CN108329398A - A kind of bletilla polysaccharide and its extracting method - Google Patents
A kind of bletilla polysaccharide and its extracting method Download PDFInfo
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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Abstract
The present invention relates to biopharmacy technical fields, are related to the extracting method of medicinal material, and in particular to a kind of bletilla polysaccharide and its extracting method.Described method includes following steps：1) before extraction, by bletilla, ultrasonic vibration crushes in a cold or frozen state, obtains bletilla solid particle；Can also it further comprise：2) gained bletilla solid particle is extracted using continuous dynamic countercurrent, obtains extracting solution；3) purified to the extracting solution, crystallize to obtain the final product.The present invention achieves following technique effect：Method is simple, improves the content of bletilla polysaccharide in extract；Production efficiency is improved, solvent dosage is reduced, reduces Extracting temperature, reduces energy consumption, improves the input efficiency of industry；Production efficiency can be improved in the effect for simplifying separation, saves efficiency；Ensure the purity and quality of bletilla polysaccharide.The extracting method processing step of the present invention is short, simple for process, is suitable for industrialized production.
Description
Technical field
The invention belongs to biopharmacy technical fields, are related to the extracting method of medicinal material, and in particular to a kind of bletilla polysaccharide and
Its extracting method.
Background technology
Bletilla (RhizomaBletillae) is orchid family bletilla platymiscium【Bletillastriata.(Thunb.)
Reichb.f.】, it is the dry tuber that a kind of Chinese medicine of medication long history is orchid bletilla, China《Compendium of Materia Medica》Have
It records：" bletilla alias even and grass, give free of charge, root white by Radix Glycyrrhizae, even and and give birth to, therefore say bletilla.Its stem tuber can be used as medicine, most early in
《Sheng Nong's herbal classic》In it is on the books, to be concealed in each version after 1963《Chinese medicine pharmacopeia》In one of herbal species, in vain
And containing a large amount of mucilage, property is bitter, puckery, sweet, is slightly cold, and enters return lung, liver, stomach, there is that astringing to arrest bleeding, heat-clearing be wet, detumescence and promoting granulation
The effect of.It is clinically widely used in treatment hemoptysis, haematemesis, sore swollen toxin, traumatic hemorrhage, chapped skin, pulmonary tuberculosis hemoptysis and bursts
Ulcer disease bleeding etc., it is significant in efficacy.
The main chemical compositions of bletilla are bibenzyl, luxuriant and rich with fragrance class and its derivative, in addition, also containing a small amount of volatile oil, mucus
Matter, bletilla mannan and starch and glucose.Modern study analysis shows, in bletilla contain a large amount of plant polysaccharides,
Content is about 40%~50% in dry bulb tissue, these polysaccharide are mainly by β-Isosorbide-5-Nitrae-mannose, β-Isosorbide-5-Nitrae-glucose
With β -1,6- glucose residues composition belongs to neutral heteropolysaccharide., and main component is two kinds of Glucomannan, mannose and glucose
The ratio of glycosyl is about 4：1.Bletilla polysaccharide has higher viscosity as natural macromolecular material, is a kind of excellent natural
Thickener and emulsion stabilizer, bletilla polysaccharide have functional slow-release, local retention, itself degradability, non-stimulated, nontoxic pair
Effect, because of its good biocompatibility and degradability, is increasingly taken seriously in medicinal preparation process, can be used as various
The load drug application material and bioprosthetic material of form.Mainly have as pharmaceutical carrier, bletilla polysaccharide category natural small molecule polymer, it can
As the capsule material of micro-capsule, the matrix of suppository, the adhesive of tablet, the insolated layer materials of sugar coated tablet, film filmogen can also
As coupling agent for medical use, the dextran product using bletilla polysaccharide as primary raw material also developed in addition；Bletilla polysaccharide is because of its uniqueness
Performance, and have higher safety, can be used as food additives, be widely used in food industry, it is more using bletilla
The excellent film forming of sugar, can prepare the antistaling agent of fruit film, it is possible to reduce and fruit moisture evaporation breathes after inhibiting picking, with
Achieve the purpose that extend insurance.
Bletilla polysaccharide is a kind of ideal cosmetic additive agent while being also active constituent, it has convergence to skin, prolongs
Slow aging, moisturizing, helps emulsification and other effects at thickening.Adding the cosmetics of bletilla polysaccharide effectively anti-frostbite and can be chapped from the cold, promote blood
Liquid cycle, moisture-keeping crease-shedding, and have no toxic side effect, someone develops the cosmetics for using bletilla polysaccharide as main active substances,
Have the function of bactericidal antiphlogistic, freckle removing and whitening, crease-resistant health care, can industrially make thickener, starches silk and cotton yarn, or apply
Material.
It is long-standing that bletilla is used as convergence, hemostatic to apply, and haemostatic effect is definitely reliable, and the mechanism of action is demonstrate,proved with it
It is bright to have a large amount of bletilla glue for shortening the clotting time, human body being inhibited to contain the dissolving work of cellulose related.Bletilla quality stick it is greasy,
Containing colloid, it can improve local blood circulation, promote epithelial cell reparation, can stop blooding, sore, moisturizing and myogenic.Bletilla carries
It takes object also to be used by experiment, artificial thrombus can be quickly formed, not only trauma hemostasis is common, also there is preferable effect to visceral hemorrhage.
Bletilla has apparent promotion keratinocyte effect, and this rush migration effect is to treatment skin trauma, hemostasis and immediate union
It has a major impact, further illustrates reparation and metabolic process that bletilla glue directly takes part in damaged tissues or cell.Adhesion shape
At with generality, it was reported that 50%~100% abdominal cavity and operation on pelvis can lead to different degrees of adhesion.Caused by adhesion
Clinical severe complication includes intestinal obstruction, sterility, chronic pelvic pain etc., increase the difficulty performed the operation again and further
The potentiality of complication occurs.The purpose of preventing adhesions is eliminated under the premise of ensureing proper wound healing and avoiding infection
Or reduce the incidence and severity of adhesion.CN103893834A is related to bletilla polysaccharide extraction purification from orchid bletilla
It obtains, for film forming for the experiment that prevents adhesion, experiment shows that there is bletilla polysaccharide good biocompatibility and prevention of postoperative adhesion to make
With the invention implies that bletilla polysaccharide may be used as a kind of prevention of postoperative adhesion new material and can be made into and is used clinically for resisting
The diversified forms product of adhesion, has a vast market foreground.Since bletilla Thick many candies molecular weight distribution is wide, solubility difference compared with
Greatly, activity and the relationship between effect and its molecular weight still need to further investigate, it is therefore desirable to provide a large amount of quality high white
And polysaccharide carries out further grading system for narrow molecular weight distribution or modified bletilla polysaccharide, meets the high-purity more to suit the requirements
Bletilla polysaccharide.CN106674370A is related to a kind of bletilla glue and its extracting method, the extracting method of the bletilla glue include：Crush step
Suddenly, screening step, extraction step and purification step.Bletilla is screened by saturated nacl aqueous solution after crushed, through adding water after screening
It is ultrasonically treated, and carries out ethanol solution purification.The first filtrate volume of this method is larger, and the amount that ethyl alcohol is added is multiplied, and causes
It is more in solvent usage amount, cause post-processing and energy consumption larger thus.During extracting bletilla polysaccharide, in pretreatment
If pulverized particles are too small, due to the interaction between the viscosity and molecule of bletilla polysaccharide, make it in subsequent separation
Process become difficult, granularity is too big again it cannot be guaranteed that the utilization rate of raw material.Therefore bletilla polysaccharide should be kept by providing one kind
Three-dimensional structure and quality, also can mild condition simple process obtain that the extracting method of the high bletilla polysaccharide of purity just becomes must
It needs.
Invention content
It is an object of the invention in view of the defects existing in the prior art and different, provide and a kind of extracting bletilla from bletilla
The method of polysaccharide and the bletilla polysaccharide extracted by this method.
The bletilla polysaccharide content that the above method extracts reaches 90% or more, and extraction rate reached to 85% or more is obtained
Bletilla polysaccharide purity height, stable quality, good drug efficacy；Method is reasonable, with short production cycle, and low energy consumption, and environmental pollution is small, can be fast
Speed efficiently extracts bletilla polysaccharide from bletilla, and can greatly improve the recovery rate of bletilla glue, improves raw material availability, drop
Low cost is suitable for mass producing.The present invention also solve together during extraction due to bletilla polysaccharide viscosity into
The process of row post-processing becomes very difficult problem.
To achieve the goals above, the present invention adopts the following technical scheme that：
A method of extraction bletilla polysaccharide includes the following steps：
1) by bletilla, ultrasonic vibration crushes in a cold or frozen state, obtains bletilla solid particle；
Extracting method of the present invention, in step 1), bletilla can be directly used for extracting, also can be after sliced or particle again
It extracts.
After bletilla is preferably cut into graininess by the present invention, then chilled ultrasonic fragmentation carries out continuous dynamic countercurrent extraction, with
Ensure farthest from wherein extracting active constituent, and ensure the size by particle after ultrasonic echography, it is ensured that mistake
When filter because viscosity is too big and caused by block.Surpass under chilled state again it is further preferred that bletilla is broken into graininess
Crushing is swung in acoustic shock.The particle volume is in 0.001cm3And 8cm3Between, and ensure the size of each dimension within 2cm.
It is further preferred that the particle volume is in (0.1~2) × (0.1~2) × (0.1~2) cm3。
The step 1) is bletilla (or bletilla particle after crushed) to be placed in 1-5min in liquid nitrogen, then again by it
It is placed in the solution of dry ice/organic solvent after carrying out ultrasonic vibration crushing 0.1-30min at once, stands and arrive solution temperature to room temperature
Bletilla particle is obtained by filtration；
Preferably, the organic solvent is acetone and/or ethyl alcohol, and acetone and ethyl alcohol volume can be any proportions, as
It is preferred that the volume ratio of ethyl alcohol and acetone is (6-8):3.The mass volume ratio of the further preferred bletilla and organic solvent is
10Kg/30~100L.
Preferably, the ultrasonic power of the ultrasound is 300-400W, and 20~40 μm of ultrasonic amplitude is (added with bletilla particle
It is loaded in above-mentioned dry ice/organic solvent 1L solution).Such parameter may insure that treated, and bletilla particle was easier to make for
Filter, and cell wall can be allowed adequately to rupture.
Further, extracting method of the present invention, including：
1) by bletilla, ultrasonic vibration crushes in a cold or frozen state, obtains bletilla solid particle；
2) gained bletilla solid particle is extracted using continuous dynamic countercurrent, obtains extracting solution；
3) purified to the extracting solution, crystallize to obtain the final product.
Preferably, extracting method of the present invention, in step 2), solid in the continuous dynamic countercurrent extraction and
The ratio of Extraction solvent is：The Extraction solvent of 80-200L/h is added in 10~20Kg/h of gained bletilla solid, which contributes to white
And it is come into full contact with solvent.
Preferably, the Extraction solvent is water or 1-5% saline solutions；
And/or 60-80 DEG C of the temperature of the extraction, extraction time 40-60min；Such extraction conditions can utmostly compel
It is that active ingredient dissolves in the solution to make cell rupture, ensures the three-dimensional structure of bletilla polysaccharide not in the environment of high temperature and soda acid
It destroys and degrades, it is ensured that extraction effect.
Extracting method of the present invention, in step 3), the purifying includes：
3.1) gained extracting solution is adjusted into pH value between 2-3；
3.2) clarified solution obtained by step 3.1) is subjected to UF membrane and concentration, obtains the trapped fluid of ultrafiltration membrane；Wherein film point
To pass through microfiltration membranes and ultrafiltration membrane from including filtered fluid；
3.3) ethyl alcohol is added in trapped fluid obtained by step 3.2), is stood to get the bletilla polysaccharide crystal.
Preferably, in step 3.1), pH value is adjusted with trichloroacetic acid, stirs 3-5h；Add 0.3~0.5% activated carbon
0.5-1h is stirred, then stands 3-5h, centrifugal filtration obtains clarified solution.
In step 3.2), it will be connected with the water inlet end of microfiltration membranes subsystem equipped with clarification liquid bath, microfiltration membranes subsystem
Water outlet is connected by pipeline with the water inlet end of ultrafiltration membrane subsystem；The water outlet of microfiltration membranes subsystem is equipped with concentrate pipeline
Lead to the processing unit for producing bletilla polysaccharide.The material membrane material for the preferred Kynoar of acid resistance film that membrane filtration system uses
Material.
It is further preferred to use micro-filtrate membrane filtration, used in micro-filtration membrane aperture at 0.4-1 μm, assist in removing by
Suspended particulates, the big impurity of the solid particles equimolecular quantity such as starch.Usually, operating pressure is 10-25bar, operation temperature 15-
35℃。
It is further preferred to use ultrafiltration membrance filter, used in membrane material be the film that can retain 10KDa, contribute to
Except impurity such as small-molecule substances.Usually, operating pressure is 30-45bar, 15-35 DEG C of operation temperature.
In step 3.3), the amount that ethyl alcohol is added so that final ethanol concentration is 60~80%；The operation of the crystallization is：It will
Ethyl alcohol be directly added into gained trapped fluid in, be then allowed to stand crystallization to get.
Gained bletilla polysaccharide carries out molecular weight determination using High Performance Gel Permeation Chromatography (HPGPC), obtains bletilla polysaccharide
Average molecular weight in 12000~320000Da.
On the basis of common knowledge of the art, above-mentioned each optimum condition can be combined with each other each preferably to get the present invention
Embodiment.
As the preferred technical solution of the present invention, a kind of extracting method of bletilla polysaccharide is provided, the specific method is as follows：
1) it is (0.5~1.5) × (0.5~1.5) × (0.2-1) cm bletilla rhizome to be sliced into volume3Specification takes 15-
20Kg is placed in 1-5min in liquid nitrogen, then slice is placed in the solution of dry ice/ethanol/acetone, wherein the volume of ethyl alcohol and acetone
Than being (6-8):3, ultrasonic vibration crushes 15-30min and takes bletilla after the solution after crushing is placed in stirring at room temperature 20-30 hours
Particle；
2) bletilla particle obtained by step 1) is subjected to continuous dynamic countercurrent extraction, with the bletilla particle of 15-20Kg/h, 150-
The speed of 180L/h saline solutions enters progress dynamic countercurrent extraction in equipment, keeps 60-80 DEG C of ultrasonic extraction tube temperature degree, extraction
It is spare to obtain the extracting solution containing bletilla polysaccharide by 50-60min；
3) trichloroacetic acid tune pH to 2~3 is added in the extracting solution obtained by step 2), adds activated carbon stirring, then stand
Centrifugation obtains supernatant liquid body after 3-5h；
4) liquid obtained by step 3) is input in acid resistance film separation system, carries out microfiltration membranes successively, ultrafiltration membrane, obtains
Trapped fluid；
5) ethyl alcohol of 3 times of volumes is slowly poured into the trapped fluid concentrated obtained by step 4), is stirred evenly, stood, is precipitated
Bletilla polysaccharide solid.
The present invention relates to the commercially available acquisition of raw material or reagent.
The present invention achieves following technique effect：
(1) after low temperature ultrasonic processing bletilla of the present invention, bletilla is extracted using continuous flow upstream ultrasonic extraction, method
Simply, the content of bletilla polysaccharide is improved；
(2) continuous flow upstream ultrasonic extraction improves production efficiency, reduces solvent dosage, reduces Extracting temperature, reduces
Energy consumption, improves the input efficiency of industry；
(3) the step of present invention uses is used in conjunction jointly with film concentration step, simplifies the effect of separation, and production effect can be improved
Rate saves efficiency；
(4) recrystallization method is used using the present invention, it is ensured that the purity and quality of bletilla polysaccharide.
(5) extracting method processing step of the invention is short, simple for process, is suitable for industrialized production.
Present invention simultaneously provides the preparation-obtained bletilla polysaccharides of any of the above-described technical solution.
Inventor is it was unexpectedly observed that viscosity has application performance more outstanding in the bletilla polysaccharide of 26000-30000cP；
More preferable 28100-30000cP；Further preferred 28600-29200cP.
The rate of bletilla polysaccharide provided by the present invention, degradation in vivo more mitigates, viscosity bigger, has good answer
Use foreground.
Specific implementation mode
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
In following embodiment, the ultrasonic power of the ultrasound is 300-400W, 20~40 μm of ultrasonic amplitude (added with
It is loaded in the above-mentioned dry ice of bletilla particle/organic solvent 1L solution).
Embodiment 1
The method that the present embodiment provides a kind of to extract bletilla polysaccharide from bletilla, includes the following steps：
1) it is 0.1 × 0.1 × 0.1cm the fresh bletilla rhizome of acquisition to be sliced into volume3Specification takes 10Kg to be placed in liquid nitrogen
Middle 1min, then chip granulate is placed in the solution 30L of dry ice/ethyl alcohol, ultrasonic vibration crushes 0.1min, and the solution after crushing is set
After stirring 12 hours at room temperature, bletilla particle is taken；
2) bletilla particle obtained by step 1) is subjected to continuous dynamic countercurrent extraction, with the bletilla particle of 10Kg/h, 80L/h's
Water carries out dynamic countercurrent extraction, keeps ultrasonic extraction pipe temperature 60 C, extracts 40min, obtains the extracting solution containing bletilla polysaccharide；It should
Extracting solution input melt cinder extracts separator, detaches filter residue, obtains the extracting solution of bletilla polysaccharide, spare；
3) trichloroacetic acid tune pH to 2~3 is added in the extracting solution obtained by step 2), adds 0.3~0.5% activated carbon and stirs
0.5h is mixed, is centrifuged after standing 3-5h and obtains supernatant liquid body；
4) liquid obtained by step 3) is input in acid resistance film separation system, carries out microfiltration membranes successively, ultrafiltration membrane, obtains
Trapped fluid；
Wherein, for the micro-filtration membrane aperture at 0.4 μm, operating pressure is 10bar, 15 DEG C of operation temperature；
The ultrafiltration membrane can retain energy molecular cut off in the film of 10kDa or more, and operating pressure is 30Bar, operation temperature
15℃；
5) ethyl alcohol is slowly poured into the trapped fluid concentrated obtained by step 4), the ethyl alcohol of addition accounts for solution 60%, and stirring is equal
It is even, it stands, bletilla polysaccharide solid 3.56Kg is precipitated.
Embodiment 2
The method that the present embodiment provides a kind of to extract bletilla polysaccharide from bletilla, includes the following steps：
1) it is 0.8 × 0.8 × 0.3cm the fresh bletilla rhizome of acquisition to be sliced into volume3Specification takes 13Kg to be placed in liquid nitrogen
Middle 2min, then slice is placed in the solution 50L of dry ice/ethanol/acetone that (volume ratio of ethyl alcohol and acetone is 3:7), ultrasound shake
It swings and crushes 10min, after the solution after crushing is placed in stirring at room temperature 18 hours, take bletilla particle；
2) bletilla particle obtained by step 1) is subjected to continuous dynamic countercurrent extraction, with the bletilla particle of 13Kg/h, 100L/h
1% brine carry out dynamic countercurrent extraction, keep 65 DEG C of ultrasonic extraction tube temperature degree, extract 45min, obtain the extraction containing bletilla polysaccharide
Liquid；Extracting solution input melt cinder is extracted into separator, filter residue is detached, obtains the extracting solution of bletilla polysaccharide, it is spare；
3) trichloroacetic acid tune pH to 2~3 is added in the extracting solution obtained by step 2), adds 0.3~0.5% activated carbon and stirs
0.5h is mixed, is centrifuged after standing 3-5h and obtains supernatant liquid body；
4) liquid obtained by step 3) is input in acid resistance film separation system, carries out microfiltration membranes successively, ultrafiltration membrane, obtains
Trapped fluid；
Wherein, for the micro-filtration membrane aperture at 0.6 μm, operating pressure is 15bar, 20 DEG C of operation temperature；
The ultrafiltration membrane can molecular cut off in the film of 10KDa or more, operating pressure is 35Bar, 20 DEG C of operation temperature；
5) ethyl alcohol slowly to be poured into the trapped fluid concentrated obtained by step 4) so that the amount of ethyl alcohol accounts for 65%, stirs evenly,
It stands, bletilla polysaccharide solid 4.56Kg is precipitated.
Embodiment 3
The method that the present embodiment provides a kind of to extract bletilla polysaccharide from bletilla, includes the following steps：
1) it is 1.5 × 1.5 × 0.8cm the fresh bletilla rhizome of acquisition to be sliced into volume3Specification takes 18Kg to be placed in liquid nitrogen
Middle 4min, then slice is placed in the solution 80L of dry ice/ethanol/acetone that (volume ratio of ethyl alcohol and acetone is 7:3), ultrasound shake
It swings and crushes 20min, after the solution after crushing is placed in stirring at room temperature 24 hours, take bletilla particle；
2) bletilla particle obtained by step 1) is subjected to continuous dynamic countercurrent extraction, with the bletilla particle of 18Kg/h, 170L/h
The speed of 3% saline solution enter in equipment progress dynamic countercurrent extraction, keep ultrasonic extraction pipe temperature 70 C, extract 55min,
The extracting solution of bletilla polysaccharide must be contained；Extracting solution input melt cinder is extracted into separator, filter residue is detached, obtains the extraction of bletilla polysaccharide
Liquid, it is spare；
3) trichloroacetic acid tune pH to 2~3 is added in the extracting solution obtained by step 2), adds 0.3~0.5% activated carbon and stirs
0.5h is mixed, is centrifuged after standing 3-5h and obtains supernatant liquid body；
4) liquid obtained by step 3) is input in acid resistance film separation system, carries out microfiltration membranes successively, ultrafiltration membrane, obtains
Trapped fluid；
Wherein, for the micro-filtration membrane aperture at 0.8 μm, operating pressure is 20bar, 30 DEG C of operation temperature；
For the ultrafiltration membrane energy molecular cut off in the film of 10KDa or more, operating pressure is 40Bar, 30 DEG C of operation temperature；
5) ethyl alcohol of 3 times of volumes is slowly poured into the trapped fluid concentrated obtained by step 4), is stirred evenly, stood, is precipitated
Bletilla polysaccharide solid 6.21Kg.
Embodiment 4
The method that the present embodiment provides a kind of to extract bletilla polysaccharide from bletilla, includes the following steps：
1) it is 2 × 2 × 1cm the fresh bletilla rhizome of acquisition to be sliced into volume3Specification takes 20Kg to be placed in liquid nitrogen
5min, then slice is placed in the solution 100L of dry ice/acetone slice, ultrasonic vibration crushes 30min, and the solution after crushing is placed in
After stirring 36 hours at room temperature, bletilla particle is taken；
2) bletilla particle obtained by step 1) is subjected to continuous dynamic countercurrent extraction, with the bletilla particle of 20Kg/h, 200L/h
5% saline solution carry out dynamic countercurrent extraction, keep 80 DEG C of ultrasonic extraction tube temperature degree, extract 60min, obtain carrying containing bletilla polysaccharide
Take liquid；Extracting solution input melt cinder is extracted into separator, filter residue is detached, obtains the extracting solution of bletilla polysaccharide, it is spare；
3) trichloroacetic acid tune pH to 2~3 is added in the extracting solution obtained by step 2), adds 0.3~0.5% activated carbon and stirs
0.5h is mixed, is centrifuged after standing 3-5h and obtains supernatant liquid body；
4) liquid obtained by step 3) is input in acid resistance film separation system, carries out microfiltration membranes successively, ultrafiltration membrane, obtains
Trapped fluid；
Wherein, for the micro-filtration membrane aperture at 1 μm, operating pressure is 25bar, 35 DEG C of operation temperature；The ultrafiltration membrane retention
For molecular weight in the film of 10KDa or more, operating pressure is 45Bar, 35 DEG C of operation temperature；
5) 4 times of volume ethanols are slowly poured into the trapped fluid concentrated obtained by step 4), so that the amount of ethyl alcohol is accounted for 80%, stirring
Uniformly, it stands, bletilla polysaccharide solid 7.08Kg is precipitated.
Comparative example 1
The operation of this comparative example is with embodiment 1, and the operation for differing only in step 1) is different, the extraction operation of the comparative example
For：The fresh bletilla rhizome of acquisition is added the water of 2 times of volumes to be ground into 10-40 mesh by step 1) with colloid mill.
Obtain bletilla polysaccharide solid 1.84kg.
Comparative example 2
The operation of this comparative example is with embodiment 1, and the operation for differing only in step 1) is different, the extraction operation of the comparative example
For：The fresh bletilla rhizome of acquisition is added the water mill of 2 times of volumes to be ground into 10-40 mesh by step 1) with colloid, and carries out ultrasonic 1-
5min。
Obtain bletilla polysaccharide solid 2.07kg.
Comparative example 3
The operation of this comparative example is with embodiment 1, and the operation for differing only in step 1) is different, the extraction operation of the comparative example
For：The fresh bletilla rhizome of acquisition is ground into 10-40 mesh by step 1) with liquid nitrogen frozen Lowtemperaturepulverizer.
Obtain bletilla polysaccharide solid 1.94kg.
Comparative example 4
The operation of this comparative example is with embodiment 1, and the operation for differing only in step 4) is different, the extraction operation of the comparative example
For extracting solution obtained by step 3) is concentrated into the 1/3 of original volume.
Obtain bletilla polysaccharide solid 2.05kg.
Comparative example 5
The operation of this comparative example is with embodiment 1, and the operation for differing only in step 3) is different, the extraction operation of the comparative example
For the extracting solution obtained by step 2) is directly carried out UF membrane.
Obtain bletilla polysaccharide solid 2.14kg.
Test example 1
This test example offer embodiment 1-4, comparative example 1-5 provide the purity and yield ratio of the method for extraction bletilla polysaccharide
Compared with.
Following yield refers to yield of the polysaccharide in wet bletilla, wherein the moisture in wet bletilla accounts for 14.9%.
Table 1
By table 1 as it can be seen that embodiment 1-4 provided by the present invention is significantly better than comparative example 1-5 on yield and purity；It proves
In the preparation method that the present invention is advocated, desirable level can reach by the control of each conditional parameter.
Test example 2
This test example provides embodiment 1-4, the anti-blocking agent experiment for the bletilla polysaccharide that comparative example 1-5 is provided and viscosity examination
It tests.
(1) embodiment 1-4, each 10g of comparative example 1-5 bletilla polysaccharides after sterilizing are weighed, 50-60 DEG C of sterilizing note is separately added into
It penetrates and uses water 40mL, stirring makes it fully dissolve, and places 50min, and ultrasound removes wherein bubble to get gelatinous bletilla polysaccharide.
(2) bletilla polysaccharide of the provided gelled of above-described embodiment 1-4, comparative example 1-5 is subjected to blood vacuum suction
Experiment, residual content is measured by microplate reader.Bletilla polysaccharide in blood vacuum suction method after confirmation is kept for 24 hours at 37 DEG C
Gel residue amount.
(3) bletilla polysaccharide of the provided gelled of above-described embodiment 1-4, comparative example 1-5 is used into Brookfield
(Brookfield) viscosimeter carries out viscosity test.
It is as shown in table 2 to investigate test result：
2 blood vacuum of table aspirates and viscosity test result
Find out from 2 data result of table, bletilla polysaccharide prepared by technical solution of the present invention is using gelling agent as antiblocking agent
In, residual quantity is higher than the index parameter of the bletilla polysaccharide obtained by other methods, illustrates the rate of degradation in vivo
More mitigate, while viscosity is with respect to bigger, other positions will not be spilt over because of mobility and cannot be effectively to operation
Play screen effect in position.
Test example 3
The bletilla polysaccharide that this test example provides embodiment 1-4, comparative example 1-5 is provided carries out hemostatic experiment.
In order to analyze the blood coagulation time of the bletilla polysaccharide prepared according to embodiment, when using activated partial thromboplastin
Between assay kit (activated Partial thromb oplastin Time Kit) evaluated.By 0.1mL's
Blood plasma is put into testing tube, and after being cultivated 2 minutes under the conditions of 37 DEG C, adds the active partial thromboplastin time examination of 0.1mL
Agent (aPPT) is cultivated 3 minutes under the conditions of 37 DEG C.It is put into the embodiment 1-4 of 0.1mL, the bletilla polysaccharide that comparative example 1-5 is provided
The hydrosol, and setting time is determined in 10 minutes, as shown in table 3.
The hemostatic evaluation of 3 bletilla polysaccharide of table
Bletilla polysaccharide prepared by technical solution of the present invention is found out compared with comparative example from 3 data result of table, when blood clotting
Between it is short.Therefore, the bletilla polysaccharide anthemorrhagic performance that prepared by technical scheme of the present invention is more excellent.
Although above having used general explanation, specific implementation mode and experiment, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (10)
1. a kind of method of extraction bletilla polysaccharide, which is characterized in that include the following steps：
1) before extraction, by bletilla, ultrasonic vibration crushes in a cold or frozen state, obtains bletilla solid particle.
2. according to the method described in claim 1, it is characterized in that, in step 1), after the bletilla is cut to graininess, then pass through
It freezes ultrasonic fragmentation and carries out continuous dynamic countercurrent extraction；
Preferably, the volume of the particle is in 0.001cm3And 8cm3Between, and ensure the size of each dimension within 2cm.
3. according to the method described in claim 2, it is characterized in that, the step 1) is, the bletilla is placed in 1- in liquid nitrogen
Then 5min is placed it in the solution of dry ice/organic solvent after carrying out ultrasonic vibration crushing 0.1-30min at once, is stood again
Bletilla particle is obtained by filtration to solution temperature to room temperature；
Preferably, the operating condition of the ultrasound is：Ultrasonic work(is loaded in the 1L solution added with dry ice/organic solvent of bletilla
Rate 300-400W, 20~40 μm of ultrasonic amplitude.
4. according to the method described in claim 3, it is characterized in that, in step 1), the organic solvent is acetone and/or second
Alcohol；
It is preferred that the mass volume ratio of the bletilla and organic solvent is 10Kg/30~100L,
And/or the mixed solvent that the organic solvent is acetone and ethyl alcohol, the volume ratio of the ethyl alcohol and acetone is (6-8):3.
5. according to claim 1-4 any one of them methods, which is characterized in that extracting method of the present invention further includes：
2) gained bletilla solid particle is extracted using continuous dynamic countercurrent, obtains extracting solution；
3) purified to the extracting solution, crystallize to obtain the final product.
6. according to the method described in claim 5, in step 2), solid and Extraction solvent in the continuous dynamic countercurrent extraction
Ratio be：The Extraction solvent of 80-200L/h is added in 10~20Kg/h of gained bletilla solid；
Preferably, the Extraction solvent is water or 1-5% saline solutions；
And/or 60-80 DEG C of the temperature of the extraction, extraction time 40-60min.
7. method according to claim 5 or 6, which is characterized in that in step 3), the purifying includes：
3.1) gained extracting solution is adjusted into pH value between 2-3；
3.2) clarified solution obtained by step 3.1) is subjected to UF membrane and concentration, obtains the trapped fluid of ultrafiltration membrane；Wherein UF membrane packet
Microfiltration membranes and ultrafiltration membrane will be passed through by including filtered fluid；
3.3) ethyl alcohol is added in trapped fluid obtained by step 3.2), is stood to get the bletilla polysaccharide crystal.
8. the method according to the description of claim 7 is characterized in that in step 3.1), pH value is adjusted with trichloroacetic acid, stirs 3-
5h；0.3~0.5% activated carbon stirring 0.5-1h is added, then stands 3-5h, centrifugal filtration obtains clarified solution；
And/or
In step 3.3), the amount that ethyl alcohol is added so that final ethanol concentration is 60~80%.
9. method according to claim 7 or 8, which is characterized in that in step 3.2), make in the microfiltration membranes and ultrafiltration membrane
Film is the material membrane material of Kynoar；
Preferably, the micro-filtrate membrane filtration, used in micro-filtration membrane aperture at 0.4-1 μm, operating pressure is 10-25bar；
And/or the ultrafiltration membrance filter, used in membrane material be the film that can retain 10KDa, operating pressure is 30-
45bar。
10. a kind of bletilla polysaccharide, which is characterized in that obtained by any one of claim 1-9 the methods；
Preferably, the viscosity of the bletilla polysaccharide is in 26000-30000cP, preferably 28100-30000cP；More preferable 28600-
29200cP。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109847111A (en) * | 2018-12-21 | 2019-06-07 | 北京颐方生物科技有限公司 | A kind of adherence preventing material and preparation method thereof comprising bletilla polysaccharide |
| CN111803577A (en) * | 2020-06-22 | 2020-10-23 | 庆元县乾宁道地药材有限公司 | Preparation process of bletilla striata gum compound |
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|---|---|---|---|---|
| CN105521492A (en) * | 2015-12-14 | 2016-04-27 | 成都中医药大学 | Porous bletilla gum, preparation method therefor and application thereof |
| CN106674370A (en) * | 2017-01-24 | 2017-05-17 | 贵州省武陵山药用植物白芨开发有限公司 | Bletilla striata gelatin and preparation method thereof |
| CN106866835A (en) * | 2017-04-14 | 2017-06-20 | 张仕英 | A kind of ultrasonic technology extracts the preparation method of bletilla striata extract |
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2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105521492A (en) * | 2015-12-14 | 2016-04-27 | 成都中医药大学 | Porous bletilla gum, preparation method therefor and application thereof |
| CN106674370A (en) * | 2017-01-24 | 2017-05-17 | 贵州省武陵山药用植物白芨开发有限公司 | Bletilla striata gelatin and preparation method thereof |
| CN106866835A (en) * | 2017-04-14 | 2017-06-20 | 张仕英 | A kind of ultrasonic technology extracts the preparation method of bletilla striata extract |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109847111A (en) * | 2018-12-21 | 2019-06-07 | 北京颐方生物科技有限公司 | A kind of adherence preventing material and preparation method thereof comprising bletilla polysaccharide |
| CN109847111B (en) * | 2018-12-21 | 2021-07-30 | 北京颐方生物科技有限公司 | Anti-adhesion material containing bletilla striata polysaccharide and preparation method thereof |
| CN111803577A (en) * | 2020-06-22 | 2020-10-23 | 庆元县乾宁道地药材有限公司 | Preparation process of bletilla striata gum compound |
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