CN106883303A - A kind of preparation method and application of geranium wilfordii polysaccharide and oligosaccharide - Google Patents
A kind of preparation method and application of geranium wilfordii polysaccharide and oligosaccharide Download PDFInfo
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Abstract
The present invention relates to a kind of geranium wilfordii polysaccharide and the preparation method and application of oligosaccharide, ultrasound assisted extraction technique and fermentation technique are applied to the extraction purification process of geranium wilfordii polysaccharide and oligosaccharide, can effectively solve the integration system of geranium wilfordii polysaccharide and oligosaccharide for problem；Geranium wilfordii polysaccharide prepared by the present invention can be effectively used for preparing the medicine and health products of strengthen immunity, and geranium wilfordii oligosaccharide can be effectively used for preparing antifatigue medicine and health products.
Description
Technical field
The present invention relates to field of medicaments, the preparation method and application of more particularly to a kind of geranium wilfordii polysaccharide and oligosaccharide.
Background technology
Geranium wilfordii is MangNiu Er seedlings section plant Mang ox seedling Erodium stephanianum Willd., heroubill
The dry aerial parts of Geranium wilfordii Maxim. or Carolina Cranesbill Herb Geranium carolinianum L..Tool
There are wind-damp dispelling, degrading the channel, antidiarrheal dysentery, antibacterial, antiviral, anti-oxidant, antitumor, anti-inflammatory and antalgic, hypoglycemic, liver protection etc. to act on.Face
Bed is mainly used in arthralgia pain due to rheumatism, and numbness contracture, muscles and bones is ached, dysentery.The now research to its active ingredient relates generally to tan
The compositions such as matter, flavones, organic acid, volatile oil, triterpenes, sterols, lignin, the research to polysaccharide therein and oligosaccharide
There is not been reported, in particular how solves integration system and also has no report for geranium wilfordii polysaccharide and oligosaccharide.
The content of the invention
For above-mentioned situation, in order to overcome the defect of prior art, it is many that the purpose of the present invention is just to provide a kind of geranium wilfordii
The preparation method of sugar and oligosaccharide, the preparation and geranium wilfordii polysaccharide that can effectively solve geranium wilfordii polysaccharide and oligosaccharide strengthens preparing
The application of application and geranium wilfordii oligosaccharide in the medicine and health products of immunity in antifatigue medicine and health products is prepared.
The technical scheme that the present invention is solved is that the preparation method of a kind of geranium wilfordii polysaccharide and oligosaccharide is comprised the following steps：
Take geranium wilfordii and dry, pulverize into meal；Take geranium wilfordii meal, to adding distilled water in geranium wilfordii meal, geranium wilfordii meal with steam
The weight ratio of distilled water is 1：11-22, soaks 23-46min, 84 DEG C of -98 DEG C of ultrasonic extraction 41-97min, and filtering, filtrate is concentrated into
Proportion is the medicinal extract of 1.11-1.20, adds ethanol ethanol content is reached volumetric concentration for 66%-79%, 4 DEG C of standing 8-15h,
6000r/min is centrifuged 45min, obtains supernatant A and centrifugation B, and supernatant A is standby, centrifugation B freeze-dryings, obtains old stork
Careless Thick many candies powder C, the distilled water for taking geranium wilfordii Thick many candies powder C 10 times of weight of addition makes to dissolve to obtain geranium wilfordii Thick many candies solution
D, to the yeast that its 1% weight is added in geranium wilfordii Thick many candies solution D, rotating speed 150-195r/min shaking table cultures hair at room temperature
Ferment, fermentation time 26-74h, filtering adds Ca (OH) in filtrate2It is that 8.1,88 DEG C of heating water bath 66min are same to filtrate pH value
When be passed through CO2Gas, puts to room temperature, and filtering, filtrate is concentrated into the medicinal extract that proportion is 1.11-1.20, adds ethanol to contain ethanol
It is 80% that amount reaches volumetric concentration, and 4 DEG C of standing 12h, 6000r/min centrifugation 35min remove a layer pellet frozen and are drying to obtain old stork
Grass polysaccharide E；It is the medicinal extract of 1.11-1.21 to take supernatant A and ethanol is recovered under reduced pressure to be concentrated into proportion, and lower floor's extraction is collected in ether extraction
Liquid is taken, 50 DEG C are flung to ether, passes sequentially through large pore resin absorption column, gel resin post, obtain eluent, eluent is concentrated under reduced pressure,
Freeze-drying obtains geranium wilfordii oligosaccharide F；Realize preparing geranium wilfordii polysaccharide and oligosaccharide by raw material of geranium wilfordii, and geranium wilfordii is more
Sugar effective for prepare strengthen immunity medicine and health products and by geranium wilfordii oligosaccharide be used for prepare antifatigue medicine and
Application in health products.
Described macroporous absorbent resin is AB-8 type macroporous absorbent resins, and gel resin is Sephadex LH-20.
Preparation method of the present invention is simple, it is easy to operate, and has wide range of applications, and can be effectively used for preparing geranium wilfordii polysaccharide and low
Glycan, geranium wilfordii polysaccharide can be used to prepare the medicine and health products of strengthen immunity；It is anti-tired that geranium wilfordii oligosaccharide can be used for preparation
The medicine and health products of labor, have opened up geranium wilfordii medicinal material and geranium wilfordii polysaccharide, the application value of oligosaccharide, with good economy
And social benefit, it is the huge innovation on Chinese medicine.
Specific embodiment
Specific embodiment of the invention is elaborated with reference to embodiments.
The present invention can be given in specific implementation by following examples.
Embodiment 1
The present invention takes geranium wilfordii and dry, pulverize into meal in specific implementation；Geranium wilfordii meal is taken, to geranium wilfordii meal
Middle addition distilled water, geranium wilfordii meal is 1 with the weight ratio of distilled water：22, soak 46min, 98 DEG C of ultrasonic extraction 97min, mistake
Filter, filtrate is concentrated into the medicinal extract that proportion is 1.20, and it is 79% to add ethanol ethanol content is reached volumetric concentration, 4 DEG C of standings
15h, 6000r/min are centrifuged 45min, obtain supernatant A and centrifugation B, and supernatant A is standby, centrifugation B freeze-dryings, obtains
Geranium wilfordii Thick many candies powder C, the distilled water for taking geranium wilfordii Thick many candies powder C 10 times of weight of addition makes to dissolve to obtain geranium wilfordii Thick many candies
Solution D, to the yeast that its 1% weight is added in geranium wilfordii Thick many candies solution D, rotating speed 195r/min shaking table cultures hair at room temperature
Ferment, fermentation time 74h, filtering adds Ca (OH) in filtrate2To filtrate pH value be 8.1,88 DEG C of heating water bath 66min simultaneously
It is passed through CO2Gas, puts to room temperature, and filtering, filtrate is concentrated into the medicinal extract that proportion is 1.20, adds ethanol ethanol content is reached body
Product concentration is 80%, and 4 DEG C of standing 12h, 6000r/min centrifugation 35min remove a layer pellet frozen and are drying to obtain geranium wilfordii polysaccharide E；
Take supernatant A ethanol is recovered under reduced pressure and be concentrated into the medicinal extract that proportion is 1.21, lower floor's extract is collected in ether extraction, and 50 DEG C are flung to
Ether, passes sequentially through AB-8 types large pore resin absorption column, Sephadex LH-20 gel resin posts, obtains eluent, and eluent subtracts
Pressure concentration, freeze-drying obtains geranium wilfordii oligosaccharide F.
Embodiment 2
The present invention can also be and take geranium wilfordii and dry, pulverize into meal；Geranium wilfordii meal is taken, is steamed to being added in geranium wilfordii meal
Distilled water, geranium wilfordii meal is 1 with the weight ratio of distilled water：11,23min, 84 DEG C of ultrasonic extraction 41min are soaked, filtering, filtrate is dense
The medicinal extract that proportion is 1.11 is reduced to, it is 66% to add ethanol ethanol content is reached volumetric concentration, and 4 DEG C stand 8h, 6000r/min
Centrifugation 45min, obtains supernatant A and centrifugation B, and supernatant A is standby, centrifugation B freeze-dryings, obtains geranium wilfordii crude polysaccharide powder
Last C, the distilled water for taking geranium wilfordii Thick many candies powder C 10 times of weight of addition makes to dissolve to obtain geranium wilfordii Thick many candies solution D, to geranium wilfordii
The yeast of its 1% weight is added in Thick many candies solution D, at room temperature the fermentation of rotating speed 150r/min shaking table cultures, fermentation time
26h, filtering adds Ca (OH) in filtrate2It is that 8.1,88 DEG C of heating water bath 66min are passed through CO simultaneously to filtrate pH value2Gas,
Put to room temperature, filtering, filtrate is concentrated into the medicinal extract that proportion is 1.11, add ethanol to make ethanol content reach volumetric concentration to be
80%, 4 DEG C of standing 12h, 6000r/min centrifugation 35min, remove a layer pellet frozen and are drying to obtain geranium wilfordii polysaccharide E；Take supernatant
A is recovered under reduced pressure ethanol and is concentrated into the medicinal extract that proportion is 1.11, and lower floor's extract is collected in ether extraction, and 50 DEG C are flung to ether, successively
By being AB-8 types large pore resin absorption column, Sephadex LH-20 gel resin posts, eluent is obtained, eluent is concentrated under reduced pressure,
Freeze-drying obtains geranium wilfordii oligosaccharide F.
Embodiment 3
Take geranium wilfordii and dry, pulverize into meal；Geranium wilfordii meal is taken, to adding distilled water, geranium wilfordii in geranium wilfordii meal
Meal is 1 with the weight ratio of distilled water：16,35min, 91 DEG C of ultrasonic extraction 69min are soaked, filtering, filtrate is concentrated into proportion and is
1.15 medicinal extract, it is 72.5% to add ethanol ethanol content is reached volumetric concentration, 4 DEG C of standing 11h, 6000r/min centrifugations
45min, obtains supernatant A and centrifugation B, and supernatant A is standby, centrifugation B freeze-dryings, obtains geranium wilfordii Thick many candies powder C,
The distilled water for taking geranium wilfordii Thick many candies powder C 10 times of weight of addition makes to dissolve to obtain geranium wilfordii Thick many candies solution D, slightly many to geranium wilfordii
The yeast of its 1% weight is added in sugar juice D, at room temperature the fermentation of rotating speed 173r/min shaking table cultures, fermentation time 50h, mistake
Filter, adds Ca (OH) in filtrate2It is that 8.1,88 DEG C of heating water bath 66min are passed through CO simultaneously to filtrate pH value2Gas, puts to room
Temperature, filtering, filtrate is concentrated into the medicinal extract that proportion is 1.15, and it is 80% to add ethanol ethanol content is reached volumetric concentration, and 4 DEG C quiet
12h, 6000r/min centrifugation 35min are put, a layer pellet frozen is removed and is drying to obtain geranium wilfordii polysaccharide E；Supernatant A is taken to be recovered under reduced pressure
Ethanol is concentrated into the medicinal extract that proportion is 1.16, and lower floor's extract is collected in ether extraction, and 50 DEG C are flung to ether, pass sequentially through AB-8
Type large pore resin absorption column, Sephadex LH-20 gel resin posts, obtain eluent, and eluent is concentrated under reduced pressure, and freeze-drying is obtained
Geranium wilfordii oligosaccharide F.
Embodiment 4
Take geranium wilfordii and dry, pulverize into meal；Geranium wilfordii meal 11kg is taken, to adding distilled water in geranium wilfordii meal
121kg, soaks 46min, 91 DEG C of ultrasonic extraction 97min, and filtering, filtrate is concentrated into the medicinal extract that proportion is 1.11, adds ethanol to make
It is 66% that ethanol content reaches volumetric concentration, and 4 DEG C of standing 11h, 6000r/min centrifugation 45min obtain supernatant A and centrifugation
B, supernatant A is standby, centrifugation B freeze-dryings, obtains geranium wilfordii Thick many candies powder C, takes geranium wilfordii Thick many candies powder C and adds 10
The distilled water of times weight makes to dissolve to obtain geranium wilfordii Thick many candies solution D, to adding its 1% weight in geranium wilfordii Thick many candies solution D
Yeast, rotating speed 173r/min shaking table cultures fermentation at room temperature, fermentation time 26h, filtering adds Ca (OH) in filtrate2Extremely
Filtrate pH value is that 8.1,88 DEG C of heating water bath 66min are passed through CO simultaneously2Gas, puts to room temperature, and filtering, filtrate is concentrated into proportion and is
1.16 medicinal extract, it is 80% to add ethanol ethanol content is reached volumetric concentration, 4 DEG C of standing 12h, 6000r/min centrifugations
35min, removes a layer pellet frozen and is drying to obtain geranium wilfordii polysaccharide E；It is 1.21 to take supernatant A and ethanol is recovered under reduced pressure to be concentrated into proportion
Medicinal extract, ether extraction collects lower floor extract, and 50 DEG C are flung to ether, pass sequentially through AB-8 types large pore resin absorption column,
Sephadex LH-20 gel resin posts, obtain eluent, and eluent is concentrated under reduced pressure, and freeze-drying obtains geranium wilfordii oligosaccharide F.
Through Anthrone-sulfuricacid method, (conventional determining method is known skill to the geranium wilfordii polysaccharide and geranium wilfordii oligosaccharide of above-mentioned preparation
Art) determine, its sugared content is not less than 82%.Determined through HPGPC methods, the molecular weight of geranium wilfordii oligosaccharide 300-2000 it
Between.
Above-mentioned is only the several embodiments for being given, in development, inventor through it is repeated multiple times experiment achieve it is identical or
Close result, preparation method is scientific and reasonable, and effectively abundant raw material is cheap, and products application is extensive, with application valency
Value, repetition test is carried out to geranium wilfordii polysaccharide and oligosaccharide, achieves satisfied technique effect, and relevant testing data is as follows：
The immunocompetence experiment of geranium wilfordii polysaccharide
One, test materials
1. experimental animal：18.5-23g Kunming kind cleaning agent mouse, male and female half and half.Carried by Zhengzhou University's Experimental Animal Center
For.
2. reagent is tested：Geranium wilfordii polyoses content prepared by the inventive method is 86.71%, Switzerland's dye liquor, lentinan
(Hubei Guangren Pharmaceutical Co., Ltd.), endoxan (Hengrui Medicine Co., Ltd., Jiangsu Prov.), sodium citrate, sodium chloride, wine
Stone acid potassium sodium, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, phenol red etc..
Two, test methods
1. influence of the geranium wilfordii polysaccharide to normal Phagocytosis By The Peritoneal Macrophages In Mice
Mouse 50 is taken, body weight 18.5-23g, male and female half and half are uniformly divided into 5 groups at random.Gavage respectively it is small, in, it is heavy dose of
Geranium wilfordii polysaccharide solution (10mg/ml, 20mg/ml, 40mg/ml；0.2ml/10g), lentinan suspension (5mg/ml；
0.2ml/10g) and same volume physiological saline (0.2ml/10g).It is administered once daily, successive administration 7 days.It is early in being administered the 7th day
The upper equal chicken red blood cell normal saline suspension 0.5ml of intraperitoneal injection 5% of each group mouse.In the 7th day 5% was injected after gastric infusion 2h
Chicken red blood cell, takes off cervical vertebra and puts to death after 4h, Intraperitoneal injection Han Shi liquid 2.5ml gently rub mouse web portion, then abdominal cut skin,
An aperture is cut on peritonaeum, drawing peritoneal fluid 2ml with suction pipe is placed in test tube, mixes；A little abdominal cavity drop is drawn on slide,
Liquid point size is about 1.5cm × 2cm.Slide is placed in the sugared porcelain dish for being covered with wet gauze, 37 DEG C of incubation 30min, physiology salt
Water washes away the cell of attachment, and Wright's stain dyeing, running water is rinsed and dried；Basis of microscopic observation Turnover of Mouse Peritoneal Macrophages is gulped down
Situation is bitten, and calculates phagocytic percentage and phagocytic index.
The Peritoneal Macrophage Phagocytosis result of table 1
* represents with blank group that than P ＜ 0.01 * is represented with blank group than P ＜ 0.05
As it can be seen from table 1 with blank control group ratio, it is red to chicken that small dose group is remarkably improved Turnover of Mouse Peritoneal Macrophages
The phagocytic percentage of cell, can improve the phagocytic index (P ＜ 0.05) of peritoneal macrophage；Greatly, middle dose group and lentinan
Group can pole significantly improve phagocytic percentage and phagocytic index (P ＜ 0.01), wherein especially with middle dosage geranium wilfordii polysaccharide group act on
For most strong.
2. the influence that geranium wilfordii polysaccharide is formed to normal mouse hemolysin
Mouse 50 is taken, body weight 18.5-23g, male and female half and half are randomly divided into 5 groups.Gavage respectively it is small, in, it is heavy dose of old
The stork grass polysaccharide aqueous solution (10mg/ml, 20mg/ml, 40mg/ml；0.2ml/10g), lentinan suspension (5mg/ml；
0.2ml/10g) and same volume physiological saline (0.2ml/10g).It is administered once daily, successive administration 7 days.Meanwhile, administration the 1st
The equal chicken red blood cell normal saline suspension 0.2ml/ of intraperitoneal injection 5% of its each group mouse only, is immunized, after being administered in the 7th day
2h, mouse orbit takes blood, centrifugation, separates serum.After being diluted with physiological saline 1: 100,1ml dilutions and 5% chicken red blood cell are taken
Suspension 0.5ml, 10% complement 0.5ml (GPS, with the advance saturation 6h of chicken red blood cell) are mixed, 37 DEG C of incubation 30min, ice
Terminating reaction in water.Another setting is not added with the blank tube of complement and compares, and draws each pipe supernatant in UV-T1810 type spectrophotometers
Colorimetric at 540nm, determines each group hemolysin formational situation.
3. the influence that geranium wilfordii polysaccharide is formed to normal mouse hemolysis plaque
Mouse 50 is taken, body weight 18.5-23g, male and female half and half are uniformly divided into 5 groups at random.Gavage respectively it is small, in, it is heavy dose of
Geranium wilfordii polysaccharide solution (10mg/ml, 20mg/ml, 40mg/ml；0.2ml/10g), lentinan suspension (5mg/ml；
0.2ml/10g) and same volume physiological saline (0.2ml/10g).It is administered once daily, successive administration 7 days.Meanwhile, administration the 1st
The equal chicken red blood cell normal saline suspension 0.2ml/ of intraperitoneal injection 5% of its each group mouse only, is immunized, after being administered in the 7th day
2h, de- cervical vertebra is put to death and dissects mouse, takes out spleen, is one with two mouse spleens of group；Homogenate, and adjust splenocyte suspension
Spleens cell number is 5 × 10 in liquid6Individual/ml.Extracting spleen cell suspension 1.0ml, and 0.2% chicken red blood cell suspension 0.5ml and 1:
10 GPS 0.5ml is mixed.Another to set the blank tube for being not added with complement, 37 DEG C of incubation 1h, centrifugation takes supernatant in UV-T1810
Colorimetric at type spectrophotometer 413nm, surveys each group hemolysis plaque formational situation.
The hemolysin of table 2, hemolysis plaque form result
* represents with blank group that than P ＜ 0.01 * is represented with blank group than P ＜ 0.05
From table 2 it can be seen that with blank control group ratio, small dose group hemolysis plaque has and significantly raises (P ＜ 0.05), it is big,
Middle dose group and lentinan group hemolysin and hemolysis plaque pole are significantly improved (P ＜ 0.01).Wherein especially with the old stork of middle dosage
Grass polysaccharide group act as optimal.
4. the influence of the immunosuppressed mice Peritoneal Macrophage Phagocytosis of geranium wilfordii Polysaccharides On Cyclophosphamide induction
Mouse 60 is taken, body weight 18.5-23g, male and female half and half are uniformly divided into 6 groups at random.In addition to blank control group, remaining
Each group set up endoxan cause immunosuppression model totally 5 groups (dosage is 8mg/ml；Continuous intraperitoneal injection in 1st, 2,3 days), mould
Type is set up after the completion of standing, and geranium wilfordii polysaccharide solution (5mg/ml, 10mg/ml, 20mg/ml are gavaged respectively；0.2ml/10g), it is fragrant
The physiological saline (0.2ml/10g) of mushroom polysaccharide suspension (5mg/ml, 0.2ml/10g) and same volume.It is administered once daily, continuously
Administration 7 days.In the administration equal chicken red blood cell normal saline suspension 0.5ml of intraperitoneal injection 5% of each group mouse in morning last day, fill
2h after stomach administration, to 4h after chicken red blood cell, takes off cervical vertebra and puts to death mouse.Intraperitoneal injection Han Shi liquid 2.5ml, gently rub mouse web portion, so
After cut off mouse part skin, an aperture is cut on peritonaeum, with liquid-transfering gun draw peritoneal fluid 2ml be placed in test tube, mix；Draw
In on slide, liquid point size is about 1.5cm × 2cm to a little abdominal cavity drop.Slide is placed on the sugared porcelain dish for being covered with wet gauze
In, 37 DEG C of incubation 30min, physiological saline washes away the cell of attachment, and Wright's stain dyeing, running water is rinsed and dried, under microscope
The phagocytosis situation of Turnover of Mouse Peritoneal Macrophages is observed, and calculates phagocytic percentage and phagocytic index.
The Peritoneal Macrophage Phagocytosis result of table 3
* is represented with model group than P ＜ 0.01
From table 3 it can be seen that compared with blank group, phagocytic index of the model group Turnover of Mouse Peritoneal Macrophages to chicken red blood cell
(P ＜ 0.01) is significantly reduced with phagocytic percentage, modeling success is illustrated.Compared with model group, large, medium and small dosage polysaccharide group and
Lentinan group is remarkably improved phagocytic index and phagocytic percentage (P ＜ of the Turnover of Mouse Peritoneal Macrophages to chicken red blood cell
0.01) it is, especially best with middle dose group effect.
5. the influence that the immunosuppressed mice hemolysin of geranium wilfordii Polysaccharides On Cyclophosphamide induction is formed
Mouse 60 is taken, body weight 18.5-23g, male and female half and half are uniformly divided into 6 groups at random.In addition to blank control group, remaining
Each group set up endoxan cause immunosuppression model totally 5 groups (dosage is 8mg/ml；Continuous intraperitoneal injection in 1st, 2,3 days)；Mould
Type is set up after the completion of standing, each group mouse (containing blank group) the chicken red blood cell normal saline suspension 0.2ml/ of intraperitoneal injection 5%,
It is immunized；And start to gavage geranium wilfordii polysaccharide solution (5mg/ml, 10mg/ml, 20mg/ml respectively；0.2ml/10g), it is fragrant
The physiological saline (0.2ml/10g) of mushroom polysaccharide suspension (5mg/ml, 0.2ml/10g) and same volume, is administered once daily, continuously
Administration 7 days.2h after last 1 administration, mouse orbit takes blood, is centrifuged, and separates serum；After being diluted with physiological saline 1: 100, take
1ml dilutions and 5% chicken red blood cell suspension 0.5ml, 10% complement 0.5ml (GPSs, with the advance saturation of chicken red blood cell
6h) mix；37 DEG C of incubation 30min, terminating reaction in frozen water separately sets and is not added with the blank tube of complement and compares；Draw each pipe supernatant
Liquid colorimetric at UV-T1810 type spectrophotometers 540nm, determines each group hemolysin formational situation, the results are shown in Table 4.
6. the influence that the immunosuppressed mice hemolysis plaque of geranium wilfordii Polysaccharides On Cyclophosphamide induction is formed
Mouse 60 is taken, body weight 18.5-23g, male and female half and half are uniformly divided into 6 groups at random.In addition to blank control group, remaining
Each group set up endoxan cause immunosuppression model totally 5 groups (dosage is 8mg/ml；Continuous intraperitoneal injection in 1st, 2,3 days)；Mould
Type is set up after the completion of standing, each group mouse (containing blank group) the chicken red blood cell normal saline suspension 0.2ml/ of intraperitoneal injection 5%,
It is immunized；And start to gavage geranium wilfordii polysaccharide solution (5mg/ml, 10mg/ml, 20mg/ml respectively；0.2ml/10g), it is fragrant
The physiological saline (0.2ml/10g) of mushroom polysaccharide suspension (5mg/ml, 0.2ml/10g) and same volume, is administered once daily, continuously
Administration 7 days.2h after being administered in last 1 time, de- cervical vertebra is put to death and dissects mouse, takes out spleen, is with two mouse spleens in group
One, homogenate, and it is 5 × 106/ml to adjust spleens cell number in splenocyte suspension.Extracting spleen cell suspension 1.0ml, with
The GPS 0.5ml of 0.2% chicken red blood cell suspension 0.5ml and 1: 10 is mixed.The blank tube for being not added with complement separately is set, 37 DEG C incubate
1h is educated, is centrifuged, take supernatant colorimetric at UV-T1810 type spectrophotometers 413nm, survey each group hemolysis plaque formational situation, knot
Fruit is shown in Table 4.
The hemolysin of table 4, hemolysis plaque form result
* is represented with model group than P ＜ 0.01
Be can be seen that from upper table, with blank control group ratio, model group hemolysin and hemolysis plaque significantly reduce (P ＜
0.01), illustrate to make immunosuppression model success.With model group ratio, each dosage geranium wilfordii polysaccharide group and lentinan group
Remarkably promote the formation (P ＜ 0.01) of mouse hemolysin and hemolysis plaque.Wherein, especially with 0.2g/kg dosage geranium wilfordii polysaccharide groups
For optimal.
The experiment of geranium wilfordii oligosaccharide antifatigue effect：
One, test materials
1. experimental animal：18.5-23g Kunming kind cleaning agent mouse, male and female half and half.Carried by Zhengzhou University's Experimental Animal Center
For.
2. reagent is tested：Geranium wilfordii oligosaccharide content prepared by the inventive method is 89.35%, superoxide dismutase
(SOD) kit, MDA (MDA) are determined and determines kit etc..
Two, test methods
The Kunming mouse 40 of body weight 18.5-23g is taken, 4 groups are randomly divided into by body weight：Normal group, low (200mg/
Kg (500mg/kg), high dose (800mg/kg) administration group in),；Once, Normal group is with the life of equivalent for daily gastric infusion
Reason salt solution gavage, successive administration 30 days, in 30min after last dose, mouse is put in swimming trunk, and water temperature is 25 ± 5 DEG C, mouse
Bear a heavy burden the sheet lead of 5% body weight in root of the tail portion, and 8s does not go out time of the water surface during record mouse submerges water from swim to head, i.e., small
The walking weight load of mouse.After swimming terminates to recover 24h, eye socket takes blood to mouse respectively for every group of mouse, separates serum, uses super oxygen
Compound mutase (SOD) determines kit, MDA (MDA) measure kit and determines SOD in mice serum respectively, and MDA values are shown in
Table 5.
The geranium wilfordii oligosaccharide of table 5 to the mice burden swimming time, the influence of SOD and MDA in serum
* the P compared with blank group is represented<0.05；* represents the P compared with blank group<0.01
As can be seen from Table 5, geranium wilfordii oligosaccharide high and low dose is remarkably improved body SOD vigor, reduces MDA and contains
Amount (P<0.05), middle dose group can pole significantly improve body SOD vigor, reduce MDA contents (P<0.01), so as to show old stork
Careless oligosaccharide has preferable antifatigue effect.
Embodiment described above, is only presently preferred embodiments of the present invention, and any formal limit is not done to the present invention
System, although the present invention is disclosed above with preferred embodiment, but is not to limit the invention, it is any to be familiar with this specialty
Technical staff, without departing from the scope of the present invention, when the technology contents using the disclosure above make few modifications
Or the Equivalent embodiments of equivalent variations are modified to, as long as being the content without departing from technical solution of the present invention, according to skill of the invention
Art essence belongs to the scope of technical solution of the present invention to any simple modification made for any of the above embodiments, equivalent variations and modification
It is interior.
Claims (7)
1. the preparation method of a kind of geranium wilfordii polysaccharide and oligosaccharide, it is characterised in that comprise the following steps：Geranium wilfordii drying is taken,
It is ground into meal；Geranium wilfordii meal is taken, is with the weight ratio of distilled water to distilled water, geranium wilfordii meal is added in geranium wilfordii meal
1：11-22, soaks 23-46min, 84 DEG C of -98 DEG C of ultrasonic extraction 41-97min, filtering, and filtrate is concentrated into proportion for 1.11-1.20
Medicinal extract, add ethanol ethanol content is reached volumetric concentration for 66%-79%, 4 DEG C of standing 8-15h, 6000r/min centrifugations
45min, obtains supernatant A and centrifugation B, and supernatant A is standby, centrifugation B freeze-dryings, obtains geranium wilfordii Thick many candies powder C,
The distilled water for taking geranium wilfordii Thick many candies powder C 10 times of weight of addition makes to dissolve to obtain geranium wilfordii Thick many candies solution D, slightly many to geranium wilfordii
The yeast of its 1% weight is added in sugar juice D, at room temperature the fermentation of rotating speed 150-195r/min shaking table cultures, fermentation time 26-
74h, filtering adds Ca (OH) in filtrate2It is that 8.1,88 DEG C of heating water bath 66min are passed through CO simultaneously to filtrate pH value2Gas,
Put to room temperature, filtering, filtrate is concentrated into the medicinal extract that proportion is 1.11-1.20, adds ethanol ethanol content is reached volumetric concentration
It is 80%, 4 DEG C of standing 12h, 6000r/min centrifugation 35min remove a layer pellet frozen and are drying to obtain geranium wilfordii polysaccharide E；Take supernatant
Liquid A is recovered under reduced pressure ethanol and is concentrated into the medicinal extract that proportion is 1.11-1.21, and lower floor's extract is collected in ether extraction, and 50 DEG C are flung to second
Ether, passes sequentially through large pore resin absorption column, gel resin post, obtains eluent, and eluent is concentrated under reduced pressure, and freeze-drying obtains geranium wilfordii
Oligosaccharide F；Described macroporous absorbent resin is AB-8 type macroporous absorbent resins, and gel resin is SephadexLH-20.
2. geranium wilfordii polysaccharide described in claim 1 and the preparation method of oligosaccharide, it is characterised in that comprise the following steps：Take
Geranium wilfordii dry, pulverize into meal；Geranium wilfordii meal is taken, to adding distilled water in geranium wilfordii meal, geranium wilfordii meal and distillation
The weight ratio of water is 1：22,46min, 98 DEG C of ultrasonic extraction 97min are soaked, filtering, filtrate is concentrated into the medicinal extract that proportion is 1.20,
Add ethanol to make ethanol content reach volumetric concentration is 79%, and 4 DEG C of standing 15h, 6000r/min centrifugation 45min obtain supernatant A
With centrifugation B, supernatant A is standby, and centrifugation B freeze-dryings obtain geranium wilfordii Thick many candies powder C, take geranium wilfordii Thick many candies
The distilled water of powder C 10 times of weight of addition makes to dissolve to obtain geranium wilfordii Thick many candies solution D, is added in geranium wilfordii Thick many candies solution D
The yeast of its 1% weight, rotating speed 195r/min shaking table cultures fermentation at room temperature, fermentation time 74h, filtering adds in filtrate
Enter Ca (OH)2It is that 8.1,88 DEG C of heating water bath 66min are passed through CO simultaneously to filtrate pH value2Gas, puts to room temperature, and filtering, filtrate is dense
The medicinal extract that proportion is 1.20 is reduced to, it is 80% to add ethanol ethanol content is reached volumetric concentration, and 4 DEG C stand 12h, 6000r/
Min is centrifuged 35min, removes a layer pellet frozen and is drying to obtain geranium wilfordii polysaccharide E；Take supernatant A ethanol is recovered under reduced pressure and be concentrated into ratio
Weight is 1.21 medicinal extract, and lower floor's extract is collected in ether extraction, and 50 DEG C are flung to ether, passed sequentially through large pore resin absorption column, are coagulated
Gum resin post, obtains eluent, and eluent is concentrated under reduced pressure, and freeze-drying obtains geranium wilfordii oligosaccharide F；Described macroporous absorbent resin is
AB-8 type macroporous absorbent resins, gel resin is Sephadex LH-20.
3. geranium wilfordii polysaccharide described in claim 1 and the preparation method of oligosaccharide, it is characterised in that comprise the following steps：Take
Geranium wilfordii dry, pulverize into meal；Geranium wilfordii meal is taken, to adding distilled water in geranium wilfordii meal, geranium wilfordii meal and distillation
The weight ratio of water is 1：11,23min, 84 DEG C of ultrasonic extraction 41min are soaked, filtering, filtrate is concentrated into the medicinal extract that proportion is 1.11,
Adding ethanol makes ethanol content reach volumetric concentration for 66%, 4 DEG C of standing 8h, 6000r/min centrifugation 45min, obtain supernatant A and
Centrifugation B, supernatant A is standby, centrifugation B freeze-dryings, obtains geranium wilfordii Thick many candies powder C, takes geranium wilfordii crude polysaccharide powder
Last C adds the distilled water of 10 times of weight to make to dissolve to obtain geranium wilfordii Thick many candies solution D, to adding it in geranium wilfordii Thick many candies solution D
The yeast of 1% weight, rotating speed 150r/min shaking table cultures fermentation at room temperature, fermentation time 26h, filtering is added in filtrate
Ca(OH)2It is that 8.1,88 DEG C of heating water bath 66min are passed through CO simultaneously to filtrate pH value2Gas, puts to room temperature, filtering, filtrate concentration
To the medicinal extract that proportion is 1.11, it is 80% to add ethanol ethanol content is reached volumetric concentration, and 4 DEG C stand 12h, 6000r/min
Centrifugation 35min, removes a layer pellet frozen and is drying to obtain geranium wilfordii polysaccharide E；Take supernatant A ethanol is recovered under reduced pressure and be concentrated into proportion and be
1.11 medicinal extract, ether extraction, collects lower floor's extract, and 50 DEG C are flung to ether, pass sequentially through large pore resin absorption column, gel tree
Fat post, obtains eluent, and eluent is concentrated under reduced pressure, and freeze-drying obtains geranium wilfordii oligosaccharide F；Described macroporous absorbent resin is AB-8
Type macroporous absorbent resin, gel resin is Sephadex LH-20.
4. geranium wilfordii polysaccharide described in claim 1 and the preparation method of oligosaccharide, it is characterised in that comprise the following steps：Take
Geranium wilfordii dry, pulverize into meal；Geranium wilfordii meal is taken, to adding distilled water in geranium wilfordii meal, geranium wilfordii meal and distillation
The weight ratio of water is 1：16,35min, 91 DEG C of ultrasonic extraction 69min are soaked, filtering, filtrate is concentrated into the medicinal extract that proportion is 1.15,
Add ethanol to make ethanol content reach volumetric concentration is 72.5%, and 4 DEG C of standing 11h, 6000r/min centrifugation 45min obtain supernatant
A and centrifugation B, supernatant A is standby, centrifugation B freeze-dryings, obtains geranium wilfordii Thick many candies powder C, takes geranium wilfordii Thick many candies
The distilled water of powder C 10 times of weight of addition makes to dissolve to obtain geranium wilfordii Thick many candies solution D, is added in geranium wilfordii Thick many candies solution D
The yeast of its 1% weight, rotating speed 173r/min shaking table cultures fermentation at room temperature, fermentation time 50h, filtering adds in filtrate
Enter Ca (OH)2It is that 8.1,88 DEG C of heating water bath 66min are passed through CO simultaneously to filtrate pH value2Gas, puts to room temperature, and filtering, filtrate is dense
The medicinal extract that proportion is 1.15 is reduced to, it is 80% to add ethanol ethanol content is reached volumetric concentration, and 4 DEG C stand 12h, 6000r/
Min is centrifuged 35min, removes a layer pellet frozen and is drying to obtain geranium wilfordii polysaccharide E；Take supernatant A ethanol is recovered under reduced pressure and be concentrated into ratio
Weight is 1.16 medicinal extract, and lower floor's extract is collected in ether extraction, and 50 DEG C are flung to ether, passed sequentially through large pore resin absorption column, are coagulated
Gum resin post, obtains eluent, and eluent is concentrated under reduced pressure, and freeze-drying obtains geranium wilfordii oligosaccharide F；Described macroporous absorbent resin is
AB-8 type macroporous absorbent resins, gel resin is Sephadex LH-20.
5. geranium wilfordii polysaccharide described in claim 1 and the preparation method of oligosaccharide, it is characterised in that comprise the following steps：Take
Geranium wilfordii dry, pulverize into meal；Geranium wilfordii meal 11kg is taken, to distilled water 121kg is added in geranium wilfordii meal, is soaked
46min, 91 DEG C of ultrasonic extraction 97min, filtering, filtrate are concentrated into the medicinal extract that proportion is 1.11, add ethanol to reach ethanol content
It is 66% to volumetric concentration, 4 DEG C of standing 11h, 6000r/min centrifugation 45min obtain supernatant A and centrifugation B, and supernatant A is standby
With centrifugation B freeze-dryings obtain geranium wilfordii Thick many candies powder C, take geranium wilfordii Thick many candies powder C and add 10 times of steamings of weight
Distilled water makes to dissolve to obtain geranium wilfordii Thick many candies solution D, to the yeast that its 1% weight is added in geranium wilfordii Thick many candies solution D, in room temperature
Lower rotating speed 173r/min shaking table cultures fermentation, fermentation time 26h, filtering adds Ca (OH) in filtrate2It is to filtrate pH value
8.1,88 DEG C of heating water bath 66min are passed through CO simultaneously2Gas, puts to room temperature, and filtering, filtrate is concentrated into the leaching that proportion is 1.16
Cream, it is 80% to add ethanol ethanol content is reached volumetric concentration, and 4 DEG C of standing 12h, 6000r/min centrifugation 35min remove layer
Pellet frozen is drying to obtain geranium wilfordii polysaccharide E；Take supernatant A ethanol is recovered under reduced pressure and be concentrated into the medicinal extract that proportion is 1.21, ether
Extraction, collects lower floor's extract, and 50 DEG C are flung to ether, pass sequentially through large pore resin absorption column, gel resin post, obtain eluent,
Eluent is concentrated under reduced pressure, and freeze-drying obtains geranium wilfordii oligosaccharide F；Described macroporous absorbent resin is AB-8 type macroporous absorption trees
Fat, gel resin is Sephadex LH-20.
6. the geranium wilfordii polysaccharide E that in claim 1 or 2-5 prepared by any one methods described is preparing the medicine of strengthen immunity
With the application in health products.
7. the geranium wilfordii oligosaccharide F that in claim 1 or 2-5 prepared by any one methods described prepares antifatigue medicine with
Application in health products.
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| CN110066348A (en) * | 2018-01-23 | 2019-07-30 | 天津科技大学 | A method of it extracts, purification witloof oligosaccharide |
| CN111297885A (en) * | 2020-02-02 | 2020-06-19 | 云南中医药大学 | Application of geranium polysaccharide in preparing liver-protecting medicine |
| CN113667028A (en) * | 2020-05-15 | 2021-11-19 | 集美大学 | Preparation method of Kappa carrageenan glue solution |
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