CN105002230B - A kind of method of enzyme extraction Echinacea polysaccharide - Google Patents

A kind of method of enzyme extraction Echinacea polysaccharide Download PDF

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CN105002230B
CN105002230B CN201510409628.1A CN201510409628A CN105002230B CN 105002230 B CN105002230 B CN 105002230B CN 201510409628 A CN201510409628 A CN 201510409628A CN 105002230 B CN105002230 B CN 105002230B
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epps
echinacea
enzyme
medicinal material
polysaccharide
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CN105002230A (en
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郝智慧
杨芬芳
黄婷婷
赵莉
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Qingdao Agricultural University
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Abstract

The invention discloses a kind of methods of enzyme extraction Echinacea polysaccharide, Echinacea aerial part is ground into suitable for size, degreasing, extraction, enzyme preparation enzymolysis, enzyme deactivation continue high temperature extraction, centrifugation, supernatant progress rotary evaporation concentration, alcohol precipitation, precipitation is washed successively with absolute ethyl alcohol, acetone, ether, after redissolution, spray drying obtains Echinacea polysaccharide.The beneficial effects of the invention are as follows：This preparation method has many advantages, such as that reaction condition is mild, extraction time is short, recovery rate is high, and reproducibility is high, stablize, the Echinacea polysaccharide dissolubility being prepared is preferable, and appearance is uniform, good water solubility, and preferable active component is obtained, there is preferable immunocompetence.

Description

A kind of method of enzyme extraction Echinacea polysaccharide
Technical field
The present invention relates to Polyose extraction field, more particularly to a kind of method of enzyme extraction Echinacea polysaccharide.
Background technology
Echinacea (Echinacea) is world-famous immune medicinal herbs, and original producton location is located at North America and southern Canada.It is A kind of Echinacea plant, the platymiscium share 8 kinds and several mutation, mainly have 3 kinds currently as drug development: Purple coneflower (Echinacea purpurea, i.e., usually said Echinacea), narrow leaf Echinacea (Echinacea ) and white Echinacea (Echinacen pallida) augustifolia.Research shows that Echinacea contains a variety of active ingredients, Caffeic acid derivative class, polysaccharide and glycoprotein, alkyl amide, alkaloids, polyacetylene, flavonoids, volatile oil, sterol etc., Active constituent has become current research hot spot.
Echinacea is referred to as " natural antibiotics " because having significant immune-enhancing activity.It is produced in mankind's plurality kinds of health care There is application in product and compound product.And other active ingredients in opposite Echinacea, the research of Echinacea polysaccharide is relatively fewer, grinds It is complicated to study carefully active constituent extraction process based on more Cichoric acid, the active constituent products material amount obtained is few and cost Height, although being extracted from medicinal material to active constituent to it about the report of the immunocompetence research of Echinacea extraction polysaccharide System further investigation especially enzymatic treatment extraction polysaccharide system research be not reported so far, purification process, which also rarely has, to be ground Study carefully.
Invention content
In order to solve the problem of that Echinacea Polyose extraction overlong time and recovery rate are low, while can also preferably be lived Property ingredient, the present invention provides a kind of enzyme extraction Echinacea polysaccharide method.
In order to achieve the above object, the present invention provides a kind of method of enzyme extraction Echinacea polysaccharide, wherein, extracting method Include the following steps：
1. medicinal material pre-processes：Echinacea aerial part be crushed into 40-60 mesh sieve；Degreasing solvent is added in, it, will be de- after degreasing Fatsolvent volatilizes under the conditions of 20-80 DEG C, and treated, and medicinal material is spare；
2. it extracts：The water that the medicinal material 1. handled is appropriate, and 15-25 times of medicinal material of addition is measured is weighed, tune pH is 2-4, is preheated To after 40 DEG C -60 DEG C, the enzyme preparation that 0.04-0.06 times of medicinal material is measured is added in, enzymolysis time 50min-70min, 80 DEG C -100 DEG C go out Enzyme 10-20min continues stirring heat preservation 1-2h at 80 DEG C -100 DEG C；
3. it concentrates：Supernatant revolving is concentrated under reduced pressure into crude drug amount 1 by centrifugation：The concentrate of 0.9-1.5；
4. alcohol precipitation：3-4 times of absolute ethyl alcohol is added in into concentrate, 24-48 hours are stood under the conditions of 0-4 DEG C；
5. it washs：4. the precipitation solution obtained is filtered, obtained sediment is washed successively with absolute ethyl alcohol, acetone, ether；
6. it dries：Sediment with the 1-2 times of water measured is redissolved, is spray-dried under conditions of 140 DEG C -160 DEG C and obtains purple Bore chrysanthemum polysaccharide.
Wherein, the degreasing solvent is petroleum ether.
Wherein, the degreasing solvent is the mixing of petroleum ether, 1,2- propylene oxide, ether, and quality parts ratio is：1-2: 0.05-0.1:0.02-0.08。
Wherein, the enzyme preparation is pectase.
Wherein, the enzyme preparation is the mixing of cellulase, pectase, glusulase, phytase, and quality parts ratio is： 0.5-1:1-2:0.1-0.5：0.05-0.1.
Wherein, the Echinacea crude drug source is derived from full-bloom stage to the ground for spending the later stage in genus echinacea plant Echinacea Part.
The beneficial effects of the invention are as follows：This preparation method has reaction condition is mild, extraction time is short, recovery rate is high etc. Advantage, and reproducibility is high, stablizes, and the Echinacea polysaccharide dissolubility being prepared is preferable, and appearance is uniform, good water solubility, and Preferable active component is obtained, there is preferable immunocompetence.
Description of the drawings
I molecular weight determination spectrograms of Fig. 1 EPPS；
II molecular weight determination spectrograms of Fig. 2 EPPS；
III molecular weight determination spectrograms of Fig. 3 EPPS；
Fig. 4 EPPS I, EPPS II, III XRD diagram of EPPS；
I infrared spectrums of Fig. 5 EPPS；
II infrared spectrums of Fig. 6 EPPS；
III infrared spectrums of Fig. 7 EPPS；
The 1H NMR spectras of Fig. 8 EPPS I；
The 13C NMR spectras of Fig. 9 EPPS I；
Figure 10 various concentrations EPPS, EPPS I, the influence of EPPS II and EPPS III to RAW264.7 cell Proliferations；
Figure 11 various concentrations EPPS, EPPS I, the influence of EPPS II and EPPS III to RAW264.7 mode of appearance.
Specific embodiment
Embodiment 1
Extracting method includes the following steps：
1. medicinal material pre-processes：Echinacea aerial part be crushed into 60 mesh sieve；Degreasing solvent is added in, after degreasing, by oil Ether volatilizes under the conditions of 60 DEG C, and treated, and medicinal material is spare；
2. it extracts：The medicinal material 100g 1. handled is weighed, adds in 1500g water, it is 3 to adjust pH, after being preheated to 50 DEG C, is added in Medicinal material 5g pectases, enzymolysis time 60min, 80 DEG C of enzyme deactivation 10min continue stirring heat preservation 1-h at 80 DEG C；
3. it concentrates：Supernatant revolving is concentrated under reduced pressure into crude drug amount 1 by centrifugation：0.9 concentrate；
4. alcohol precipitation：3 times of absolute ethyl alcohol is added in into concentrate, 24 hours are stood under the conditions of 4 DEG C；
5. it washs：4. the precipitation solution obtained is filtered, obtained sediment is washed successively with absolute ethyl alcohol, acetone, ether；
6. it dries：The water of 1 times of amount of sediment is redissolved, is spray-dried under conditions of 140 DEG C and obtains Echinacea polysaccharide.
Embodiment 2
Extracting method includes the following steps：
1. medicinal material pre-processes：Echinacea aerial part be crushed into 40 mesh sieve；Petroleum ether is added in, after degreasing, by petroleum ether It is volatilized under the conditions of 60 DEG C, treated, and medicinal material is spare；
2. it extracts：The medicinal material 100g 1. handled is weighed, adds in 1500g water, it is 2 to adjust pH, after being preheated to 40 DEG C, is added in Medicinal material 4g pectases, enzymolysis time 50min, 80 DEG C of enzyme deactivation 10min continue stirring heat preservation 1h at 80 DEG C；
3. it concentrates：Supernatant revolving is concentrated under reduced pressure into crude drug amount 1 by centrifugation：0.9 concentrate；
4. alcohol precipitation：3 times of absolute ethyl alcohol is added in into concentrate, 24 hours are stood under the conditions of 0 DEG C；
5. it washs：4. the precipitation solution obtained is filtered, obtained sediment is washed successively with absolute ethyl alcohol, acetone, ether；
6. it dries：The water of 1 times of amount of sediment is redissolved, is spray-dried under conditions of 140 DEG C and obtains Echinacea polysaccharide.
Embodiment 3
Extracting method includes the following steps：
1. medicinal material pre-processes：Echinacea aerial part be crushed into 60 mesh sieve；Petroleum ether degreasing solvent is added in, after degreasing, Petroleum ether is volatilized under the conditions of 60 DEG C, treated, and medicinal material is spare；
2. it extracts：The medicinal material 100g 1. handled is weighed, adds in 2500g water, it is 4 to adjust pH, after being preheated to 60 DEG C, is added in Medicinal material 6g pectases, enzymolysis time 70min, 100 DEG C of enzyme deactivation 20min continue stirring heat preservation 2h at 100 DEG C；
3. it concentrates：Supernatant revolving is concentrated under reduced pressure into crude drug amount 1 by centrifugation：1.5 concentrate；
4. alcohol precipitation：4 times of absolute ethyl alcohol is added in into concentrate, 48 hours are stood under the conditions of 4 DEG C；
5. it washs：4. the precipitation solution obtained is filtered, obtained sediment is washed successively with absolute ethyl alcohol, acetone, ether；
6. it dries：The water of 2 times of amounts of sediment is redissolved, is spray-dried under conditions of 160 DEG C and obtains Echinacea polysaccharide.
Embodiment 4
Extracting method includes the following steps：
1. medicinal material pre-processes：Echinacea aerial part be crushed into 60 mesh sieve；Degreasing solvent is added in, after degreasing, by degreasing Solvent volatilizes under the conditions of 60 DEG C, and treated, and medicinal material is spare；
2. it extracts：The medicinal material 100g 1. handled is weighed, adds in 1500g water, it is 3 to adjust pH, after being preheated to 60 DEG C, is added in Medicinal material 5g enzyme preparations, enzymolysis time 60min, 80 DEG C of enzyme deactivation 10min continue stirring heat preservation 1h at 80 DEG C；
3. it concentrates：Supernatant revolving is concentrated under reduced pressure into crude drug amount 1 by centrifugation：1 concentrate；
4. alcohol precipitation：3 times of absolute ethyl alcohol is added in into concentrate, 24 hours are stood under the conditions of 4 DEG C；
5. it washs：4. the precipitation solution obtained is filtered, obtained sediment is washed successively with absolute ethyl alcohol, acetone, ether；
6. it dries：The water of 2 times of amounts of sediment is redissolved, is spray-dried under conditions of 150 DEG C and obtains Echinacea polysaccharide.
Wherein, the degreasing solvent is the mixing of petroleum ether, 1,2- propylene oxide, ether, and quality parts ratio is：1-2: 0.05-0.1:0.02-0.08。
Wherein, enzyme preparation is the mixing of 1.19g cellulases, 2.38g pectases, 1.19g glusulases, 0.24g phytases.
Embodiment 5
Extracting method includes the following steps：
1. medicinal material pre-processes：Echinacea aerial part be crushed into 60 mesh sieve；Degreasing solvent is added in, after degreasing, by degreasing Solvent volatilizes under the conditions of 60 DEG C, and treated, and medicinal material is spare；
2. it extracts：The medicinal material 100g 1. handled is weighed, adds in 1500g water, it is 3 to adjust pH, after being preheated to 60 DEG C, is added in Medicinal material 5g enzyme preparations, enzymolysis time 60min, 80 DEG C of enzyme deactivation 10min continue stirring heat preservation 1h at 80 DEG C；
3. it concentrates：Supernatant revolving is concentrated under reduced pressure into crude drug amount 1 by centrifugation：1 concentrate；
4. alcohol precipitation：3 times of absolute ethyl alcohol is added in into concentrate, 24 hours are stood under the conditions of 4 DEG C；
5. it washs：4. the precipitation solution obtained is filtered, obtained sediment is washed successively with absolute ethyl alcohol, acetone, ether；
6. it dries：The water of 2 times of amounts of sediment is redissolved, is spray-dried under conditions of 150 DEG C and obtains Echinacea polysaccharide.
Wherein, degreasing solvent is the mixing of petroleum ether, 1,2- propylene oxide, ether, and quality parts ratio is：1:0.05: 0.02。
Wherein, enzyme preparation is the mixing of 1.52g cellulases, 3.03g pectases, 0.3g glusulases, 0.15g phytases.
Embodiment 6
Extracting method includes the following steps：
1. medicinal material pre-processes：Echinacea aerial part be crushed into 60 mesh sieve；Degreasing solvent is added in, after degreasing, by degreasing Solvent volatilizes under the conditions of 60 DEG C, and treated, and medicinal material is spare；
2. it extracts：The medicinal material 100g 1. handled is weighed, adds in 1500g water, it is 3 to adjust pH, after being preheated to 60 DEG C, is added in Medicinal material 5g enzyme preparations, enzymolysis time 60min, 80 DEG C of enzyme deactivation 10min continue stirring heat preservation 1h at 80 DEG C；
3. it concentrates：Supernatant revolving is concentrated under reduced pressure into crude drug amount 1 by centrifugation：1 concentrate；
4. alcohol precipitation：3 times of absolute ethyl alcohol is added in into concentrate, 24 hours are stood under the conditions of 4 DEG C；
5. it washs：4. the precipitation solution obtained is filtered, obtained sediment is washed successively with absolute ethyl alcohol, acetone, ether；
6. it dries：The water of 2 times of amounts of sediment is redissolved, is spray-dried under conditions of 150 DEG C and obtains Echinacea polysaccharide.
Wherein, the degreasing solvent is the mixing of petroleum ether, 1,2- propylene oxide, ether, and quality parts ratio is：2: 0.1:0.08。
Wherein, enzyme preparation is the mixing of 1.39g cellulases, 2.78g pectases, 0.7g glusulases, 0.13g phytases.
Embodiment 7
Extracting method includes the following steps：
1. medicinal material pre-processes：Echinacea aerial part be crushed into 60 mesh sieve；Degreasing solvent is added in, after degreasing, by degreasing Solvent volatilizes under the conditions of 60 DEG C, and treated, and medicinal material is spare；
2. it extracts：The medicinal material 100g 1. handled is weighed, adds in 1500g water, it is 3 to adjust pH, after being preheated to 60 DEG C, is added in 5g enzyme preparations, enzymolysis time 60min, 80 DEG C of enzyme deactivation 10min continue stirring heat preservation 1h at 80 DEG C；
3. it concentrates：Supernatant revolving is concentrated under reduced pressure into crude drug amount 1 by centrifugation：1 concentrate；
4. alcohol precipitation：3 times of absolute ethyl alcohol is added in into concentrate, 24 hours are stood under the conditions of 4 DEG C；
5. it washs：4. the precipitation solution obtained is filtered, obtained sediment is washed successively with absolute ethyl alcohol, acetone, ether；
6. it dries：The water of 2 times of amounts of sediment is redissolved, is spray-dried under conditions of 150 DEG C and obtains Echinacea polysaccharide.
Wherein, the degreasing solvent is the mixing of petroleum ether, 1,2- propylene oxide, ether, and quality parts ratio is：1.5: 0.07:0.05。
Wherein, enzyme preparation is the mixing of 1.36g cellulases, 2.95g pectases, 0.58g glusulases, 0.14g phytases.
First, the Active Components of Echinacea polysaccharide obtained
The processing procedure of polysaccharide：
1. it pre-processes：Polysaccharide is made to the solution of 5mg/mL, with Sevage method removing proteins, with macroreticular resin AB-8 cold-macerations Decolourize 1d.
2. it purifies：Using DEAE-Cellulose (DE-52) chromatographic column, with NaCl solution gradient elution.
3. sample collection：Each pipe polyoses content is detected with Phenol-sulphate acid method, merges peak position according to elution curve, concentrates, thoroughly Analysis.
4. it dries:Dialyzate is concentrated, freeze-drying obtains the pure component of Echinacea.
1.1 materials and instrument
1.1.1 material and reagent
Material needed for experiment is shown in Table 1-1 with reagent.
Table 1-1 experiment materials and reagent
1.1.2 instrument and equipment
Instrument and equipment needed for experiment is shown in Table 1-2.
Table 1-2 laboratory apparatus and equipment
1.2 experimental method
1.2.1 polysaccharide molecular weight measures
This part measures the molecular weight of each EPPS I, EPPS II, EPPS III using efficient liquid phase color method.Chromatographic condition：
Mobile phase：0.1mol/L NaNO3Solution；
Flow velocity：1.0ml/min；
Pillar：Shodex OHpak 806 and 802 connect；
Detector：Differential refraction detector (Optilab rex) and laser detector (DAWN HELEOS-II) combination are (beautiful Huai Yate companies of state)；
Sample pre-treatments：With mobile phase dissolved dilution to 5000ppm sample introductions.
1.2.2 XRD analysis
EPPS I, EPPS II, EPPS III are pressed on progress X ray polycrystalline diffraction on specimen holder.Condition：Ceramic X-ray Pipe, Cu targets, rated voltage 40kV, rated current 50mA, 2 θ 5-60 of scanning range.
1.2.3 infrared analysis
Take after KBr is dried and mixed in right amount with 500 μ g purification of samples EPPS I, EPPS II, EPPS III, under infrared lamp in It is ground uniformly to no crystal grain in agate mortar, 5min is pressed on grease press with 10 × 107 pressure, the thin slice of pressure is in infrared spectrum It is scanned in the range of instrument and 400-4000cm-1, spectra re-recorded data and spectrogram.
1.2.4 nuclear magnetic resonance
It weighs polysaccharide component 30mg and is dissolved in 0.5mL D respectively2In O, 1H NMR and 13C NMR nmr analysis is carried out.
1.3 experimental results and analysis
1.3.1 polysaccharide molecular weight measures
EPPS I, EPPS II, EPPS III molecular weight determination see Fig. 1,2,3 respectively.
By Fig. 1 to 3, it is seen that EPPS I, EPPS II, EPPS III molecular weight be respectively 21.5KDa, 12.0KDa, 13.0KDa.The difference of molecular weight may lead to the difference of activity, it is therefore necessary to further EPPS I, EPPS made from research IIth, the activity of EPPS III.
1.3.2 XRD analysis
EPPS I, EPPS II, EPPS III XRD analysis result see Fig. 4.
From fig. 4, it can be seen that EPPS I, EPPS II, EPPS III have weak diffraction maximum, almost amorphous region in 2 θ for 24 or so, Illustrate that their crystallinity are low, be in amorphous state.The diffraction peak intensity of EPPS II is a little, may be compared with its crystallization of EPPS I, EPPS III Property is quite a lot of.
1.3.3 infrared analysis
EPPS I, EPPS II, EPPS III infrared analysis see Fig. 5,6,7 respectively.
By Fig. 5 to 7 it is found that EPPS I, EPPS II, the group of EPPS III are closely similar.3371.9cm-1 is O-H's in Fig. 5 Stretching vibration illustrates hydroxyl in molecule.2933.27cm-1 is C-H stretching vibrations.1152.76、1080.21、1026.52cm- 1 three peaks are pyranose ring characteristic absorption peak, are the nonsymmetrical vibration peaks of its glycosidic bond C-O-C, are pyrrole at 669.51cm-1 The symmetric vibration peak of saccharide ring of muttering C-O-C.It is pyranose to illustrate EPPS I.3400.72cm-1 is the stretching vibration of O-H in Fig. 6, is said Hydroxyl in bright molecule.2936.81cm-1 is C-H stretching vibrations.Stretching vibrations of the 3431.66cm-1 for O-H, explanation in Fig. 7 Hydroxyl in molecule.2937.81cm-1 is C-H stretching vibrations.
1.3.4 nuclear magnetic resonance
It chooses the obvious EPPS I in infrared signature peak and carries out 1H NMR, 13C NMR analysis, as a result see Fig. 8,9 respectively. There is the hydrogen shift of proton on anomeric carbon C1 in 1H NMR, at 5.306ppm, it is α type pyranoses to show these glucose residues, This is consistent with infrared spectrum analysis.In 13C NMR, at 99.641ppm chemical shift show that C-l replaces, Chemical shift shows there is C-2, C-3, the C-4 not replaced at 76.745ppm, 73.332ppm, 71.514ppm；Without 78- Chemical shift in the range of 85ppm shows that there is no C-2, C-3, the C-4 replaced；69.305ppm the chemical shift at place shows There is the C-6 replaced.The chemical shift of 60.457ppm shows that also there are unsubstituted C-6, it is thus possible to also with other α Branched structure.
1.4 brief summary
This part experiment obtains drawing a conclusion by simple Spectrum Analysis：(1) molecule of EPPS I, EPPS II, EPPS III Amount is respectively 21.5KDa, 12.0KDa, 13.0KDa.(2) EPPS I, EPPS II, the crystallinity of EPPS III are all very low, are in nothing Amorphous condition.(3) infrared to show that EPPS I, EPPS II, the structure of EPPS III are similar, more apparent I characteristic peaks of EPPS are pyranose. (4) the nuclear-magnetism result of EPPS I demonstrates EPPS I as pyranose, and C-1, C-6 substitution occurs.
2nd, the immunocompetence research for the Echinacea polysaccharide being prepared
This experiment is object with NK cells in mice and mouse monokaryon macrophage RAW 264.7, research Echinacea polysaccharide Immunocompetence.
2.1 experiment material
2.1.1 material and reagent
Material needed for experiment is shown in Table 2-1 with reagent.
Table 2-1 experiment materials and reagent
2.1.2 instrument and equipment
Instrument and equipment needed for experiment is shown in Table 2-2.
Table 2-2 laboratory apparatus and equipment
2.2 experimental method
2.2.1 NK cell activity is influenced
NK cells are natural killer cells, are the important immunocytes of body, have non-specific lethal effect, immune It plays an important role in system.It is not only related with antitumor, viral infection resisting and immunological regulation, but also in some cases Participate in hypersensitivity and the generation of autoimmune disease.K562 is leukaemia cell, in the height undifferentiated stage, from white blood Separation is established in the hydrothorax of the patient of sick rapid change period.K562 is the external target of the Sensitive altitude sensitivity of NK cells.Detect NK The killing activity of cell does target cell through commonly using it.
MTT colorimetric methods are a kind of methods for detecting cell survival and growth.Its testing principle is in living cells mitochondria Succinate dehydrogenase can make exogenous MTT be reduced to the bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) of water-insoluble and be deposited on cell In, and dead cell is without this function.Dimethyl sulfoxide (DMSO) (DMSO) can dissolve the first a ceremonial jade-ladle, used in libation in cell, it is measured in 570nm with microplate reader Absorbance value can reflect living cells quantity indirectly.
The mouse spleen lower differentiation that can promote NK cells of IL-3 inductions in vivo.It tests to divide from mouse spleen in this part It is effector cell from NK cells, using K562 cells as target cell, determines that NK cells are thin to target using MTT colorimetric determination cell numbers The killing rate of born of the same parents K562.
2.2.1.1 the preparation of mouse boosting cell suspension
De- neck puts to death mouse, 75% alcohol soaking disinfection 2-3min, and spleen is taken in super-clean bench, rinses 1 time with PBS, then It is ground in 150 mesh screens, the RPMI1640 culture solutions of 20%FBS is added in grinding, are screened in 50mL centrifuge tubes. 800rpm centrifuges 5min, abandons supernatant, adds in Tris-NH4Cl buffer solution 5mL, and piping and druming is uniform, stands 3-4min.1000rpm from Heart 5min, abandons supernatant, is washed 2 times with PBSs of the 5mL containing 1%P/S, is washed 1 time with 5mlRPMI1640, and Trypan Blue, counting are abandoned Clearly, cell concentration is adjusted to 5 × 106/mL with the RPMI1640 complete culture solutions of 10%FBS, 1%P/S, for use.
2.2.1.2 the preparation of target cell
K562 cells are put in centrifuge tube, 1200rpm/min, centrifuge 3min, complete with the RPMI1640 of 10%FBS, 1%P/S Full nutrient solution is adjusted to the cell suspension of a concentration of 5 × 105/ml.
2.2.1.3NK the measure of cell activity
Mouse boosting cell suspension is added in 96 orifice plates, per 50 μ L of hole, sequentially add containing EPPS, EPPS I, EPPS II, The RPMI1640 complete culture solutions of III 50,100,250 μ g/ml of EPPS, respectively do 4 multiple holes.In 37 DEG C, 5%CO2 incubator cultures After 12h, 50 μ l of K562 cell suspensions are added in, while set blank control group, target cell control group and effector cell's control group.It trains again It supports and abandons supernatant afterwards for 24 hours, the 50 μ L of MTT of 0.5mg/mL are added in per hole, continue after cultivating 4h, DMSO150 μ L, culture are added in per hole After 1h, 5min is shaken on microwell plate fast oscillator, makes bluish violet first a ceremonial jade-ladle, used in libation crystallization fully dissolving, measuring each hole with microplate reader exists Absorbance value at 570nm.As a result it is represented with NK cell killing rates, NK cell killings rate is calculated by formula (5-1)：
NK cell activity (%)=[1- (experimental group OD570nmEffector cell's control group OD570nm)/target cell control group OD Value] X100% (5-1)
2.2.2 RAW264.7 cell activity is influenced
RAW 264.7 is that mouse monokaryon macrophage is that monocytes/macrophages have various biological functions, mainly may be used To be summarised as the following aspects：1. nonspecific immunity is defendd.After foreign pathogen enters body, before immune response is excited It can be swallowed and remove by monocytes/macrophages, but a small number of pathogen can be bred in its intracellular.2. remove foreign cell.3. non-spy Different immunosurveillance.4. present antigen.I.e. after exotic antigen enters body, swallowed, digested by monocytes/macrophages first, it will Effective antigenic determinant and mhc class ii molecule are combined into complex, this species complex is identified by T cell, immune so as to excite Response.5. secrete medium IL-1, interferon, complement (C1, C4, C2, C3, C, Factor B) etc..
This part experiment with RAW 264.7 is research object, by detecting the proliferation activity of cell, NO emission levels and thin The secretion level of born of the same parents' immune factor inquires into ion vitro immunization regulating and controlling effect of the Echinacea polysaccharide to macrophage.
2.2.2.1 the influence to RAW264.7 cell Proliferations
The RAW264.7 cell recoveries of purchase, are cultivated in 50ml culture bottles, etc. bottom of bottle cell density reach 80% or so When, original fluid is discarded, is rinsed one time without DMEM culture solutions of the FBS containing 1%P/S with 4mL, is discarded washing lotion, add in 2mL pancreatin TE digests 3-4min, sees cell rounding under the microscope, and 5mL DMEM complete culture solutions are added in when gap increases and terminate digestion, Cell is blown down with suction pipe, is moved in centrifuge tube, 1200rpm, centrifuges 3min.Add DMEM complete culture solutions, Trypan Blue, It counts, is adjusted to the cell suspension of a concentration of 2 × 105/ml.More than cell suspension is laid in 96 orifice plates, per 100 μ L of hole, in 37 DEG C, 5%CO2Incubator culture 12h, after cell is adherent, discards supernatant, and is separately added into 50,100,250,500 μ g/mL's EPPS, EPPS I, EPPS II and EPPS III, blank control group are DMEM complete culture solutions, and every group sets 8 repetitions.In 37 DEG C, 5% CO2Continue culture in incubator for 24 hours, collect supernatant (measure for being used for the later stage) in sterile centrifugation tube, 50 μ are added in per hole The MTT of l0.5mg/ml continues after cultivating 4h, DMSO150 μ L is added in per hole, after cultivating 1h, shakes on microwell plate fast oscillator 5min is shaken, makes bluish violet first a ceremonial jade-ladle, used in libation crystallization fully dissolving, absorbance value of each hole at 570nm is measured with microplate reader.RAW264.7 Proliferative capacity represented with value added index, by formula (5-2) calculate：
Proliferating antigen Ki67=(experimental group OD570nm/ blank control group OD570nm) X100% (5-2)
2.2.2.2 to the influence of RAW264.7 cells secretion NO
By 2.2.2.1 lower methods, after cell conditioned medium is received sterile centrifugation tube, measure NO's by NO kit specifications Content.
2.2.2.3 the influence to RAW264.7 cell secretion of cytokines
By 2.2.2.1 lower methods, after cell conditioned medium is received sterile centrifugation tube, measured by ELISA kit specification The content of IFN-γ.
2.2.2.4 to the influence of RAW264.7 cell mode of appearance
By 2.2.2.1 lower method processing cells, observation various concentration EPPS, EPPS I, EPPS II and EPPS III are right The influence of RAW264.7 mode of appearance.
2.3 experimental results and analysis
2.3.1 NK cell activity is influenced
Various concentration Echinacea polysaccharide (Echinacea Purpurea Polysaccharide, EPPS) and its purifying institute It obtains the influence of component EPPS I, EPPS II, EPPS III to isolated mouse NK cell activity and is shown in Table 2-3.
Table 2-3 various concentrations EPPS, EPPS I, the influence of EPPS II and EPPS III to isolated mouse NK cell activity
By table 2-3, the result shows that, EPPS, EPPS I, EPPS II, EPPS III have facilitation to NK cells in mice activity, and And there is a certain amount to imitate relationship.EPPS, EPPS III reduces in the range of 50-250 μ g/mL with the raising of concentration, facilitation； EPPS I, EPPS II are in the range of 50-250 μ g/mL with the raising of concentration, facilitation increase.
2.3.2 to RAW264.7 activity influences
2.3.2.1 the influence to RAW264.7 cell Proliferations
Various concentration Echinacea polysaccharide (Echinacea Purpurea Polysaccharide, EPPS) and its purifying institute It obtains the influence of component EPPS I, EPPS II, EPPS III to RAW264.7 cell Proliferations and sees Figure 10.
Figure 10 the result shows that, EPPS, EPPS I, EPPS II, EPPS III have facilitation to RAW264.7 cell activity. EPPS promotes the ability of proliferation first increase in the range of concentration 50-500 μ g/mL and reduce afterwards, facilitation maximum during 250 μ g/mL, EPPS I, EPPS II promote the ability of proliferation to promote proliferation in the trend of attenuating, EPPS III in the range of concentration 50-500 μ g/mL Ability first increase in the range of concentration 50-500 μ g/mL and reduce afterwards, facilitation is maximum during 100 μ g/mL.
2.3.2.2 to the influence of RAW264.7 cells secretion NO
The influence of EPPS, EPPS I, EPPS II and EPPS III to RAW264.7 cells secretion NO is shown in Table 2-4.
The influence of table 2-4 various concentrations EPPS, EPPS I, EPPS II and EPPS III to RAW264.7 cells secretion NO
By table 2-4 as it can be seen that EPPS, EPPS I, EPPS II and EPPS III can promote releases of the RAW264.7 to NO, and in 50- It is in dose-effect relationship in the range of 250 μ g/mL.
2.3.2.3 the influence to RAW264.7 cell secretion of cytokines
The influence of EPPS, EPPS I, EPPS II and EPPS III to RAW264.7 cell secretion of gamma-IFN is shown in Table 2-5 respectively.
Table 2-5 various concentrations EPPS, EPPS I, the influence of EPPS II and EPPS III to RAW264.7 cell secretion of gamma-IFN
By table 2-5 as it can be seen that EPPS, EPPS I, EPPS II and EPPS III can promote releases of the RAW264.7 to IFN-γ, and It is in dose-effect relationship in the range of 50-250 μ g/ml.
2.3.2.4 to the influence of RAW264.7 cell mode of appearance
Figure 11 is shown in the influence of various concentration EPPS, EPPS I, EPPS II and EPPS III to RAW264.7 mode of appearance.
As seen from Figure 11, the cell of space management is rounded, and the cell that EPPS, EPPS I, EPPS II and EPPS III are handled has Elongated trend, volume become larger, become less regular.The influence of EPPS, EPPS I is more apparent.
2.4 brief summary
This experiment shows that EPPS, EPPS I, EPPS II, EPPS III have facilitation to NK cells in mice activity, and have one Fixed dose-effect relationship.EPPS, EPPS I, EPPS II, EPPS III have apparent facilitation to RAW264.7 cell Proliferations, simultaneously Secretory volume of the RAW264.7 cells to NO, IFN-γ can be increased.
Contrast test one
The recovery rate of Echinacea polysaccharide is influenced according to hydrolysis temperature, pH, enzyme dosage, enzymolysis time.Design orthogonal test. Test result analysis is shown in Tables 1 and 2.
1 Echinacea polysaccharide Enzymatic Extraction experimental design factor level table of table
2 Echinacea polysaccharide Enzymatic Extraction orthogonal experiments of table and analysis
According to result of the test, it is known that in Echinacea polysaccharide enzyme process extraction process, the influence sequence of 4 factors on extraction rate For A hydrolysis temperature ＞ D enzymolysis time ＞ C enzyme dosage ＞ B pH.According to analysis, i.e. hydrolysis temperature is 50 DEG C, pH 3.0, and enzyme is used It is 5% to measure, enzymolysis time 60min.
Contrast test two
The Echinacea polysaccharide extract rate of comparative example 1-7.Table 3
Group Echinacea 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%
As shown in Table 3, the extraction process of the Echinacea polysaccharide of seven embodiment groups is obtained for higher recovery rate.
To sum up, present invention extraction Echinacea polysaccharide, while extraction time is shortened, substantially increases Echinacea polysaccharide Recovery rate, and the preferable Echinacea polysaccharide of dissolubility has been obtained, there is preferable active constituent.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.

Claims (4)

  1. A kind of 1. method of enzyme extraction Echinacea polysaccharide, which is characterized in that extracting method includes the following steps：
    1. medicinal material pre-processes：Echinacea aerial part be crushed into 40-60 mesh sieve；Degreasing solvent is added in, it is after degreasing, degreasing is molten Agent volatilizes under the conditions of 20-80 DEG C, and treated, and medicinal material is spare；
    2. it extracts：The water that 1. medicinal material that step is handled is appropriate, and 15-25 times of medicinal material of addition is measured is weighed, tune pH is 2-4, is preheated To after 40 DEG C -60 DEG C, the enzyme preparation that 0.04-0.06 times of medicinal material is measured is added in, enzymolysis time 50min-70min, 80 DEG C -100 DEG C go out Enzyme 10-20min continues stirring heat preservation 1-2h at 80 DEG C -100 DEG C；
    3. it concentrates：Supernatant revolving is concentrated under reduced pressure into crude drug amount 1 by centrifugation：The concentrate of 0.9-1.5；
    4. alcohol precipitation：3-4 times of absolute ethyl alcohol is added in into concentrate, 24-48 hours are stood under the conditions of 0-4 DEG C；
    5. it washs：4. precipitation solution that step is obtained filters, and obtained sediment is washed successively with absolute ethyl alcohol, acetone, ether；
    6. it dries：Sediment with the 1-2 times of water measured is redissolved, is spray-dried under conditions of 140 DEG C -160 DEG C and obtains Echinacea Polysaccharide；
    Wherein, the enzyme preparation is the mixing of cellulase, pectase, glusulase, phytase, and quality parts ratio is：0.5- 1:1-2:0.1-0.5：0.05-0.1.
  2. 2. the method for enzyme extraction Echinacea polysaccharide according to claim 1, which is characterized in that the degreasing solvent is oil Ether.
  3. 3. the method for enzyme extraction Echinacea polysaccharide according to claim 1, which is characterized in that the degreasing solvent is oil The mixing of ether, 1,2- propylene oxide, ether, quality parts ratio are：1-2:0.05-0.1:0.02-0.08.
  4. 4. the method for enzyme extraction Echinacea polysaccharide according to any one of claims 1 to 3, which is characterized in that the purple cone Chrysanthemum crude drug source is derived from full-bloom stage to the aerial part for spending the later stage in genus echinacea plant Echinacea.
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