL ammonium hydroxide is taken to be mixed with the ultra-pure water of 20mL, it is preferably now with the current.
C. the configuration of sulfate standard solution storing solution:Precision is weighed through 105 DEG C of dry anhydrous sodium sulfates to constant weight
0.7352g is dissolved in ultra-pure water, is settled to 1L, is made into the sulfate titer storing solution of 0.5mg/mL.
D. sample pretreatment precision weighs 10mgSEEP, is dissolved in 1mol/L dilute hydrochloric acid, is settled to 10mL, is transferred to tool plug
In test tube, boiling water bath hydrolyzes 4h, if hydrolysis has evaporation, plus the dilute hydrochloric acid of 1mol/L complements to 10mL.
E. the drafting of standard curve：Be respectively configured 0 with 0.5mg/mL sulfate titer storing solutions, 0.05,0.01,
0.015,0.02,0.025mg/mL sulfate titers, draw in each concentration standard liquid 50mL tool plug test tubes, Plus acidic chromium respectively
Sour barium solution 3mL, 60 DEG C of ultrasonic water bath 10min stand, are cooled to room temperature, and are added 1：1 ammonia spirit 1mL, after shaking mixing,
0.45um membrane filtrations are discarded 5mL just liquid, continue to collect filtrate, be returned to zero with ultra-pure water, at spectrophotometric determination 375nm
Absorption photometric value with a concentration of abscissa of sulfate standard solution, it is bent to draw standard using standard items absorbance as ordinate
Line obtains equation of linear regression Y=5.3828X+0.0218（R2=0.9995, n=3）
F. the measurement of sample size:SEEP sample sour water solution treatment fluids are measured, 50mL is settled to ultra-pure water, are transferred to tool plug
In conical flask, be configured to the sample solution of 0.02mg/mL for measuring, specific method with standard curve operation, with absorbance
Value brings the sulfate concentration that regression equation calculates Yang Pin into.
Embodiment 1
A kind of sulphation Echinacea polysaccharide, preparation process are as follows：
S1. chlorosulfonic acid-pyridine esterifying reagent is prepared；
S2. the uniform solution of Echinacea polysaccharide is prepared using the Echinacea polysaccharide of purifying：Weigh the purified Echinacea polysaccharide of 3g
It sets and the uniform solution that Echinacea polysaccharide is prepared in appropriate N,N-dimethylformamide is added in conical flask；
S3. esterifying reagent is added in the uniform solution of Echinacea polysaccharide, confined reaction 3h is stirred at 80 DEG C, the reaction was complete
Afterwards, it is cooled to room temperature, 100 mL ice water of precooling is added, it is neutral to be neutralized to pH values with the NaOH solution of 5mol/L, it is added 3 ~
A concentration of 80% ethyl alcohol of 4 times of volumes, staticly settles for 24 hours；
S5. it filters and takes precipitation, after adding appropriate water dissolution to precipitate, be packed into bag filter, first use tap water flowing water 48 h of dialysis, then use
Distilled water 24 h of dialysis, concentration dialyzate, freeze-drying obtain sulphation Echinacea polysaccharide.
Wherein, the preparation process of the Echinacea polysaccharide of purifying is as follows：
S21. water decoction-alcohol sedimentation prepares Echinacea Thick many candies：A certain amount of complete stool Echinacea is weighed, with 80% ethyl alcohol soaked overnight, 70
DEG C water-bath flows back 2 times, 1h 1 time, with gauze wrapped medicinal material, is decocted 2 times with water（It boils by intense fire, slow boiling maintains boiling）, filter out
The dregs of a decoction merge and decoct gained filtrate twice, filtrate is concentrated, crude drug concentration is made to reach 1g/mL, 4500 rpm/min, centrifuge
5min takes supernatant alcohol precipitation 3 times repeatedly, freeze-drying to obtain Echinacea Thick many candies sample；
S22. Sevage methods removing protein prepares the Echinacea polysaccharide of purifying：Using albumen in Sevage method Polysaccharide removings, weigh
The Echinacea polysaccharide of 30g dryings, is dissolved in 3000mL distilled water, and 1600mL Sevage reagents are added（Chloroform：N-butanol=4:
1）, fully shaking, 2500rpm/min, 15min discard lower layer's albuminate, take supernatant, be concentrated into 500mL, adds 95% second
Alcohol makes alcohol content reach 70%, stands 24 hours, precipitation is taken to be freeze-dried.
The preparation process of chlorosulfonic acid-pyridine esterifying reagent is as follows：
A. the three-neck flask with stirrer and condensing unit is set in ice-water bath；
B. the anhydrous pyridine 100mL of precooling is first added, is vigorously stirred, is allowed to be fully cooled；
C. 16.7mL chlorosulfonic acids are added dropwise again（It is slowly added dropwise, in 30 min）, see in flask a large amount of pale yellow colored solids occur
Body stops reaction, and prepared by esterifying reagent completes.
Embodiment 2
Sulphation Echinacea polysaccharide prepared by above-described embodiment 1 is applied to the immunological regulation of chicken, uses a concentration of 2-7 mg/
mL。
Embodiment 3
Sulphation Echinacea polysaccharide prepared by above-described embodiment 1 is applied to the immunological regulation of chicken, uses a concentration of 2-8 mg/
mL。
Implement real 4
Sulphation Echinacea polysaccharide prepared by above-described embodiment 1 is applied to the immunological regulation of chicken, uses a concentration of 2-9mg/mL。
Comparative example 1
Essentially identical with the operating procedure of embodiment 1, the reaction temperature in wherein S2 is 25 DEG C.By the sulphation Echinacea of preparation
Polysaccharide is applied to the immunological regulation of chicken, uses a concentration of 2-8 mg/mL。
Comparative example 2
It is essentially identical with the operating procedure of embodiment 1, being staticly settled without ethyl alcohol in wherein S4.By the sulphation Echinacea of preparation
Polysaccharide is applied to the immunological regulation of chicken, uses a concentration of 2-8 mg/mL。
Structure detection
1, the measurement of SEEP prepared by embodiment 1 to lymphocyte safe concentration
Assay method：
Take 30 ages in days, sterile Culling heart blood, heparin sodium anti-freezing, after adding Hank ' s liquid doubling dilutions, by the lymph after doubling dilution
Cell is carefully slowly with 1:1 is added in lymphocyte separation medium upper layer, and 2000r/min centrifuges 10min, draws intermediate milky cloud
Misty cell is washed 2 times, 1500r/min with Hank ' s, centrifuges 10min, and the RPMI1640 containing 10% fetal calf serum is added and cultivates
Liquid.
The concentration of lymphocyte obtained above is adjusted to 2 × 10696 hole cell versions are added in a/mL, per hole 100ul.
By 11 concentration of SEEP doubling dilutions for a concentration of 2mg/mL that is prepared in advance, each concentration sets 4 multiple holes, while cell is arranged
Control wells are separately added into 96 porocyte culture plates containing lymphocyte, are placed in 37 DEG C of 5%CO2Culture 44 is small in incubator
When, it is added per hole a concentration of（5mg/mL）30 μ L of MTT continue to cultivate 4h, in microplate reader absorbanceA 570 Upper measurement cell OD values,
Select OD values more than maximum 3 concentration in cell control well as safe concentration.
Testing result：
Testing result is as shown in table 2, as shown in Table 1, in 11 concentration with 2mg/mL doubling dilutions, 2-2-2-10Mg/mL is to chicken
The increment pole of periphery hemolymph is significantly higher than cell control well, and 1mg/mL differences are not notable.Marginal value generally no longer limit of consideration
Within, it is the upper limit value for selecting concentration that can reach maximum OD values Cmin, therefore deduces that Echinacea Polysaccharides in Chicken
Peripheral blood lymphocytes best safety a concentration of 2-7~2-9mg/mL。
Measurement of 1 SEEP of table to chicken peripheral blood lymphocytes safe concentration
| mg/mL | OD values |
| 20 | 0.137±0.004 |
| 2-1 | 0.188±0.004** |
| 2-2 | 0.182±0.004** |
| 2-3 | 0.196±0.005** |
| 2-4 | 0.196±0.004** |
| 2-5 | 0.207±0.004** |
| 2-6 | 0.21±0.008** |
| 2-7 | 0.21±0.005** |
| 2-8 | 0.204±0.009** |
| 2-9 | 0.202±0.008** |
| 2-10 | 0.203±0.01** |
| CC | 0.143±0.006 |
Note：Compared with CC groups, * * indicate that difference is extremely notable, i.e.,P<0.01, * indicates significant difference, i.e.,P<0.05, no mark person is poor
It is different notable, that is,P≥0.05.CC indicates cell control well.
2, the analysis of the infrared spectrum of SEEP prepared by embodiment 1
Assay method：
With polysaccharide：KBr=1:100, the KBr powder of 1mgSEEP and 100mg is weighed respectively, is fully ground, tabletting, 400 ~
4000cm-1Wave-number range carry out IR spectrum scanning, obtain the infrared spectrogram of Echinacea polysaccharide and sulfated polysaccharides.
Testing result
Obtained infrared spectrogram is detected as shown in Figure 1, the infrared spectrum of polysaccharide shows 3 sugared characteristic vibration peaks：
3600~3200 cm-1There is the last one at place and wide absorption peak, is the stretching vibration of O-H and intramolecular or intermolecular hydrogen bonding；3200
~2800 cm-1There is an absorption peak at place, is the stretching vibration of C-H；In 1400~1200 cm-1There is an absorption peak at place, is C-H, C-
O and C-C vibration absorption peaks.In 1300~1200 cm-1There is strong absorption peak in place, is the characteristic peak of S=O stretching vibrations；900
~800 cm-1There is strong stretching vibration peak in place, is C-O-SO3Related symmetry C-O-S absorption peaks.
3, the influence that SEEP activates Dendritic Cells Induced allogeneic T cells
Detection method
Stimulate the preparation of cell：Isolating dendritic cells, cultivate immature Dendritic Cells I-DCs and ripe dendron shape is thin
Born of the same parents M-DCs, is separately added into SEEP, concurrently sets blank group and control group, and control group is handled with lipopolysaccharides, and silk is used after cultivating 48h
Rimocidin C（50ug/mL）At 37 DEG C, 5%CO230min is handled, treated cell, 2000r/min, centrifugation is resuspended in PBS
10min abandons supernatant, is repeated 2 times, with the DMEM cells being resuspended and adjust cell concentration be 2 × 106A/mL, as stimulation
Cell.
The preparation of reacting cells：Take 30 ages in days, sterile Culling heart blood, heparin sodium anti-freezing, after adding Hank ' s liquid doubling dilutions,
By the lymphocyte after doubling dilution carefully slowly with 1:1 is added in lymphocyte separation medium upper layer, and 2000r/min centrifuges min,
Intermediate milky cloud cell is drawn, is washed 2 times, 1500r/min with Hank ' s, 10min is centrifuged, is added and contains 10% tire ox blood
Clear RPMI1640 culture solutions.
Mixed lymphocyte reaction (MLP)：It is 1 by stimulation cell obtained above and reacting cells ratio：1、1：5、1：10、1：
20 are separately added into each hole, and experiment is divided into embodiment group, LPS（Lipopolysaccharides）Group, without stimulation group (Blank, B), to concurrently set T thin
Born of the same parents' group is negative control group, and every group sets 4 repetitions, in 37 DEG C, CO2After cultivating 44 h in incubator, 30 μ L MTT are added per hole,
After 37 DEG C are incubated 4h, supernatant is abandoned, 100 μ L DMSO are added per hole, cell plates, which are placed in concussion 5min on micro oscillator, makes it sink
Shallow lake is completely dissolved, and is measured in microplate readerA 570 Value.
Dendritic Cells through different disposal is to the stimulus index calculation formula of allogeneic T cells：SI=(A Processing group-A Blank group)/（A Negative control group-A Blank group）.
Testing result
The influence testing result that SEEP activates Dendritic Cells Induced allogeneic T cells is as shown in table 2 and table 3.
Influences of 2 SEEP of table to non-I-DCs induction allogeneic T cells activation
| Group/SI | 1:1 | 1:5 | 1:10 | 1:20 |
| Embodiment 2 | -0.59±0.11d | -0.78±0.09b | -0.85±0.07c | -0.96±0.12c |
| Embodiment 3 | -0.51±0.10bcd | -0.43±0.07a | -0.49±0.07c | -0.62±0.07bc |
| Embodiment 4 | -0.55±0.15cd | -0.33±0.08a | -0.43±0.07ab | -0.51±0.08ab |
| Comparative example 1 | -0.29±0.07a | -0.32±0.02a | -0.43±0.12ab | -0.25±0.03ab |
| Comparative example 2 | -0.28±0.03a | -0.37±0.08a | -0.39±0.07ab | -0.55±0.12ab |
| LPS | -0.39±0.12abc | -0.47±0.14a | -0.27±0.14a | -0.15±0.05a |
Note：Same column shoulder mark same letter indicates that difference is not notable without letter person（P＞ 0.05）, the different letter person's expressions of shoulder mark
Significant difference（P<0.05）.
As shown in Table 2, SEEP and lipopolysaccharides（LPS）It is equal to the I-DCs induction allogeneic T cells activation of different proportion
Inhibiting effect is shown, and the inhibiting effect of 2 ~ 4 each group of embodiment is higher than LPS（P<0.05）, the inhibiting effect of comparative example 1 ~ 2 is low
In applying 2 ~ 4 each group of example, I-DCs induces the inhibiting effect that allogeneic T cells activation is presented, causes I-DC activated T lymphocytes
Ability decline, to be conducive to the treatment of autoimmune disease, SEEP is to non-I-DCs induction allogeneic T cells activation
It is that the stronger immune effect of inhibiting effect is better.
3 SEEP of table induces non-M-DCs born of the same parents the influence of allogeneic T cells activation
| Group/SI | 1:1 | 1:5 | 1:10 | 1:20 |
| Embodiment 2 | 1.04±0.08a | 3.11±0.82a | 3.31±0.58a | 5.72±0.53a |
| Embodiment 3 | 1.07±0.13a | 3.18±0.39a | 2.79±0.47a | 4.23±1.04b |
| Embodiment 4 | 0.74±0.24b | 1.96±0.33ab | 1.76±0.36b | 2.59±0.53c |
| Comparative example 1 | 0.69±0.07b | 1.85±0.18ab | 1.49±0.14b | 1.66±0.19c |
| Comparative example 2 | 0.66±0.04b | 1.82±0.27ab | 1.43±0.24b | 1.86±0.15c |
| LPS | 1.01±0.31a | 1.25±0.32c | 0.70±0.69c | 2.46±1.46c |
As shown in Table 3, SEEP and LPS shows promotion to the M-DCs induction allogeneic T cells activation of different proportion and makees
With the facilitation of embodiment 2 ~ 4 is significantly higher than LPS（P<0.05）, other differences are notableP>0.05）, SEPP co-cultivations M-
DC shows that SEPP co-cultures M-DC and can enhance M-DC activated T lymphocytes to Allogeneic T lymphocyte activation facilitation
Ability, to improve the immune response ability of body.
4, influences of the SEEP to dendritic cell phenotypic and cell factor
Detection method：
It is sterile to take its shin bone and femur with 30 age in days Lingnan Yellows, it is first impregnated 15 minutes with bromogeramine solution for disinfection, then with 75% wine
Essence is impregnated 5 minutes, and remaining meat bits are removed under gnotobasis, is drawn PBS with 10mL syringes and is rinsed ossis, until ossis becomes
White for this purpose, collecting cell, 2000r/min centrifuges 10min, cell is resuspended with PBS, by 1：1 ratio contains re-suspension liquid addition
In the 10mL centrifuge tubes of Histopaque-1119,2500r/min centrifuges 30min, collects the nebulous cell of middle layer white,
It is resuspended 2 times, 2000r/min with PBS, centrifuges 10min, outwell supernatant liquid, obtained precipitation is chicken marrow source DCs, is added
DMEM culture solutions containing 10% fetal calf serum, adjustment cell concentration are 2 × 106A/mL.
The detection that I-DCs and M-DCs carries out secreting function is cultivated, concrete operations are as follows：The above-mentioned DCs that obtains is inoculated into 6
Porocyte plates, per hole 3mL, 37 DEG C, full dose changes liquid difference after 5%CO2 cultures for 24 hours, and SEEP is added, concurrently sets blank group（B）With
Control group（LPS）, after cultivating 48h, collect DCs and supernatant, the Dendritic Cells of collection washed 2 times with PBS, FBS processing is thin
Born of the same parents 10min adds monoclonal antibody PE-CD11c, FITC-MHCII of fluorescent marker, is protected from light at 4 DEG C and is incubated 30min, PBS
Washing 3 times, the expression of flow cytomery cell surface CD11c, MHCII molecule, cell supernatant for measure NO,
Rtanes, IL-4, IL-10 and IFN-γ.
The above-mentioned Dendritic Cells that obtains is inoculated into 6 porocyte plates, per hole 3mL, 37 DEG C, full dose is changed after 5%CO2 cultures for 24 hours
Liquid, and chicken recombination rh GM-CSF (50ng/mL) and rh IL-4 are added into cell（50ng/mL）It is seen daily with inverted microscope
Examine cellular morphology variation.Partly amount changed liquid respectively with the 5th day on day 3, while supplementing rhGM-CSF and rh IL-4, meanwhile, Xiang Pei
LPS and SEEP stimulations Dendritic Cells is added in nutrient solution makes its maturation, 7 days whens take Dendritic Cells cell and supernatant, dendron
Shape cell is used to measure the expression quantity of CD11c and MHCII.
The culture of DCs cells collects cell supernatant for measuring NO, Rtanes, IL-4, IL-10 and IFN-γ.
Testing result
Influences of the SEEP to dendritic cell phenotypic developed by molecule is as shown in table 4, table 5.
Influences of 4 SEEP of table to I-DCs phenotype developed by molecule
| Group | CD11c | MHCII |
| Embodiment 2 | 83.37±1.02a | 77.67±0.84ab |
| Embodiment 3 | 84.2±1.15a | 79.36±0.96a |
| Embodiment 4 | 83.8±0.92a | 75±0.71ab |
| Comparative example 1 | 75.333±0.52bc | 62.967±3.72d |
| Comparative example 2 | 72.9±4.05c | 65.967±3.92cd |
| LPS | 79.8±1.15ab | 71.03±1.20bc |
| B | 77.43±1.03bc | 49.97±2.09e |
Influences of 5 SEEP of table to M-DCs phenotype developed by molecule
| Group | CD11c | MHCII |
| Embodiment 2 | 85.03±1.85b | 37.6±0.99e |
| Embodiment 3 | 92.6±0.53a | 31.6±1.07f |
| Embodiment 4 | 77.97±1.42c | 44.77±1.39c |
| Comparative example 1 | 72.333±1.68 de | 41.3±0.81d |
| Comparative example 2 | 74.7±1.21cd | 34.767±0.83ef |
| LPS | 89.5±0.95a | 66.13±1.24a |
| B | 70.07±1.04e | 53.5±1.64b |
As shown in Table 4, significantly high to the expression quantity of I-DCs phenotype molecules CD11c in embodiment 2, embodiment 3 and embodiment 4
In B groups (P<0.05), at the same embodiment 3 and embodiment 4 be all remarkably higher than LPS groups (P<0.05)；The embodiment in MHCII expression
2 ~ 4 and LPS be significantly higher than B groups (P<0.05), at the same 2 ~ 4 groups of embodiment be significantly higher than LPS groups (P＜ 0.05).
Known by table 5, in the expression of MHII, the substantially less than B groups of embodiment 2 ~ 4 (P<0.05)。
CD11 is the significant albumen of bone marrow Dendritic Cells, by experimental result it is found that SEPP can be different degrees of rush
It is converted into DCs into bone marrow precursor, as DC quantity increases, the immunoregulation capability enhancing of animal body.MHCII is located at anti-
For original on delivery cell, antigenic fragments are prompted to T cell by antigen presenting cell after antigen is swallowed using MHCII, are started immune
Reaction, SEPP have significant facilitation to the expression of the MHCII of I-DCs, show that SEPP offers to that can enhance I-DC antigens
Ability, and it is inhibited to the expression of the MHCII of M-DCs, it is double to show that SEPP has the immune response of Dendritic Cells
Weight adjustment effect.
The influence of SEEP Dendritic Cells secrete cytokines, as shown in table 6 and table 7.
Influences of 6 SEEP of table to M-DCs secrete cytokines
| Group | IL-4（ng/L） | IL-10（ng/L） | IFN-γ（pg/mL） |
| Embodiment 2 | 51.41±6.34cd | 19.38±0.94d | 23.67±7.45a |
| Embodiment 3 | 37.83±2.75d | 23.98±1.37bc | 17.18±2.5ab |
| Embodiment 4 | 46.37±3.84cd | 23.47±1.2abc | 15.01±0.99ab |
| Comparative example 1 | 58.8±1.72bc | 25.93±1b | 14.47±0.65b |
| Comparative example 2 | 55.96±10.4c | 27.65±1.2ab | 13.71±0.6b |
| LPS | 57.99±1.52bc | 20.1±1.87cd | 22.81±0.99ab |
| B | 79.46±3.65a | 30.15±1.2a | 16.75±0.87ab |
Influences of 7 SEEP of table to I-DCs secrete cytokines
| Group | IL-4（ng/L） | IL-10（ng/L） | IFN-γ（pg/mL） |
| Embodiment 2 | 68.94±8.66bc | 23.55±1.16b | 58.46±1.88a |
| Embodiment 3 | 72.89±5.91abc | 26.99±1.23ab | 43.81±6.38abc |
| Embodiment 4 | 59.74±4.27c | 26.85±2.23ab | 41.65±7.8abc |
| Comparative example 1 | 76.13±0.8abc | 28.6±1.02ab | 41.07±1.11abc |
| Comparative example 2 | 80.4±1.43ab | 28.5±0.64sb | 40.89±0.74abc |
| LPS | 63.03±8.35bc | 25.41±3.59b | 45.98±8.56ab |
| B | 88.89±7.62a | 32.31±1.02a | 26.27±8.43c |
In the immune response of animal body, helper T lymphocyte（Th）It plays an important role, IFN-γ is mainly by Th1 type cells
Secretion, and IL-4, IL-10 are mainly secreted by Th2 type cells, by experimental result it is found that SEPP is to DCs secretion IL-4, IL-10 tools
There is inhibiting effect, and there is facilitation to the secretion of IFN-γ, promotes immune response to be biased to Th1 states, as IL-4, IL-10
When expression reduces, it is Th1 type cells that the IFN-γ of expression, which promotes Th cell differentiations, and IFN-γ is secreted by Th1 type cells, is adjusted
Cellular immunity, IL-4, IL-10 are secreted by Th2 types, adjust humoral immunity, Th1/Th1 is a dynamic equilibrium, IL-4, IL-
10 reduce, and IFN-γ increases, and immune response is promoted to be biased to humoral immunity, enhance the immune of body fight Intracellular bacterial and protozoon
Respond.
The influence of Echinacea polysaccharide and SEEP Dendritic Cells secretion chemotactic factor (CF), as shown in table 8.
8 SEEP of table secretes M-DCs the influence of chemotactic factor (CF)
| Group | M-DCs Rantes（pg/mL） | I-DCs Rantes（pg/mL） |
| Embodiment 2 | 515.93±10.44c | 828.37±51.36a |
| Embodiment 3 | 518.82±8.04c | 835.13±77.26a |
| Embodiment 4 | 613.01±27.09b | 520.39±21.07c |
| Comparative example 1 | 511.67±1.81c | 513.15±3.63c |
| Comparative example 2 | 491.96±5.11c | 533.00±8.85c |
| LPS | 684.2±23.8a | 708.47±41.58b |
| B | 513.5±13.06c | 680.09±26.61b |
As shown in Table 8, M-DCs secretes in chemotactic factor (CF) result, and SEPPH, SEPPM and SEPPL and LPS groups are above B groups, wherein
LPS be significantly higher than B groups (P<0.05), remaining difference significantly (P>0.05)；In I-DCs secretes chemotactic factor (CF) result, SEPPH
Be significantly higher than B groups with SEPPM, but SEPPL be substantially less than B groups (P<0.05), LPS groups there are no significant compared with B groups variation (P
＜ 0.05).
Rantes has different biological functions, it not only has chemotaxis to various kinds of cell, but also can be strong
Activated lymphocyte, participate in the generation of inflammatory reaction, adjust the growth and differentiation of cell, the up-regulation of the Rantes factors can lure
Activated leukocyte cell is led, by experimental result it is found that SEEP is by promoting DCs to secrete Rantes, to which the correlation for enhancing Rantes is anti-
Should and function.
DCs is sole duty APC in animal body, and animal body generates immune response to immunogene and is somewhat dependent on
DCs.DCs is widely present in blood and immune organ, and I-DCs has powerful antigen phagocytic function, M-DCs expression high levels
Costimulating factor and adhesion factor, during maturation moving into peripheral tissues enters secondary lymphatic organ, is connect with T cell
It touches, and excites immune response.
The I-DCs and M-DCs of SEEP stimulation cultures mix training after mitomycin C is handled with Allogenic Lymphocytes
It supports, I-DCs is inhibited to the proliferation of lymphocyte, and M-DCs has facilitation, I- to the proliferation of lymphocyte
DCs can inducing immune tolerance, T cell mixed lymphocytes breeder reaction of the same race can be reduced and weakened, autoimmunity disease is prevented
The generation of disease；And M-DCs being capable of high expressing cell surface co-stimulatory molecules while inducer T lymphocyte proliferation.By experimental result
It is found that I-DCs is inhibited to the proliferation of lymphocyte, handled through SEPP the I-DC of culture to lymphopoietic
Inhibiting effect is better than LPS, and therefore, SEPP promotes I-DCs inducing immune tolerance abilities to be better than LPS, controls autoimmune disease
Tool is treated to have very great help, and M-DCs has facilitation to the proliferation of Allogeneic T lymphocyte, and with DC:The ratio of T
Increase, this facilitations of SEPP are better than LPS, and lymphocyte is the important cells of immune response, and SEPP promotes lymph
Cel l proliferation enhances the immune response ability of body, to enhance resistivity of the body to disease.
In conclusion SEPP has dual regulation to chicken DCs partial functions, and chicken DCs is to chicken vivo immunization reaction
It plays a crucial role, its state determines that immune response is biased to and power, SEPP enough promote bone to a certain extent
Marrow precursor is converted into DCs, promotes the secretion of IFN-γ, NO, Rantes, inhibits the secretion of IL-4, IL-10, comprehensive adjustment
DCs functions integrate least cost and various indexs to be conducive to DCs in chicken vivo immunization adjustment effect from experimental result
Most strong facilitation, analysis obtain optium concentration be 2-8mg/mL.
Claims (10)
1. a kind of preparation method of sulphation Echinacea polysaccharide, which is characterized in that include the following steps：
S1. chlorosulfonic acid-pyridine esterifying reagent is prepared；
S2. the uniform solution of Echinacea polysaccharide is prepared using the Echinacea polysaccharide of purifying；
S3. esterifying reagent is added in the uniform solution of Echinacea polysaccharide, 2.5 ~ 3.5h of confined reaction is stirred at 75 ~ 85 DEG C,
After the reaction was complete, it is cooled to room temperature and adjusts pH to neutrality, alcohol precipitation；
S4. it takes and precipitates the dialysis that is dissolved in water in S3, concentrate dialysate, freeze-drying obtains sulphation Echinacea polysaccharide.
2. the preparation method of sulphation Echinacea polysaccharide as described in claim 1, which is characterized in that Echinacea polysaccharide described in S2
Uniform solution be Echinacea polysaccharide be dissolved in the solution formed in N,N-dimethylformamide.
3. the preparation method of sulphation Echinacea polysaccharide as described in claim 1, which is characterized in that Echinacea polysaccharide described in S2
Uniform solution a concentration of 9.5 ~ 10.5mg/mL.
4. the preparation method of sulphation Echinacea polysaccharide as described in claim 1, which is characterized in that chlorosulfonic acid-pyrrole described in S1
The preparation process of pyridine esterifying reagent is：Anhydrous pyridine is fully cooled at 0 DEG C, is added dropwise chlorosulfonic acid, drop finish formed it is yellowish
Color solid obtains esterifying reagent, and the wherein volume ratio of anhydrous pyridine and chlorosulfonic acid is 100:16.7.
5. the preparation method of sulphation Echinacea polysaccharide as described in claim 1, which is characterized in that the purple cone purified described in S2
The preparation method of chrysanthemum polysaccharide is：Water decoction-alcohol sedimentation prepares Echinacea Thick many candies, then the purple of purifying is prepared by Sevage method removing proteins
Bore chrysanthemum polysaccharide.
6. a kind of sulphation Echinacea polysaccharide being prepared by any one of claim 1 ~ 5 the method.
7. sulphation Echinacea polysaccharide as claimed in claim 6, which is characterized in that the polyoses content and sulfuric acid of Echinacea polysaccharide
Radical content uses spectrophotometry, and the polyoses content of Echinacea polysaccharide is 0.27 ~ 0.32 mg/mL, and sulfate radical content is
0.25~0.3 mg/mL。
8. application of the sulphation Echinacea polysaccharide as claimed in claim 6 in preparing chicken immune and adjusting drug.
9. application as claimed in claim 8, which is characterized in that the use a concentration of 2 of the sulphation Echinacea polysaccharide-7~2-9mg/
mL。
10. application as claimed in claim 8, which is characterized in that the use a concentration of 2 of the sulphation Echinacea polysaccharide-8mg/
mL。
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