CN105002230A - Method for enzyme extraction of echinacea polysaccharide - Google Patents
Method for enzyme extraction of echinacea polysaccharide Download PDFInfo
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Abstract
The invention discloses a method for enzyme extraction of echinacea polysaccharide. The method comprises crushing the aboveground part of echinacea to obtain pieces with suitable sizes, carrying out degreasing and extraction, adding an enzyme preparation into the extract, carrying out enzymolysis, carrying out enzyme deactivation, carrying out high temperature extraction, carrying out centrifugation, carrying out rotary evaporation condensation on the supernatant, carrying out alcohol precipitation, washing the precipitates orderly by waterless ethanol, acetone and ether, carrying out redissolution, and carrying out spray-drying to obtain echinacea polysaccharide. The method has the advantages of mild reaction conditions, short extraction time, high recovery rate, good reappearance and good stability. The prepared echinacea polysaccharide has the advantages of good dissolvability, unified appearance, good water solubility, good active components and good immunization activity.
Description
Technical field
The present invention relates to Polyose extraction field, particularly a kind of method of enzyme extraction echinacea purpurea polysaccharide.
Background technology
Echinacea purpurea (Echinacea) is world-famous immune medicinal herbs, and country of origin is positioned at North America and southern Canada.It is a kind of Echinacea plant, this platymiscium has 8 kinds and several mutation, 3 kinds are mainly contained: Echinacea purpurea Moench (Echinacea purpurea at present as drug development, namely usually said echinacea purpurea), narrow leaf echinacea purpurea (Echinacea augustifolia) and white echinacea purpurea (Echinacen pallida).Research shows that echinacea purpurea contains various active composition, and caffeic acid derivative's class, polysaccharide and glycoprotein, alkyl amide, alkaloids, polyyne class, flavonoid, volatile oil, sterol etc., its activeconstituents has become current research focus.
Echinacea purpurea because having significant immune-enhancing activity, and is called as " natural antibiotics ".Application is had in mankind's plurality kinds of health care product and compound product.And other effective constituents in relative echinacea purpurea, the research of echinacea purpurea polysaccharide is relatively less, studying more chicoric acid is main activeconstituents leaching process complexity, the activeconstituents products material amount obtained is few and cost is high, although the existing report extracting the immunocompetence research of polysaccharide about echinacea purpurea, but the systematic study its system extracted from medicinal material to activeconstituents being furtherd investigate to especially enzymatic treatment extraction polysaccharide is not reported so far, and purification process also rarely has research.
Summary of the invention
In order to solve echinacea purpurea Polyose extraction overlong time, and the problem that extraction yield is low, better activeconstituents can also be obtained simultaneously, the invention provides a kind of method of enzyme extraction echinacea purpurea polysaccharide.
In order to achieve the above object, the invention provides a kind of method of enzyme extraction echinacea purpurea polysaccharide, wherein, extracting method comprises the following steps:
1. medicinal material pre-treatment: echinacea purpurea over-ground part was pulverized 40-60 mesh sieve; Add degreasing solvent, after degreasing, volatilized by degreasing solvent under 20-80 DEG C of condition, the medicinal material after process is for subsequent use;
2. extract: take that 1. to process the medicinal material obtained appropriate, add the water of medicinal material 15-25 times amount, tune pH is 2-4, after being preheated to 40 DEG C-60 DEG C, add the zymin of medicinal material 0.04-0.06 times amount, enzymolysis time 50min-70min, 80 DEG C-100 DEG C enzyme 10-20min that go out, continue to stir insulation 1-2h at 80 DEG C-100 DEG C;
3. concentrate: centrifugal, supernatant liquor is revolved the concentrated solution steaming and be evaporated to crude drug amount 1:0.9-1.5;
4. alcohol precipitation: add 3-4 dehydrated alcohol doubly in concentrated solution, leave standstill 24-48 hour under 0-4 DEG C of condition;
5. wash: by the precipitation solution suction filtration 4. obtained, the throw out obtained uses dehydrated alcohol, acetone, washed with diethylether successively;
6. dry: redissolved by the water of throw out by 1-2 times amount, under the condition of 140 DEG C-160 DEG C, spraying dry obtains echinacea purpurea polysaccharide.
Wherein, described degreasing solvent is sherwood oil.
Wherein, described degreasing solvent is the mixing of sherwood oil, 1,2 epoxy prapane, ether, and its ratio of quality and the number of copies is: 1-2:0.05-0.1:0.02-0.08.
Wherein, described zymin is polygalacturonase.
Wherein, described zymin is the mixing of cellulase, polygalacturonase, helicase, phytase, and its ratio of quality and the number of copies is: 0.5-1:1-2:0.1-0.5:0.05-0.1.
Wherein, described echinacea purpurea crude drug source, in genus echinacea plant echinacea purpurea, takes from full-bloom stage to the over-ground part spending the later stage.
The invention has the beneficial effects as follows: this preparation method, there is reaction conditions gentleness, extraction time short, extraction yield advantages of higher, and circulation ratio is high, stable, the echinacea purpurea polysaccharide solvability prepared is better, outward appearance is homogeneous, good water solubility, and obtains good active ingredient, has good immunocompetence.
Specification sheets
accompanying drawing
fig. 1 EpPS I molecular weight determination is composed
figure;
fig. 2 EpPS II molecular weight determination is composed
figure;
fig. 3 EpPS III molecular weight determination is composed
figure;
fig. 4 EpPS I, EPPS II, EPPS III XRD
figure;
fig. 5 EpPS I INFRARED SPECTRUM
figure;
fig. 6 EpPS II INFRARED SPECTRUM
figure;
fig. 7 EpPS III INFRARED SPECTRUM
figure;
fig. 8 Ethe 13C NMR of PPS I composes
figure;
fig. 9 Ethe 13C NMR of PPS I composes
figure;
figure 10different concns EPPS, EPPS I, EPPS II and EPPS III are on the impact of RAW264.7 cell;
figure 11different concns EPPS, EPPS I, EPPS II and EPPS III are on the impact of RAW264.7 mode of appearance.
Embodiment
Embodiment 1
Extracting method comprises the following steps:
1. medicinal material pre-treatment: echinacea purpurea over-ground part was pulverized 60 mesh sieves; Add degreasing solvent, after degreasing, volatilized by sherwood oil under 60 DEG C of conditions, the medicinal material after process is for subsequent use;
2. extract: take the medicinal material 100g 1. processing and obtain, add 1500g water, adjust pH to be 3, after being preheated to 50 DEG C, add medicinal material 5g polygalacturonase, enzymolysis time 60min, 80 DEG C of enzyme 10min that go out, continue to stir insulation 1-h at 80 DEG C;
3. concentrate: centrifugal, supernatant liquor is revolved the concentrated solution steaming and be evaporated to crude drug amount 1:0.9;
4. alcohol precipitation: the dehydrated alcohol adding 3 times in concentrated solution, leaves standstill 24 hours under 4 DEG C of conditions;
5. wash: by the precipitation solution suction filtration 4. obtained, the throw out obtained uses dehydrated alcohol, acetone, washed with diethylether successively;
6. dry: redissolved by the water of throw out by 1 times amount, under the condition of 140 DEG C, spraying dry obtains echinacea purpurea polysaccharide.
Embodiment 2
Extracting method comprises the following steps:
1. medicinal material pre-treatment: echinacea purpurea over-ground part was pulverized 40 mesh sieves; Add sherwood oil, after degreasing, volatilized by sherwood oil under 60 DEG C of conditions, the medicinal material after process is for subsequent use;
2. extract: take the medicinal material 100g 1. processing and obtain, add 1500g water, adjust pH to be 2, after being preheated to 40 DEG C, add medicinal material 4g polygalacturonase, enzymolysis time 50min, 80 DEG C of enzyme 10min that go out, continue to stir insulation 1h at 80 DEG C;
3. concentrate: centrifugal, supernatant liquor is revolved the concentrated solution steaming and be evaporated to crude drug amount 1:0.9;
4. alcohol precipitation: the dehydrated alcohol adding 3 times in concentrated solution, leaves standstill 24 hours under 0 DEG C of condition;
5. wash: by the precipitation solution suction filtration 4. obtained, the throw out obtained uses dehydrated alcohol, acetone, washed with diethylether successively;
6. dry: redissolved by the water of throw out by 1 times amount, under the condition of 140 DEG C, spraying dry obtains echinacea purpurea polysaccharide.
Embodiment 3
Extracting method comprises the following steps:
1. medicinal material pre-treatment: echinacea purpurea over-ground part was pulverized 60 mesh sieves; Add petroleum ether degreasing solvent, after degreasing, volatilized by sherwood oil under 60 DEG C of conditions, the medicinal material after process is for subsequent use;
2. extract: take the medicinal material 100g 1. processing and obtain, add 2500g water, adjust pH to be 4, after being preheated to 60 DEG C, add medicinal material 6g polygalacturonase, enzymolysis time 70min, 100 DEG C of enzyme 20min that go out, continue to stir insulation 2h at 100 DEG C;
3. concentrate: centrifugal, supernatant liquor is revolved the concentrated solution steaming and be evaporated to crude drug amount 1:1.5;
4. alcohol precipitation: the dehydrated alcohol adding 4 times in concentrated solution, leaves standstill 48 hours under 4 DEG C of conditions;
5. wash: by the precipitation solution suction filtration 4. obtained, the throw out obtained uses dehydrated alcohol, acetone, washed with diethylether successively;
6. dry: redissolved by the water of throw out by 2 times amount, under the condition of 160 DEG C, spraying dry obtains echinacea purpurea polysaccharide.
Embodiment 4
Extracting method comprises the following steps:
1. medicinal material pre-treatment: echinacea purpurea over-ground part was pulverized 60 mesh sieves; Add degreasing solvent, after degreasing, volatilized by degreasing solvent under 60 DEG C of conditions, the medicinal material after process is for subsequent use;
2. extract: take the medicinal material 100g 1. processing and obtain, add 1500g water, adjust pH to be 3, after being preheated to 60 DEG C, add medicinal material 5g zymin, enzymolysis time 60min, 80 DEG C of enzyme 10min that go out, continue to stir insulation 1h at 80 DEG C;
3. concentrate: centrifugal, supernatant liquor is revolved the concentrated solution steaming and be evaporated to crude drug amount 1:1;
4. alcohol precipitation: the dehydrated alcohol adding 3 times in concentrated solution, leaves standstill 24 hours under 4 DEG C of conditions;
5. wash: by the precipitation solution suction filtration 4. obtained, the throw out obtained uses dehydrated alcohol, acetone, washed with diethylether successively;
6. dry: redissolved by the water of throw out by 2 times amount, under the condition of 150 DEG C, spraying dry obtains echinacea purpurea polysaccharide.
Wherein, described degreasing solvent is the mixing of sherwood oil, 1,2 epoxy prapane, ether, and its ratio of quality and the number of copies is: 1-2:0.05-0.1:0.02-0.08.
Wherein, zymin is the mixing of 1.19g cellulase, 2.38g polygalacturonase, 1.19g helicase, 0.24g phytase.
Embodiment 5
Extracting method comprises the following steps:
1. medicinal material pre-treatment: echinacea purpurea over-ground part was pulverized 60 mesh sieves; Add degreasing solvent, after degreasing, volatilized by degreasing solvent under 60 DEG C of conditions, the medicinal material after process is for subsequent use;
2. extract: take the medicinal material 100g 1. processing and obtain, add 1500g water, adjust pH to be 3, after being preheated to 60 DEG C, add medicinal material 5g zymin, enzymolysis time 60min, 80 DEG C of enzyme 10min that go out, continue to stir insulation 1h at 80 DEG C;
3. concentrate: centrifugal, supernatant liquor is revolved the concentrated solution steaming and be evaporated to crude drug amount 1:1;
4. alcohol precipitation: the dehydrated alcohol adding 3 times in concentrated solution, leaves standstill 24 hours under 4 DEG C of conditions;
5. wash: by the precipitation solution suction filtration 4. obtained, the throw out obtained uses dehydrated alcohol, acetone, washed with diethylether successively;
6. dry: redissolved by the water of throw out by 2 times amount, under the condition of 150 DEG C, spraying dry obtains echinacea purpurea polysaccharide.
Wherein, degreasing solvent is the mixing of sherwood oil, 1,2 epoxy prapane, ether, and its ratio of quality and the number of copies is: 1:0.05:0.02.
Wherein, zymin is the mixing of 1.52g cellulase, 3.03g polygalacturonase, 0.3g helicase, 0.15g phytase.
Embodiment 6
Extracting method comprises the following steps:
1. medicinal material pre-treatment: echinacea purpurea over-ground part was pulverized 60 mesh sieves; Add degreasing solvent, after degreasing, volatilized by degreasing solvent under 60 DEG C of conditions, the medicinal material after process is for subsequent use;
2. extract: take the medicinal material 100g 1. processing and obtain, add 1500g water, adjust pH to be 3, after being preheated to 60 DEG C, add medicinal material 5g zymin, enzymolysis time 60min, 80 DEG C of enzyme 10min that go out, continue to stir insulation 1h at 80 DEG C;
3. concentrate: centrifugal, supernatant liquor is revolved the concentrated solution steaming and be evaporated to crude drug amount 1:1;
4. alcohol precipitation: the dehydrated alcohol adding 3 times in concentrated solution, leaves standstill 24 hours under 4 DEG C of conditions;
5. wash: by the precipitation solution suction filtration 4. obtained, the throw out obtained uses dehydrated alcohol, acetone, washed with diethylether successively;
6. dry: redissolved by the water of throw out by 2 times amount, under the condition of 150 DEG C, spraying dry obtains echinacea purpurea polysaccharide.
Wherein, described degreasing solvent is the mixing of sherwood oil, 1,2 epoxy prapane, ether, and its ratio of quality and the number of copies is: 2:0.1:0.08.
Wherein, zymin is the mixing of 1.39g cellulase, 2.78g polygalacturonase, 0.7g helicase, 0.13g phytase.
Embodiment 7
Extracting method comprises the following steps:
1. medicinal material pre-treatment: echinacea purpurea over-ground part was pulverized 60 mesh sieves; Add degreasing solvent, after degreasing, volatilized by degreasing solvent under 60 DEG C of conditions, the medicinal material after process is for subsequent use;
2. extract: take the medicinal material 100g 1. processing and obtain, add 1500g water, adjust pH to be 3, after being preheated to 60 DEG C, add 5g zymin, enzymolysis time 60min, 80 DEG C of enzyme 10min that go out, continue to stir insulation 1h at 80 DEG C;
3. concentrate: centrifugal, supernatant liquor is revolved the concentrated solution steaming and be evaporated to crude drug amount 1:1;
4. alcohol precipitation: the dehydrated alcohol adding 3 times in concentrated solution, leaves standstill 24 hours under 4 DEG C of conditions;
5. wash: by the precipitation solution suction filtration 4. obtained, the throw out obtained uses dehydrated alcohol, acetone, washed with diethylether successively;
6. dry: redissolved by the water of throw out by 2 times amount, under the condition of 150 DEG C, spraying dry obtains echinacea purpurea polysaccharide.
Wherein, described degreasing solvent is the mixing of sherwood oil, 1,2 epoxy prapane, ether, and its ratio of quality and the number of copies is: 1.5:0.07:0.05.
Wherein, zymin is the mixing of 1.36g cellulase, 2.95g polygalacturonase, 0.58g helicase, 0.14g phytase.
The Active Components of the echinacea purpurea polysaccharide one, obtained
The treating processes of polysaccharide:
1. pre-treatment: solution polysaccharide being made 5mg/mL, with Sevage method removing protein, with macroporous resin AB-8 cold-maceration decolouring 1d.
2. purifying: adopt DEAE-Cellulose (DE-52) chromatography column, with NaCl solution gradient elution.
3. sample collection: detect each pipe polysaccharide content with Phenol-sulphate acid method, merges peak position according to elution curve, concentrated, dialysis.
4. dry: concentrated by dialyzate, lyophilize obtains the pure component of echinacea purpurea.
1.1 materials and instrument
1.1.1 material and reagent
Material needed for experiment and reagent are shown in
table 1-1.
table 1-1 experiment material and reagent
1.1.2 instrument and equipment
Instrument and equipment needed for experiment is shown in
table 1-2.
table 1-2 laboratory apparatuss and equipment
1.2 experimental technique
1.2.1 polysaccharide molecule flow measurement
This part adopts high performance liquid phase color method to measure the molecular weight of each EPPS I, EPPS II, EPPS III.Chromatographic condition:
Moving phase: 0.1mol/L NaNO
3solution;
Flow velocity: 1.0ml/min;
Pillar: Shodex OHpak 806 and 802 connects;
Detector: differential refraction detector (Optilab rex) and laser detector (DAWN HELEOS-II) coupling (Huai Yate company of the U.S.);
Sample pre-treatments: with moving phase dissolved dilution to 5000 ppm sample introduction.
1.2.2 XRD analysis
EPPS I, EPPS II, EPPS III are pressed on specimen holder and carry out X-ray polycrystalline diffraction.Condition: ceramic X-ray tube, Cu target, voltage rating 40 kV, rated current 50 mA, sweep limit 2 θ 5-60.
1.2.3 infrared analysis
Mix getting after KBr drying with 500 μ g purification of samples EPPS I, EPPS II, EPPS III in right amount, under infrared lamp, in agate mortar, grinding is even extremely without crystal grain, on grease press, 5 min are pressed with 10 × 107 pressure, the thin slice of pressure in infrared spectrometer and the interscan of 400-4000cm-1 scope, spectra re-recorded data and spectrum
figure.
1.2.4 nucleus magnetic resonance
Take polysaccharide fraction 30 mg and be dissolved in 0.5 mL D respectively
2in O, carry out 1H NMR and 13C NMR nmr analysis.
1.3 experimental results and analysis
1.3.1 polysaccharide molecule flow measurement
The molecular weight determination of EPPS I, EPPS II, EPPS III is shown in respectively
fig. 1,2, 3.
By
fig. 1 to 3, the molecular weight of visible EPPS I, EPPS II, EPPS III is respectively 21.5KDa, 12.0KDa, 13.0KDa.The difference of molecular weight may cause active difference, is therefore necessary to study obtained EPPS I, EPPS II, the activity of EPPS III further.
1.3.2XRD analyze
The XRD analysis of EPPS I, EPPS II, EPPS III the results are shown in
fig. 4.
By
fig. 4visible, EPPS I, EPPS II, EPPS III are about 24 have weak diffraction peak at 2 θ, are almost pars amorpha, illustrate that their degree of crystallinity is low, be all in metamict.The diffraction peak of EPPS II more by force, may be quite a lot of compared with EPPS I, its crystallinity of EPPS III.
1.3.3 infrared analysis
The infrared analysis of EPPS I, EPPS II, EPPS III is shown in respectively
fig. 5,6, 7.
By
fig. 5 to 7known, the group of EPPS I, EPPS II, EPPS III is closely similar.
fig. 5middle 3371.9cm-1 is the stretching vibration of O-H, and hydroxyl in molecule is described.2933.27cm-1 is C-H stretching vibration.1152.76,1080.21,1026.52cm-1 tri-peaks are pyranose ring charateristic avsorption band, and being the nonsymmetrical vibration peak of its glycosidic link C-O-C, is the symmetric vibration peak of pyranose ring C-O-C at 669.51cm-1 place.Illustrate that EPPS I is for pyranose.
fig. 6middle 3400.72cm-1 is the stretching vibration of O-H, and hydroxyl in molecule is described.2936.81cm-1 is C-H stretching vibration.
fig. 6middle 3431.66cm-1 is the stretching vibration of O-H, and hydroxyl in molecule is described.2937.81cm-1 is C-H stretching vibration.
1.3.4 nucleus magnetic resonance
1.4 brief summary
This part experiment obtains drawing a conclusion by simple Spectrum Analysis: the molecular weight of (1) EPPS I, EPPS II, EPPS III is respectively 21.5 KDa, 12.0 KDa, 13.0 KDa.(2) degree of crystallinity of EPPS I, EPPS II, EPPS III is all very low, is all in metamict.(3) the infrared structural similitude showing EPPS I, EPPS II, EPPS III, EPPS I characteristic peak is comparatively evident as pyranose.(4) the nuclear-magnetism result of EPPS I demonstrates EPPS I for pyranose, and C-1, C-6 replacement occurs.
The immunocompetence research of the echinacea purpurea polysaccharide two, prepared
This experiment with NK cells in mice and mouse monokaryon scavenger cell RAW 264.7 for object, the immunocompetence of research echinacea purpurea polysaccharide.
2.1 experiment material
2.1.1 material and reagent
Material needed for experiment and reagent are shown in
table 2-1.
table 2-1 experiment material and reagent
| Title | Producer | Specification |
| Mouse | Kunming small white mouse | Male |
| RPMI-1640 | Gibco | Biological reagent |
| DMEM | Gibco | Biological reagent |
| Foetal calf serum FBS | Gibco | Biological reagent |
| P/S | HyClone company of the U.S. | Biological reagent |
| TE | Shanghai Yan Yu commerce and trade company limited | Biological reagent |
| Tetramethyl-azo azoles salt (MTT) | Sigma Co., USA | Biological reagent |
| Dimethyl sulfoxide (DMSO) (DMSO) | Chemical Reagent Co., Ltd., Sinopharm Group | Analytical pure |
| Tris | Sigma Co., USA | Biological reagent |
| Ammonium chloride | Chemical Reagent Co., Ltd., Sinopharm Group | Analytical pure |
| Trypan blue | Sigma Co., USA | Biological reagent |
2.1.2 instrument and equipment
Instrument and equipment needed for experiment is shown in
table 2-2.
table 2-2 laboratory apparatuss and equipment
| Title | Producer | Model |
| CO2 incubator | The rich news in Shanghai | BC-J80S |
| Vertical pressure Sterilizers | The rich news in Shanghai | YXQ-LS-30SII |
| Inverted microscope | Olympus | CKX41 |
| Super clean bench | Beijing Ha Donglian | DL-CJ-1NDII |
| 96 porocyte culture plates | Costar | 3599 |
| 6 porocyte culture plates | Costar | 3516 |
| Whizzer | Town in Shanghai booth | TDL-40C |
| Whizzer | Eppendorf | 5415R |
| Micropore sample injector | Eppendorf | All size between 2.5 μ l-1ml |
| Microwell plate fast oscillator | The east of a river, Suzhou precision instrument company limited | QB-9001 |
| Microplate reader | Thermo Multiskan | MK3 |
2.2 experimental technique
2.2.1 NK cytoactive is affected
NK cell is nature
kill and woundcell, is the important immunocyte of body, has non-specific
kill and woundeffect, plays an important role in immunity system.Not only with antitumor, anti-virus infection is relevant with immunomodulatory, and participates in the generation of allergy and autoimmune disorder in some cases.K562 is leukemia cell, is in highly non-differential period, and from the hydrothorax of the patient of leukemia acute transformation phase, separation is set up.K562 is the external target of the Sensitive altitude sensitivity of NK cell.Detection NK cell
kill and woundactive warp is commonly used it and is done target cell.
MTT colorimetry is a kind of method detecting cell survival and growth.Its Cleaning Principle is that the succinodehydrogenase in viable cell plastosome can make exogenous MTT be reduced to water-insoluble bluish voilet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) (DMSO) energy dissolved cell, measures its absorbance value by microplate reader at 570nm, can indirectly reflect viable cell quantity.
The mouse spleen lower differentiation that can promote NK cell of IL-3 induction in vivo.This part experiment, to be separated NK cell for effector cell from mouse spleen, with K562 cell for target cell, adopts MTT colorimetric determination cell count to determine that NK cell is to target cell K562's
kill and woundrate.
2.2.1.1 the preparation of mouse boosting cell suspension
De-neck puts to death mouse, and 75% alcohol-pickled sterilization 2-3 min, get spleen in super clean bench, rinse 1 time with PBS, then grind in 150 eye mesh screens, grinding limit, limit adds the RPMI1640 nutrient solution of 20%FBS, is screened in 50 mL centrifuge tubes.800 rpm, centrifugal 5 min, abandon supernatant, add Tris-NH4Cl damping fluid 5 mL, and piping and druming evenly, leaves standstill 3-4 min.Centrifugal 5 min of 1000 rpm, abandon supernatant, and the PBS containing 1%P/S with 5 mL washes 2 times, and wash 1 time with 5mlRPMI1640, Trypan Blue, counting, abandon supernatant, with RPMI1640 complete culture solution adjustment cell concn to 5 × 10 of 10%FBS, 1%P/S
6/ mL, stand-by.
2.2.1.2 the preparation of target cell
K562 cell is put in centrifuge tube, 1200 rpm/min, centrifugal 3 min, and being adjusted to concentration with the RPMI1640 complete culture solution of 10%FBS, 1%P/S is 5 × 10
5the cell suspension of/ml.
2.2.1.3 the mensuration of NK cytoactive
Mouse boosting cell suspension is added in 96 orifice plates, every hole 50 μ L, then add the RPMI1640 complete culture solution containing EPPS, EPPS I, EPPS II, EPPS III 50,100,250 μ g/ml successively, respectively do 4 multiple holes.In 37 DEG C, 5%CO
2after 12 h cultivated by incubator, add K562 cell suspension 50 μ l, establish blank group, target cell control group and effector cell's control group simultaneously.Supernatant is abandoned after cultivating 24 h again, every hole adds the MTT 50 μ L of 0.5 mg/mL, after continuing cultivation 4 h, every hole adds DMSO150 μ L, after cultivating 1 h, jolting 5 min on microwell plate fast oscillator, makes the crystallization of bluish voilet first a ceremonial jade-ladle, used in libation fully dissolve, measures the absorbance of each hole at 570 nm places by microplate reader.Result is with NK cell
kill and woundrate represents, NK cell
kill and woundrate is pressed formula (5-1) and is calculated:
NK cytoactive (%)=[1-(experimental group OD
570nm-effector cell control group OD
570nm)/target cell control group OD value] X100% (5-1)
2.2.2 RAW264.7 cytoactive is affected
RAW 264.7 is mouse monokaryon scavenger cells is that monocytes/macrophages has many-sided biological function, mainly may be summarized to be the following aspects: 1. nonspecific immunity defence.After foreign pathogen enters body, just can be removed by monokaryon-macrophage phagocytic before exciting immunne response, but minority pathogenic agent can be bred in its born of the same parents.2. foreign cell is removed.3. nonspecific immunity monitors.4. present antigen.Namely after exotic antigen enters body, first engulfed by monocytes/macrophages, digest, by effective antigenic determinant and mhc class ii molecular juction synthesis complex body, this species complex by T cell identification, thus excites immunne response.5. medium IL-1, Interferon, rabbit, complement (C1, C4, C2, C3, C, Factor B) etc. are secreted.
This part experiment for research object, inquires into echinacea purpurea polysaccharide ion vitro immunization regulating and controlling effect to scavenger cell by the secretion level detecting the proliferation activity of cell, NO emission levels and the cellular immunization factor with RAW 264.7.
2.2.2.1 on the impact of RAW264.7 cell proliferation
The RAW264.7 cell recovery bought, cultivates in 50ml culturing bottle, when waiting bottle floor cells density to reach about 80%, discard original fluid, with 4 mL containing the DMEM nutrient solution rinsing one time of FBS containing 1%P/S, discard washing lotion, add 2mL pancreatin TE and digest 3-4 min, see cell rounding under the microscope, gap adds 5 mL DMEM complete culture solutions and stops digestion when increasing, blown down by cell, move in centrifuge tube with suction pipe, 1200 rpm, centrifugal 3 min.Add DMEM complete culture solution, Trypan Blue, counting, is adjusted to the cell suspension that concentration is 2 × 105/ml.Be laid on by above cell suspension in 96 orifice plates, every hole 100 μ L, in 37 DEG C, 5%CO
212 h cultivated by incubator, and after cell attachment, supernatant discarded, adds EPPS, EPPS I of 50,100,250,500 μ g/mL, EPPS II and EPPS III respectively, and blank group is DMEM complete culture solution, and often group establishes 8 repetitions.In 37 DEG C, 5%CO
2continue in incubator to cultivate 24h, collect supernatant liquor (mensuration for the later stage) in sterile centrifugation tube, every hole adds the MTT of 50 μ L0.5mg/mL, after continuing to cultivate 4h, every hole adds DMSO150 μ L, after cultivating 1 h, and jolting 5 min on microwell plate fast oscillator, the crystallization of bluish voilet first a ceremonial jade-ladle, used in libation is fully dissolved, measures the absorbance of each hole at 570 nm places by microplate reader.The multiplication capacity of RAW264.7 represents with value added index, calculates by formula (5-2):
Proliferating antigen Ki67=(experimental group OD
570nm/ blank group OD
570nm) X100% (5-2)
2.2.2.2 on the impact of RAW264.7 emiocytosis NO
By method under 2.2.2.1 item, after cell conditioned medium is received sterile centrifugation tube, measure the content of NO by NO test kit specification sheets.
2.2.2.3 on the impact of RAW264.7 cell secretion of cytokines
By method under 2.2.2.1 item, after cell conditioned medium is received sterile centrifugation tube, measure the content of IFN-γ by ELISA kit specification sheets.
2.2.2.4 on the impact of RAW264.7 cell mode of appearance
By method process cell under 2.2.2.1 item, observe different concns EPPS, EPPS I, EPPS II and EPPS III to the impact of RAW264.7 mode of appearance.
2.3 experimental result and analysis
2.3.1 NK cytoactive is affected
Different concns echinacea purpurea polysaccharide (Echinacea Purpurea Polysaccharide, EPPS) and purifying obtained component EPPS I, EPPS II, the impact of EPPS III on isolated mouse NK cytoactive are shown in
table 2-3.
table 2-3 different concns EPPS, EPPS I, EPPS II and EPPS III are on the impact of isolated mouse NK cytoactive
By
table 2-3 results show, EPPS, EPPS I, EPPS II, EPPS III pair of NK cells in mice activity have promoter action, and have certain dose-effect relationship.EPPS, EPPS III is with the rising of concentration within the scope of 50-250 μ g/mL, and promoter action reduces; EPPS I, EPPS II are with the rising of concentration within the scope of 50-250 μ g/mL, and promoter action increases.
2.3.2 to RAW264.7 activity influence
2.3.2.1 on the impact of RAW264.7 cell proliferation
Different concns echinacea purpurea polysaccharide (Echinacea Purpurea Polysaccharide, EPPS) and purifying obtained component EPPS I, EPPS II, the impact of EPPS III on RAW264.7 cell proliferation are shown in
figure 10.
figure 10result shows, EPPS, EPPS I, EPPS II, EPPS III pair of RAW264.7 cytoactive have promoter action.EPPS promotes that the ability of propagation first increases within the scope of concentration 50-500 μ g/mL and reduces afterwards, during 250 μ g/mL, promoter action is maximum, EPPS I, EPPS II promote that the ability of propagation is the trend of attenuating within the scope of concentration 50-500 μ g/mL, the ability of EPPS III promotion propagation first increases and reduces afterwards within the scope of concentration 50-500 μ g/mL, and during 100 μ g/mL, promoter action is maximum.
2.3.2.2 on the impact of RAW264.7 emiocytosis NO
EPPS, EPPS I, EPPS II and EPPS III impact on RAW264.7 emiocytosis NO is shown in
table 2-5.
table 2-5 different concns EPPS, EPPS I, EPPS II and EPPS III are on the impact of RAW264.7 emiocytosis NO
By
figure 11visible, EPPS, EPPS I, EPPS II and EPPS III can promote that RAW264.7 is to the release of NO, and in dose-effect relationship within the scope of 50-250 μ g/mL.
2.3.2.3 on the impact of RAW264.7 cell secretion of cytokines
EPPS, EPPS I, EPPS II and EPPS III impact on RAW264.7 emiocytosis IFN-γ is shown in respectively
table 2-6.
table 2-6 different concns EPPS, EPPS I, EPPS II and EPPS III are on the impact of RAW264.7 emiocytosis IFN-γ
By
figure 11visible, EPPS, EPPS I, EPPS II and EPPS III can promote that RAW264.7 is to the release of IFN-γ, and in dose-effect relationship within the scope of 50-250 μ g/ml.
2.2.2.4 on the impact of RAW264.7 cell mode of appearance
Different concns EPPS, EPPS I, EPPS II and the impact of EPPS III on RAW264.7 mode of appearance are shown in
figure 11.
By
figure 11visible, the cell of space management is rounded, and the cell of EPPS, EPPS I, EPPS II and EPPS III process has elongated trend, the not too rule that volume becomes large, becomes.The impact of EPPS, EPPS I is comparatively obvious.
2.4 brief summary
This experiment shows EPPS, EPPS I, EPPS II, EPPS III pair of NK cells in mice activity has promoter action, and have certain dose-effect relationship.EPPS, EPPS I, EPPS II, EPPS III pair of RAW264.7 cell proliferation have obvious promoter action, can increase the secretory volume of RAW264.7 cell to NO, IFN-γ simultaneously.
Simultaneous test one
According to hydrolysis temperature, pH, enzyme dosage, enzymolysis time, the extraction yield of echinacea purpurea polysaccharide is affected.Design orthogonal test.Test result analysis is shown in
table 1with
table 2.
table 1the test design level of factor table of echinacea purpurea polysaccharide Enzymatic Extraction
| Level | Hydrolysis temperature (DEG C) | pH | Enzyme dosage (%) | Enzymolysis time (min) |
| Levels | Enzymolysis temperature（℃） | pH | Enzymy addition（%） | Enzymolysis time（min） |
| 1 | 30 | 2 | 3 | 30 |
| 2 | 40 | 3 | 4 | 45 |
| 3 | 50 | 4 | 5 | 60 |
| 4 | 60 | 5 | 6 | 120 |
| 5 | 70 | 6 | 7 | 180 |
table 2the orthogonal experiments of echinacea purpurea polysaccharide Enzymatic Extraction and analysis
| Level | Hydrolysis temperature (DEG C) | pH | Enzyme dosage (%) | Enzymolysis time (min) | Extraction yield |
| Number | Enzymolysis temperature（℃） | pH | Enzymy addition（%） | Enzymolysis time（min） | Extraction yield(%) |
| 1 | 1 | 1 | 1 | 1 | 8.8 |
| 2 | 1 | 2 | 2 | 2 | 9.19 |
| 3 | 1 | 3 | 3 | 3 | 9.81 |
| 4 | 2 | 1 | 2 | 3 | 10.42 |
| 5 | 2 | 2 | 3 | 1 | 10.38 |
| 6 | 2 | 3 | 1 | 2 | 9.36 |
| 7 | 3 | 1 | 3 | 2 | 9.3 |
| 8 | 3 | 2 | 1 | 3 | 9.81 |
| 9 | 3 | 3 | 2 | 1 | 9.98 |
| k 1 | 9.267 | 9.507 | 9.323 | 9.720 | |
| k 2 | 10.053 | 9.793 | 9.863 | 9.283 | |
| k 3 | 9.697 | 9.717 | 9.830 | 10.013 | |
| R | 0.786 | 0.286 | 0.540 | 0.730 |
According to test-results, known in echinacea purpurea polysaccharide enzyme process leaching process, the impact order of 4 factors on extraction rate is A hydrolysis temperature > D enzymolysis time > C enzyme dosage > B pH.According to analysis, namely hydrolysis temperature is 50 DEG C, and pH is 3.0, and enzyme dosage is 5%, and enzymolysis time is 60 min.
Simultaneous test two
The echinacea purpurea polysaccharide extract rate of comparative example 1-7.
table 3
| Group | Echinacea purpurea polysaccharide extract rate/% |
| Embodiment 1 | 10.42% |
| Embodiment 2 | 10.23% |
| Embodiment 3 | 10.35% |
| Embodiment 4 | 10.41% |
| Embodiment 5 | 10.42% |
| Embodiment 6 | 10.438% |
| Embodiment 7 | 10.421% |
By
table 3known, the leaching process of the echinacea purpurea polysaccharide of seven embodiment groups is obtained for higher extraction yield.
To sum up, the present invention extracts echinacea purpurea polysaccharide, while shortening extraction time, substantially increases the extraction yield of echinacea purpurea polysaccharide, and obtains the good echinacea purpurea polysaccharide of solvability, have good activeconstituents.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. a method for enzyme extraction echinacea purpurea polysaccharide, is characterized in that, extracting method comprises the following steps:
1. medicinal material pre-treatment: echinacea purpurea over-ground part was pulverized 40-60 mesh sieve; Add degreasing solvent, after degreasing, volatilized by degreasing solvent under 20-80 DEG C of condition, the medicinal material after process is for subsequent use;
2. extract: taking step, 1. to process the medicinal material obtained appropriate, add the water of medicinal material 15-25 times amount, tune pH is 2-4, after being preheated to 40 DEG C-60 DEG C, add the zymin of medicinal material 0.04-0.06 times amount, enzymolysis time 50min-70min, 80 DEG C-100 DEG C enzyme 10-20min that go out, continue to stir insulation 1-2h at 80 DEG C-100 DEG C;
3. concentrate: centrifugal, supernatant liquor is revolved the concentrated solution steaming and be evaporated to crude drug amount 1:0.9-1.5;
4. alcohol precipitation: add 3-4 dehydrated alcohol doubly in concentrated solution, leave standstill 24-48 hour under 0-4 DEG C of condition;
5. wash: precipitation solution suction filtration step 4. obtained, the throw out obtained uses dehydrated alcohol, acetone, washed with diethylether successively;
6. dry: redissolved by the water of throw out by 1-2 times amount, under the condition of 140 DEG C-160 DEG C, spraying dry obtains echinacea purpurea polysaccharide.
2. the method for enzyme extraction echinacea purpurea polysaccharide according to claim 1, is characterized in that, described degreasing solvent is sherwood oil.
3. the method for enzyme extraction echinacea purpurea polysaccharide according to claim 1 and 2, is characterized in that, described degreasing solvent is the mixing of sherwood oil, 1,2 epoxy prapane, ether, and its ratio of quality and the number of copies is: 1-2:0.05-0.1:0.02-0.08.
4. the method for the enzyme extraction echinacea purpurea polysaccharide according to any one of claims 1 to 3, is characterized in that, described zymin is polygalacturonase.
5. the method for the enzyme extraction echinacea purpurea polysaccharide according to any one of Claims 1-4, it is characterized in that, described zymin is the mixing of cellulase, polygalacturonase, helicase, phytase, and its ratio of quality and the number of copies is: 0.5-1:1-2:0.1-0.5:0.05-0.1.
6. the method for the enzyme extraction echinacea purpurea polysaccharide according to any one of claim 1 to 5, is characterized in that, described echinacea purpurea crude drug source, in genus echinacea plant echinacea purpurea, takes from full-bloom stage to the over-ground part spending the later stage.
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|---|---|---|---|---|
| CN106389788A (en) * | 2016-11-14 | 2017-02-15 | 青岛农业大学 | Medicine extract for strengthening anti-virus capability and immune function of livestock, preparation, and preparation method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102101892A (en) * | 2009-12-16 | 2011-06-22 | 青岛康地恩药业有限公司 | Method for extracting polysaccharide from echinacea |
| CN102716164A (en) * | 2012-06-28 | 2012-10-10 | 齐鲁动物保健品有限公司 | Echinacea extract and preparation method of echinacea extract |
| CN104127463A (en) * | 2014-07-01 | 2014-11-05 | 青岛蔚蓝生物股份有限公司 | Echinacea extract product, preparation method and application thereof |
-
2015
- 2015-07-13 CN CN201510409628.1A patent/CN105002230B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102101892A (en) * | 2009-12-16 | 2011-06-22 | 青岛康地恩药业有限公司 | Method for extracting polysaccharide from echinacea |
| CN102716164A (en) * | 2012-06-28 | 2012-10-10 | 齐鲁动物保健品有限公司 | Echinacea extract and preparation method of echinacea extract |
| CN104127463A (en) * | 2014-07-01 | 2014-11-05 | 青岛蔚蓝生物股份有限公司 | Echinacea extract product, preparation method and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| ROSARIA COZZOLINO等: "Structural analysis of the polysaccharides from Echinacea angustifolia radix", 《CARBOHYDRATE POLYMERS》 * |
| 刘汉珍等: "酶法提取滁菊多糖的工艺优化", 《中药材》 * |
| 熊先锋等: "超声波酶法提取水溶性灵芝多糖的研究", 《山东轻工业学院学报》 * |
| 熊磊等: "纤维素酶解滁菊提取多糖工艺及滁菊多糖抗氧化性研究", 《皖南医学院学报》 * |
| 陈练等: "添加植酸酶对米糠消化酶水解产物化学组成及理化性质的影响", 《江苏农业科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106389788A (en) * | 2016-11-14 | 2017-02-15 | 青岛农业大学 | Medicine extract for strengthening anti-virus capability and immune function of livestock, preparation, and preparation method |
| CN106389788B (en) * | 2016-11-14 | 2019-09-13 | 青岛农业大学 | It is a kind of for enhancing the drug extract, preparation and preparation method of immunologic function of livestock and birds and anti-virus ability |
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