CN109400740A - A kind of Dendrobium denneanum acidic polysaccharose and the preparation method and application thereof - Google Patents
A kind of Dendrobium denneanum acidic polysaccharose and the preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of Dendrobium denneanum acidic polysaccharoses and the preparation method and application thereof, this method is using Dendrobium denneanum stem as material, drying, which crushes, obtains Dendrobium denneanum dry powder, it is extracted using the method for alkali water extract-alcohol precipitation, through degreasing, buck extraction, alcohol precipitation, deproteinized, removes small molecule, freeze-drying acquisition Dendrobium denneanum acidic polysaccharose.Gained polysaccharide is α-configuration, and molecular weight is Mw=5.78 × 104 Da, and wherein glucuronic acid content is 292.2 mg/g.The polysaccharide has significant immune-enhancing activity, can be used for improving in immune drug and apply.
Description
Technical field
The invention belongs to technical field of extraction of Chinese traditional medicine, specifically, being related to a kind of Dendrobium denneanum acidic polysaccharose and its preparation
Method and application.
Background technique
The name of dendrobium nobile (Dendrobium) sees the Classic of Mountains and Rivers earliest, medicinal first recorded in Shennong's Herbal, is listed in
Product are common rare traditional Chinese medicines.Its medicinal history is long, has the function of nourishing Yin and clearing heat, promote the production of body fluid beneficial stomach, moistening lung to arrest cough etc., is praised
For one of Chinese nine big mesonas.China's Dendrobium Plants are abundant, share 76 kinds of Dendrobium Sw, 2 mutation.Referred to as Chinese medicine treasure-house
Sichuan be also the main product of dendrobium nobile one of, share 11 kinds of Dendrobium Sw, wherein Dendrobium denneanum yield is especially abundant.Research hair
It is existing, polysaccharide rich in Dendrobium Sw, and mostly have compared with strong biological activity, such as anti-oxidant, anti-aging, drop blood
Sugared, antitumor and immunological regulation etc..Wherein the anti-immunity of Dendrobium Sw polysaccharide adjusts activity and is concerned, but is concentrated mainly on
The neutral polysaccharide of dendrobium nobile, and scientific discovery, plant acidic polysaccharose usually has stronger bioactivity, and plays biology in polysaccharide
When active, the acidic-groups such as uronic acid in structure are played a crucial role.
Less for the material base research of Dendrobium denneanum plant polyose effect at present, the extracting and developing of acidic polysaccharose is pure
Acidic-group exposure basis in change and polysaccharide, which is studied, to be lacked, and has no the report of relevant document or patent.
Summary of the invention
In view of this, the present invention provides a kind of Dendrobium denneanum acidic polysaccharoses and the preparation method and application thereof.
In order to solve the above-mentioned technical problem, the invention discloses a kind of preparation methods of Dendrobium denneanum acidic polysaccharose, including
Following steps:
Step 1 takes Dendrobium denneanum powder in a round bottom flask, and the slightly boiled refluxing extraction in water-bath is added after petroleum ether, filters,
Filtrate is abandoned, drying to constant weight to remove liposoluble substance；The dregs of a decoction continue with the slightly boiled refluxing extraction of ethyl alcohol, filtering, abandoning filtrate；It is dry
The dregs of a decoction afterwards move into again is added sodium hydroxide in beaker, magnetic agitation extracts at room temperature, collects filtrate, repeats to extract once, close
And be adjusted to neutrality after filtrate with hydrochloric acid twice, rotary evaporation, it with Sevag reagent deproteinized, is repeated 4 times, collects and merge supernatant
Liquid rotary evaporation；
Step 2, supernatant are slowly added to dehydrated alcohol with separatory funnel, centrifugation is abandoned heavy in stirring on magnetic stirring apparatus thereto
It forms sediment；Continue in filtrate plus dehydrated alcohol, collection precipitate；It is centrifuged after successively washing precipitating with acetone, ethyl acetate, dehydrated alcohol,
It is saved backup in 4 DEG C of refrigerators；
Step 3, by after above-mentioned sample deionized water dissolving, be fitted into bag filter in beaker, under conditions of magnetic agitation
Use deionized water dialysis；Sample solution is freeze-dried up to Dendrobium denneanum acidic polysaccharose after collecting dialysis.
Optionally, the boiling point of the petroleum ether in the step 1 is 60-90 DEG C, the quality of Dendrobium denneanum powder and petroleum ether
Volume ratio (g/ml) is 1:4-3:4；Reflux extracting time is 0.5h-1.5h；The volumn concentration of the ethyl alcohol is 80%.
Optionally, the molal weight ratio (mmol/g) of the sodium hydroxide in the step 1 and Dendrobium denneanum powder is 1:1-
5:1；Magnetic agitation extraction time is 4-8h.
Optionally, the substance of the first time rotary evaporation in the step 1 and Dendrobium denneanum powder volume mass ratio are 3:
2-7:2, Sevag reagent are the n-butanol and chloroform of volume ratio 1:4；Sevag reagent and Dendrobium denneanum powder volume quality
Than for 3:2-7:2, the supernatant of second of rotary evaporation and the volume mass ratio (mL/g) of Dendrobium denneanum powder are 5:2-10:2.
Optionally, the additive amount of the first time dehydrated alcohol in the step 2 is supernatant and dehydrated alcohol total volume
40%；The additive amount of second of dehydrated alcohol is the 60% of filtrate and dehydrated alcohol total volume.
Optionally, the dialysis time in the step 3 is 64h-80h, and 8h changes a water.
The invention also discloses a kind of Dendrobium denneanum acidic polysaccharoses that above-mentioned preparation method is prepared.
It optionally, is α-configuration, molecular weight is Mw=5.78 × 104 Da, and wherein glucuronic acid content is 292.2 mg/g.
It improves in immune drug and applies in preparation the invention also discloses a kind of above-mentioned Dendrobium denneanum acidic polysaccharose.
Compared with prior art, the present invention can be obtained including following technical effect:
1) the Dendrobium denneanum acidic polysaccharose content of technique preparation is high, stable and controllable for quality；Using being stirred at room temperature, extraction reduction is more
Sugared structural damage；Separation is extracted using the method that alkali carries take alcohol to precipitate for the first time and obtains Dendrobium denneanum acidic polysaccharose.
2) Dendrobium denneanum acidic polysaccharose is tested using Macrophage Model for the first time, it is found that it is thin macrophage can be remarkably reinforced in the polysaccharide
The phagocytic activity of born of the same parents promotes secrete cytokines, improves immunocompetence.
3) active constituent Dendrobium denneanum acidic polysaccharose prepared by the present invention is quality controllable, and content is high, specific chemical components,
Drug activity is significant.The separation purifying technique design of the preparation method of extract of the present invention is rationally, easy to operate.Present invention test
Dendrobium denneanum acidic polysaccharose on macrophage immunity active influence, for Dendrobium denneanum acidic polysaccharose exploitation provide it is necessary
Theoretical foundation, while improving the value of exploiting and utilizing and economic value of Dendrobium denneanum.
Certainly, it implements any of the products of the present invention it is not absolutely required to while reaching all the above technical effect.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present invention, constitutes a part of the invention, this hair
Bright illustrative embodiments and their description are used to explain the present invention, and are not constituted improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is polysaccharide infrared spectrogram of the embodiment of the present invention；
Fig. 2 is influence of the polysaccharide of the embodiment of the present invention to macrophage phagocytosis；
Fig. 3 is influence of the polysaccharide of the embodiment of the present invention to macrophages secrete TNF-α；
Fig. 4 is influence of the polysaccharide of the embodiment of the present invention to macrophages secrete IL-6.
Specific embodiment
Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, whereby to the present invention how application technology hand
Section solves technical problem and reaches the realization process of technical effect to fully understand and implement.
The invention discloses a kind of preparation methods of Dendrobium denneanum acidic polysaccharose, comprising the following steps:
Step 1 takes Dendrobium denneanum powder in a round bottom flask, and the slightly boiled reflux in water-bath after petroleum ether (60-90 DEG C) is added
Extract 0.5h-1.5h, wherein the mass volume ratio (g/ml) of Dendrobium denneanum powder and petroleum ether is 1:4-3:4；Filter is abandoned in filtering
Liquid, drying to constant weight to remove liposoluble substance；The slightly boiled refluxing extraction of ethyl alcohol that it is 80% with volumn concentration that the dregs of a decoction, which continue,
Filtrate is abandoned in 0.5h-1.5h, filtering；The dregs of a decoction after drying move into again is added sodium hydroxide in beaker, wherein sodium hydroxide with repeatedly
The molal weight ratio (mmol/g) of sheath Dendrobium is 1:1-5:1；Magnetic agitation extracts 4-8h at room temperature, collects filtrate, repeats
It extracts once, is adjusted to neutrality after merging filtrate twice with hydrochloric acid, rotary evaporation to certain volume, wherein after rotary evaporation
Substance and Dendrobium denneanum powder volume mass ratio are 3:2-7:2, are gone with Sevag reagent (n-butanol: chloroform (v:v)=1:4)
Albumen, Sevag reagent and Dendrobium denneanum powder volume mass ratio are 3:2-7:2, are repeated 4 times, and collect merging supernatant rotation and steam
Hair, the supernatant of rotary evaporation and the volume mass ratio (mL/g) of Dendrobium denneanum powder are 5:2-10:2；
Step 2, supernatant are slowly added to dehydrated alcohol with separatory funnel thereto, make ethyl alcohol body in stirring on magnetic stirring apparatus
Product reaches 40%, and precipitating is abandoned in centrifugation.Continue in filtrate plus dehydrated alcohol makes in system, ethyl alcohol volume is made to reach 60%, collects precipitating.
It is centrifuged after successively washing precipitating with acetone, ethyl acetate, dehydrated alcohol, is saved backup in 4 DEG C of refrigerators.
Step 3, by after above-mentioned sample deionized water dissolving, be fitted into bag filter in beaker, in the item of magnetic agitation
Deionized water dialysis 64h-80h, 8h is used to change a water under part；Sample solution is freeze-dried up to Dendrobium denneanum acid after collecting dialysis
Property polysaccharide.
Embodiment 1
It takes 20g Dendrobium denneanum powder in a round bottom flask every time, is added after 40mL petroleum ether (boiling point is 60-90 DEG C) in water-bath
In slightly boiled refluxing extraction 1h, filtering, abandon filtrate, drying to constant weight to remove liposoluble substance.The dregs of a decoction continue to be contained with volume basis
Filtrate is abandoned in the slightly boiled refluxing extraction 1h of ethyl alcohol that amount is 80%, filtering.The dregs of a decoction after drying move into addition 0.2M hydrogen in large beaker again
Sodium oxide molybdena 300mL, magnetic agitation extracts 6h at room temperature, collects filtrate, repeats to extract primary, merges after filtrate twice with hydrochloric acid tune
For section to neutrality, rotary evaporation to certain volume 50mL is heavy with 50mLSevag reagent (n-butanol: chloroform=1:4) deproteinized
It is 4 times multiple, it collects and merges supernatant rotary evaporation to 50-100mL.
Supernatant is slowly added to dehydrated alcohol with separatory funnel in stirring on magnetic stirring apparatus thereto, makes ethyl alcohol volume
Reach 40%, precipitating is abandoned in centrifugation.Continue in filtrate plus dehydrated alcohol makes ethyl alcohol volume in system reach 60%, collects precipitating.Successively
It is centrifuged after washing precipitating with acetone, ethyl acetate, dehydrated alcohol, is saved backup in 4 DEG C of refrigerators.
After a small amount of deionized water dissolving of above-mentioned sample, it is fitted into bag filter in beaker with deionized water dialysis (magnetic
Power stirring) 72h, 8h changes a water.Sample solution is freeze-dried up to Dendrobium denneanum acidic polysaccharose DDP-S after collecting dialysis.
A, to the measurement of glucuronic acid content:
Precision measures 0,0.1,0.2,0.4,0.6,0.8 mL of uronic acid reference substance solution in test tube, and each test tube is mended with distilled water
Enough to 1mL.The 6 mL concentrated sulfuric acids are added to each test tube under the conditions of ice-water bath, after shaking up, the water-bath 20min in 85 DEG C of water-baths,
It is cooled to room temperature after taking-up, each pipe plus 0.2 mL, 0.1% carbazole liquid are kept 2h, returned to zero with distilled water, at room temperature in 530nm wavelength
Lower measurement light absorption value, three parallel processing.Drawing using standard concentration is X-axis light absorption value as the standard curve of Y-axis, is obtained linear
Regression equation.
Sample is dissolved with distilled water, is configured to the sample solution of 1mg/mL.Take polysaccharide solution 1mL, in ice-water bath to
The 6 mL concentrated sulfuric acids are added in each pipe, and after shaking up, the water-bath 20min in 85 DEG C of water-baths is cooled to room temperature after taking-up, each pipe plus 0.2mL
0.1% carbazole liquid, is kept 2h at room temperature, is returned to zero with distilled water, measures light absorption value under 530nm wavelength.Three parallel processing, meter
Calculate glucuronic acid content in sample.Glucuronic acid content is 292.2 mg/g.
B, molecular weight and infrared analysis:
Appropriate polysaccharide sample is taken, the sample solution of 5mg/mL is configured to.Each polysaccharide sample is detected using efficient liquid phase exclusion chromatography
The molecular weight of product.Molecular weight detection result is Mw=5.78 × 104 Da.
1mg polysaccharide sample and tabletting after potassium bromide ground and mixed are taken, blank and sample tabletting are put into Fourier transform
Infrared spectrometer.Scanning analysis under the conditions of beam area 4000-500cm-1 and resolution ratio 4cm-1, obtains the red of each sample
External spectrum figure (Fig. 1).
Polysaccharide is-OH in the absorption peak that 3426 cm-1 occur, and the absorption peak that 2930 cm-1 occur represents C-H.?
Occurs the characteristic peak of C-O stretching vibration in-COOH at 1644cm-1.Occurs the flexible vibration of C-O in-COOH at 1422cm-1
Dynamic characteristic peak；Continuously occur three pyranoside absorption peaks at 1157,1079 and 1018 cm 1；Go out at 858cm-1
The characteristic peak of α-configuration glycosidic bond is showed.
C, immunocompetence is tested
The RAW264.7 cell of logarithmic growth phase is collected, is inoculated in 96 orifice plates after adjusting concentration of cell suspension, is guaranteeing every hole 6
Every hole is inoculated with the cell suspension of 100 μ L in the case where × 103 cells.Blank group is set up simultaneously, the fresh training of equivalent is only added
Base is supported, to exclude influence of the culture medium for light absorption value.3 parallel holes are arranged in each concentration gradient, as repetition.Cell is put
It is placed in 37 DEG C, cultivates 24 hours in the constant incubator of 5%CO2.
Each polysaccharide sample solution that 100 μ L various concentrations are added in every hole after supernatant is carefully sucked, blank and control group add
The fresh culture for entering equivalent continues culture 24 hours.It is washed twice after carefully sucking supernatant with PBS solution, every hole is added
0.075% neutral red solution of 100 μ L is dyed, and is cultivated 1 hour.Dye liquor is carefully sucked, it is molten that 300 μ L PBS are added in every hole
Liquid is washed, and is repeated three times.100 μ L of ethanol acetate (V/V=1:1) cytolysate is added in every hole after the completion of washing, in room
Temperature is lower overnight.Each hole absorbance OD value is measured at 492 nm using microplate reader.Cell is calculated to dimethyl diaminophenazine chloride according to following formula
Phagocytic activity:
Macrophage as internal maximum phagocyte, major function first is that identification, swallowing and to eliminate external cause of disease micro-
The cell of aging in biology and organism is the important component for constituting organism innate immunity defence line, therefore, macrophage
Phagocytic activity be one of the important indicator for measuring body innate immune function, the phagocytic activity for improving macrophage is thin to macrophage
The Immunity regulation of born of the same parents is extremely important.The result shows that polysaccharide sample can effectively improve the phagocytosis of RAW264.7 cell
Ability, sample show certain concentration dependent to the raising of RAW264.7 cell phagocytic activity, with the increase of concentration,
Facilitation is more obvious, but when 200 μ g/mL of arrival, facilitation is no longer obviously increased.Phagocytic index is maximum when to 800 μ g/mL
(as shown in Figure 2).
Tumor necrosis factor TNF-alpha energy direct killing inhibits tumour cell, can also pass through the tune to body's immunity
Section effect, promotes the killing of T cell and its homicidal wound cells against tumor cells.The main mononuclear macrophage by activating of TNF-α
It is discharged with endothelial cell.Hematopoiesis, immune and inflammatory process can be adjusted in tumor necrosis factor.Test uses ELISA reagent
Box double antibody sandwich method detects the content of TNF-α in RAW264.7 cell culture supernatant.Result is it is found that sample as shown in Figure 3
In DDP-S processing group, each dosage has certain facilitation to TNF-α, and with the increase of dosage, to TNF-α
Facilitation is more obvious.The yield of TNF-α is higher than control group in high dose processing group, shows that sample can promote to a certain extent
RAW264.7 cell TNF secretion-α enhances immune function.
Interleukin-6 (IL-6) is generated by cells such as Monocytes/Macrophages, T lymphocyte and bone-marrow-derived lymphocytes.
IL-6 can stimulate the cell Proliferation for participating in being immunoreacted, differentiation and improve its function, mainly have the differentiation of induction B cell, induction single
The biological natures such as monocyte differentiation, induction IL-2 and IL-2 expression of receptor and enhancing NK cell activity.Test uses ELISA reagent
Box double antibody sandwich method detects the content of IL-6 in RAW264.7 cell culture supernatant.As shown in Figure 4, low dosage polysaccharide sample
There was no significant difference compared with control group for the IL-6 content of processing, when dosage increases to 600 μ g/mL, IL- in polysaccharide sample processing group
6 yield is apparently higher than control group, shows that polysaccharide sample can promote RAW264.7 cell point in higher concentration to a certain extent
Secrete IL-6.
Embodiment 2
Step 1 takes Dendrobium denneanum powder in a round bottom flask, and the slightly boiled reflux in water-bath after petroleum ether (60-90 DEG C) is added
Extract 0.5h, wherein the mass volume ratio (g/ml) of Dendrobium denneanum powder and petroleum ether is 3:4；Filtering is abandoned filtrate, is dried to
Constant weight is to remove liposoluble substance；The slightly boiled refluxing extraction 0.5h of ethyl alcohol that it is 80% with volumn concentration that the dregs of a decoction, which continue, filtering,
Abandon filtrate；The dregs of a decoction after drying move into again is added sodium hydroxide in beaker, wherein mole of sodium hydroxide and Dendrobium denneanum powder
Mass ratio (mmol/g) is 5:1；Magnetic agitation extracts 4h at room temperature, collects filtrate, repeats to extract once, after merging filtrate twice
It is adjusted to neutrality with hydrochloric acid, rotary evaporation to certain volume, wherein substance and Dendrobium denneanum powder volume matter after rotary evaporation
Amount is than being 3:2, with Sevag reagent (n-butanol: chloroform (v:v)=1:4) deproteinized, Sevag reagent and Dendrobium denneanum powder
Volume mass ratio is 3:2, is repeated 4 times, and collects and merges supernatant rotary evaporation, the supernatant and Dendrobium denneanum powder of rotary evaporation
Volume mass ratio (mL/g) be 10:2；
Step 2, supernatant are slowly added to dehydrated alcohol with separatory funnel thereto, make ethyl alcohol body in stirring on magnetic stirring apparatus
Product reaches 40%, and precipitating is abandoned in centrifugation.Continue in filtrate plus dehydrated alcohol makes in system, ethyl alcohol volume is made to reach 60%, collects precipitating.
It is centrifuged after successively washing precipitating with acetone, ethyl acetate, dehydrated alcohol, is saved backup in 4 DEG C of refrigerators.
Step 3, by after above-mentioned sample deionized water dissolving, be fitted into bag filter in beaker, in the item of magnetic agitation
Deionized water dialysis 64h, 8h is used to change a water under part；Sample solution is freeze-dried acid more up to Dendrobium denneanum after collection dialysis
Sugar.
Measurement using the A in embodiment 1 to glucuronic acid content, glucuronic acid content are 291.4 mg/g.
It is thin that the Dendrobium denneanum acidic polysaccharose (600 μ g/mL) being prepared using the present embodiment 2 can effectively improve RAW264.7
Born of the same parents' phagocytic activity, phagocytic index 1.92.
The Dendrobium denneanum acidic polysaccharose (600 μ g/mL) being prepared using the present embodiment 2 can promote RAW264.7 cell
TNF secretion-α, the yield of TNF-α are 1655pg/mL.
Embodiment 3
Step 1 takes Dendrobium denneanum powder in a round bottom flask, and the slightly boiled reflux in water-bath after petroleum ether (60-90 DEG C) is added
Extract 1.5h, wherein the mass volume ratio (g/ml) of Dendrobium denneanum powder and petroleum ether is 1:4；Filtering is abandoned filtrate, is dried to
Constant weight is to remove liposoluble substance；The slightly boiled refluxing extraction 1.5h of ethyl alcohol that it is 80% with volumn concentration that the dregs of a decoction, which continue, filtering,
Abandon filtrate；The dregs of a decoction after drying move into again is added sodium hydroxide in beaker, wherein mole of sodium hydroxide and Dendrobium denneanum powder
Mass ratio (mmol/g) is 1:1；Magnetic agitation extracts 8h at room temperature, collects filtrate, repeats to extract once, after merging filtrate twice
It is adjusted to neutrality with hydrochloric acid, rotary evaporation to certain volume, wherein substance and Dendrobium denneanum powder volume matter after rotary evaporation
Amount is than being 7:2, with Sevag reagent (n-butanol: chloroform (v:v)=1:4) deproteinized, Sevag reagent with Dendrobium denneanum powder
Opisthosoma product mass ratio is 7:2, is repeated 4 times, and collects and merges supernatant rotary evaporation, the supernatant and Dendrobium denneanum powder of rotary evaporation
The volume mass ratio (mL/g) at end is 5:2；
Step 2, supernatant are slowly added to dehydrated alcohol with separatory funnel thereto, make ethyl alcohol body in stirring on magnetic stirring apparatus
Product reaches 40%, and precipitating is abandoned in centrifugation；Continue in filtrate plus dehydrated alcohol makes ethyl alcohol volume in system reach 60%, collects precipitating；According to
It is secondary with acetone, ethyl acetate, dehydrated alcohol wash precipitating after be centrifuged, saved backup in 4 DEG C of refrigerators.
Step 3, by after above-mentioned sample deionized water dissolving, be fitted into bag filter in beaker, in the item of magnetic agitation
Deionized water dialysis 80h, 8h is used to change a water under part；Sample solution is freeze-dried acid more up to Dendrobium denneanum after collection dialysis
Sugar.
Measurement using the A in embodiment 1 to glucuronic acid content, glucuronic acid content are 290.5 mg/g.
It is thin that the Dendrobium denneanum acidic polysaccharose (600 μ g/mL) being prepared using the present embodiment 3 can effectively improve RAW264.7
Born of the same parents' phagocytic activity, phagocytic index 1.94.
The Dendrobium denneanum acidic polysaccharose (600 μ g/mL) being prepared using the present embodiment 2 can promote RAW264.7 cell
TNF secretion-α, the yield of TNF-α are 1705pg/mL.
Above description has shown and described several preferred embodiments of invention, but as previously described, it should be understood that invention is not
It is confined to form disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations, modification
And environment, and can be carried out within that scope of the inventive concept describe herein by the above teachings or related fields of technology or knowledge
Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then it all should be in the appended power of invention
In the protection scope that benefit requires.
Claims (9)
1. a kind of preparation method of Dendrobium denneanum acidic polysaccharose, which comprises the following steps:
Step 1 takes Dendrobium denneanum powder in a round bottom flask, and the slightly boiled refluxing extraction in water-bath is added after petroleum ether, filters,
Filtrate is abandoned, drying to constant weight to remove liposoluble substance；The dregs of a decoction continue with the slightly boiled refluxing extraction of ethyl alcohol, filtering, abandoning filtrate；It is dry
The dregs of a decoction afterwards move into again is added sodium hydroxide in beaker, magnetic agitation extracts at room temperature, collects filtrate, repeats to extract once, close
And be adjusted to neutrality after filtrate with hydrochloric acid twice, rotation is evaporated to certain volume after abandoning precipitating, Sevag reagent deproteinized is added,
It is repeated 4 times, collects and merge supernatant rotary evaporation；
Step 2, supernatant are slowly added to dehydrated alcohol with separatory funnel, centrifugation is abandoned heavy in stirring on magnetic stirring apparatus thereto
It forms sediment；Continue in filtrate plus dehydrated alcohol, collection precipitate；It is centrifuged after successively washing precipitating with acetone, ethyl acetate, dehydrated alcohol,
It is saved backup in 4 DEG C of refrigerators；
Step 3, by after above-mentioned sample deionized water dissolving, be fitted into bag filter in beaker, under conditions of magnetic agitation
Use deionized water dialysis；Sample solution is freeze-dried up to Dendrobium denneanum acidic polysaccharose after collecting dialysis.
2. preparation method according to claim 1, which is characterized in that the boiling point of the petroleum ether in the step 1 is 60-90
DEG C, the mass volume ratio (g/ml) of Dendrobium denneanum powder and petroleum ether is 1:4-3:4；Reflux extracting time is 0.5h-1.5h；Institute
The volumn concentration for stating ethyl alcohol is 80%.
3. preparation method according to claim 1, which is characterized in that sodium hydroxide and Dendrobium denneanum in the step 1
The molal weight ratio (mmol/g) of powder is 1:1-5:1；Magnetic agitation extraction time is 4-8h.
4. preparation method according to claim 1, which is characterized in that the object of the first time rotary evaporation in the step 1
Matter and Dendrobium denneanum powder volume mass ratio are 3:2-7:2, and Sevag reagent is the n-butanol and chloroform that volume ratio is 1:4；
Sevag reagent with Dendrobium denneanum powder volume mass ratio be 3:2-7:2, the supernatant and Dendrobium denneanum of second of rotary evaporation
The volume mass ratio (mL/g) of powder is 5:2-10:2.
5. preparation method according to claim 1, which is characterized in that first time dehydrated alcohol in the step 2 adds
Dosage is the 40% of supernatant and dehydrated alcohol total volume；The additive amount of second of dehydrated alcohol is that filtrate and dehydrated alcohol are overall
Long-pending 60%.
6. preparation method according to claim 1, which is characterized in that the dialysis time in the step 3 is 64h-80h,
8h changes a water.
7. the Dendrobium denneanum acidic polysaccharose that preparation method described in claim 1-6 any claim is prepared.
8. Dendrobium denneanum acidic polysaccharose according to claim 7, which is characterized in that be α-configuration, molecular weight is Mw=5.78
× 104 Da, wherein glucuronic acid content is 292.2 mg/g.
9. Dendrobium denneanum acidic polysaccharose as claimed in claim 7 is improved in immune drug in preparation and is applied.
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| CN105348408A (en) * | 2015-12-15 | 2016-02-24 | 云南民族大学 | Method for increasing leaching rate of polysaccharide in dendrobium denndanum |
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| CN105348408A (en) * | 2015-12-15 | 2016-02-24 | 云南民族大学 | Method for increasing leaching rate of polysaccharide in dendrobium denndanum |
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