CN106883303B - 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 the preparation method and application of a kind of geranium wilfordii polysaccharide and 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 problems, such as the integration system of geranium wilfordii polysaccharide and oligosaccharide for；Geranium wilfordii polysaccharide prepared by the present invention can be effectively used for the drug and health care product of preparation strengthen immunity, and geranium wilfordii oligosaccharide can be effectively used for preparing antifatigue drug and health care product.
Description
Technical field
The present invention relates to field of medicaments, more particularly to the preparation method and application of a kind of geranium wilfordii polysaccharide and oligosaccharide.
Background technique
Geranium wilfordii is Mang ox Miao Ke 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 is the effects of wind-damp dispelling, degrading the channel, antidiarrheal dysentery, antibacterial, antiviral, anti-oxidant, antitumor, anti-inflammatory and antalgic, hypoglycemic, liver protection.Face
Bed is mainly used for rheumatic arthralgia, numbness contracture, and muscles and bones is ached, dysentery.Tan now is related generally to the research of its effective component
The ingredients 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 not been reported for geranium wilfordii polysaccharide and oligosaccharide.
Summary of the invention
For above situation, in order to overcome the defect of the prior art, it is more 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 enhance in preparation
Application and geranium wilfordii oligosaccharide in the drug and health care product of immunity are preparing the application in antifatigue drug and health care product.
The technical solution that the present invention solves is, the preparation method of a kind of geranium wilfordii polysaccharide and oligosaccharide, comprising the following steps:
Geranium wilfordii is taken to be dried, crushed into coarse powder；Geranium wilfordii coarse powder is taken, distilled water, geranium wilfordii coarse powder and steaming are added into geranium wilfordii coarse powder
The weight ratio of distilled water is 1:11-22, impregnates 23-46min, 84 DEG C of -98 DEG C of ultrasonic extraction 41-97min, filtering, and filtrate is concentrated into
Specific gravity is the medicinal extract of 1.11-1.20, and ethyl alcohol, which is added, makes ethanol content reach volumetric concentration 66%-79%, 4 DEG C of standing 8-15h,
6000r/min is centrifuged 45min, obtains supernatant A and centrifugation B, and supernatant A is spare, and centrifugation B freeze-drying obtains old stork
Careless Thick many candies powder C, the distilled water for taking geranium wilfordii Thick many candies powder C that 10 times of weight are added make to dissolve to obtain geranium wilfordii Thick many candies solution
The yeast of its 1% weight is added into geranium wilfordii Thick many candies solution D by D, and revolving speed 150-195r/min shaking table culture is sent out at room temperature
Ca (OH) is added in filtrate in ferment, fermentation time 26-74h, filtering2It is that 8.1,88 DEG C of heating water bath 66min are same to filtrate pH value
When be passed through CO2Gas is put to room temperature, filtering, and filtrate is concentrated into the medicinal extract that specific gravity is 1.11-1.20, and ethyl alcohol, which is added, contains ethyl alcohol
It is 80% that amount, which reaches volumetric concentration, and 4 DEG C of standing 12h, 6000r/min centrifugation 35min take lower layer's pellet frozen to be drying to obtain old stork
Grass polysaccharide E；It takes supernatant A that ethyl alcohol is recovered under reduced pressure and is concentrated into the medicinal extract that specific gravity is 1.11-1.21, lower layer's extraction is collected in ether extraction
Liquid is taken, 50 DEG C are flung to ether, pass sequentially through large pore resin absorption column, gel resin column, obtain eluent, and eluent is concentrated under reduced pressure,
It is freeze-dried to obtain geranium wilfordii oligosaccharide F；It realizes and prepares geranium wilfordii polysaccharide and oligosaccharide by raw material of geranium wilfordii, and geranium wilfordii is more
Sugar effective for prepare strengthen immunity drug and health care product and by geranium wilfordii oligosaccharide be used to prepare antifatigue drug and
Application in health care product.
The macroporous absorbent resin is AB-8 type macroporous absorbent resin, and gel resin is Sephadex LH-20.
Preparation method of the present invention is simple, easily operated, has wide range of applications, and can be effectively used for preparation geranium wilfordii polysaccharide and low
Glycan, geranium wilfordii polysaccharide can be used for preparing the drug and health care product of strengthen immunity；Geranium wilfordii oligosaccharide can be used for preparing resist it is tired
The drug and health care product of labor, have opened up the application value of geranium wilfordii medicinal material and geranium wilfordii polysaccharide, oligosaccharide, have good economy
And social benefit, it is the huge innovation on Chinese medicine.
Specific embodiment
It elaborates with reference to embodiments to a specific embodiment of the invention.
The present invention in specific implementation, can be provided by following embodiment.
Embodiment 1
The present invention in specific implementation, takes geranium wilfordii to be dried, crushed into coarse powder；Geranium wilfordii coarse powder is taken, to geranium wilfordii coarse powder
The weight ratio of middle addition distilled water, geranium wilfordii coarse powder and distilled water is 1:22, impregnates 46min, 98 DEG C of ultrasonic extraction 97min, mistake
Filter, filtrate are concentrated into the medicinal extract that specific gravity is 1.20, and ethyl alcohol, which is added, makes ethanol content reach volumetric concentration 79%, 4 DEG C of standings
15h, 6000r/min are centrifuged 45min, obtain supernatant A and centrifugation B, and supernatant A is spare, and centrifugation B freeze-drying obtains
Geranium wilfordii Thick many candies powder C, the distilled water for taking geranium wilfordii Thick many candies powder C that 10 times of weight are added make to dissolve to obtain geranium wilfordii Thick many candies
The yeast of its 1% weight is added into geranium wilfordii Thick many candies solution D for solution D, and revolving speed 195r/min shaking table culture is sent out at room temperature
Ca (OH) is added in filtrate in ferment, fermentation time 74h, filtering2To filtrate pH value be 8.1,88 DEG C of heating water bath 66min simultaneously
It is passed through CO2Gas is put to room temperature, filtering, and filtrate is concentrated into the medicinal extract that specific gravity is 1.20, and ethyl alcohol, which is added, makes ethanol content reach body
Product concentration is 80%, and 4 DEG C of standing 12h, 6000r/min centrifugation 35min take lower layer's pellet frozen to be drying to obtain geranium wilfordii polysaccharide E；
It takes supernatant A that ethyl alcohol is recovered under reduced pressure and is concentrated into the medicinal extract that specific gravity is 1.21, ether extraction collects lower layer's extract liquor, 50 DEG C are flung to
Ether passes sequentially through AB-8 type large pore resin absorption column, Sephadex LH-20 gel resin column, obtains eluent, eluent subtracts
Pressure concentration, is freeze-dried to obtain geranium wilfordii oligosaccharide F.
Embodiment 2
The present invention, which can also be, takes geranium wilfordii to be dried, crushed into coarse powder；Geranium wilfordii coarse powder is taken, is added and steams into geranium wilfordii coarse powder
The weight ratio of distilled water, geranium wilfordii coarse powder and distilled water is 1:11, impregnates 23min, 84 DEG C of ultrasonic extraction 41min, is filtered, and filtrate is dense
It is reduced to the medicinal extract that specific gravity is 1.11, ethyl alcohol, which is added, makes ethanol content reach volumetric concentration 66%, 4 DEG C of standings 8h, 6000r/min
It is centrifuged 45min, obtains supernatant A and centrifugation B, supernatant A is spare, and centrifugation B freeze-drying obtains geranium wilfordii crude polysaccharide powder
Last C, the distilled water for taking geranium wilfordii Thick many candies powder C that 10 times of weight are added 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, revolving speed 150r/min shaking table culture is fermented at room temperature, fermentation time
Ca (OH) is added in filtrate in 26h, filtering2It is that 8.1,88 DEG C of heating water bath 66min are passed through CO simultaneously to filtrate pH value2Gas,
It puts to room temperature, filtering, filtrate is concentrated into the medicinal extract that specific gravity is 1.11, and ethyl alcohol, which is added, makes ethanol content reach volumetric concentration
80%, 4 DEG C of standing 12h, 6000r/min centrifugation 35min take lower layer's pellet frozen to be drying to obtain geranium wilfordii polysaccharide E；Take supernatant
A is recovered under reduced pressure ethyl alcohol and is concentrated into the medicinal extract that specific gravity is 1.11, and ether extraction collects lower layer's extract liquor, 50 DEG C are flung to ether, successively
By for AB-8 type large pore resin absorption column, Sephadex LH-20 gel resin column, obtaining eluent, eluent is concentrated under reduced pressure,
It is freeze-dried to obtain geranium wilfordii oligosaccharide F.
Embodiment 3
Geranium wilfordii is taken to be dried, crushed into coarse powder；Geranium wilfordii coarse powder is taken, distilled water, geranium wilfordii are added into geranium wilfordii coarse powder
The weight ratio of coarse powder and distilled water is 1:16, impregnates 35min, 91 DEG C of ultrasonic extraction 69min, filtering, and filtrate is concentrated into specific gravity and is
1.15 medicinal extract, ethyl alcohol, which is added, makes ethanol content reach volumetric concentration 72.5%, 4 DEG C of standing 11h, 6000r/min centrifugations
45min obtains supernatant A and centrifugation B, and supernatant A is spare, and centrifugation B freeze-drying obtains geranium wilfordii Thick many candies powder C,
The distilled water for taking geranium wilfordii Thick many candies powder C that 10 times of weight are added makes to dissolve to obtain geranium wilfordii Thick many candies solution D, slightly more to geranium wilfordii
The yeast of its 1% weight is added in sugar juice D, revolving speed 173r/min shaking table culture is fermented at room temperature, fermentation time 50h, mistake
Ca (OH) is added in filtrate in filter2It is that 8.1,88 DEG C of heating water bath 66min are passed through CO simultaneously to filtrate pH value2Gas is put to room
Temperature, filtering, filtrate are concentrated into the medicinal extract that specific gravity is 1.15, and ethyl alcohol, which is added, makes ethanol content reach volumetric concentration 80%, and 4 DEG C quiet
12h is set, 6000r/min is centrifuged 35min, lower layer's pellet frozen is taken to be drying to obtain geranium wilfordii polysaccharide E；Supernatant A is taken to be recovered under reduced pressure
Ethyl alcohol is concentrated into the medicinal extract that specific gravity is 1.16, and ether extraction collects lower layer's extract liquor, 50 DEG C are flung to ether, pass sequentially through AB-8
Type large pore resin absorption column, Sephadex LH-20 gel resin column, obtain eluent, and eluent is concentrated under reduced pressure, is freeze-dried
Geranium wilfordii oligosaccharide F.
Embodiment 4
Geranium wilfordii is taken to be dried, crushed into coarse powder；Geranium wilfordii coarse powder 11kg is taken, distilled water is added into geranium wilfordii coarse powder
121kg impregnates 46min, 91 DEG C of ultrasonic extraction 97min, filtering, and filtrate is concentrated into the medicinal extract that specific gravity is 1.11, and ethyl alcohol, which is added, to be made
It is 66% that ethanol content, which reaches volumetric concentration, and 4 DEG C of standing 11h, 6000r/min centrifugation 45min obtain supernatant A and centrifugation
B, supernatant A is spare, and centrifugation B freeze-drying obtains geranium wilfordii Thick many candies powder C, geranium wilfordii Thick many candies powder C is taken to be added 10
The distilled water of times weight makes to dissolve to obtain geranium wilfordii Thick many candies solution D, its 1% weight is added into geranium wilfordii Thick many candies solution D
Yeast, revolving speed 173r/min shaking table culture is fermented at room temperature, fermentation time 26h, and Ca (OH) is added in filtrate in filtering2Extremely
Filtrate pH value is that 8.1,88 DEG C of heating water bath 66min are passed through CO simultaneously2Gas is put to room temperature, filtering, and filtrate is concentrated into specific gravity and is
1.16 medicinal extract, ethyl alcohol, which is added, makes ethanol content reach volumetric concentration 80%, 4 DEG C of standing 12h, 6000r/min centrifugations
35min takes lower layer's pellet frozen to be drying to obtain geranium wilfordii polysaccharide E；Taking supernatant A that ethyl alcohol is recovered under reduced pressure to be concentrated into specific gravity is 1.21
Medicinal extract, ether extraction, collect lower layer's extract liquor, 50 DEG C are flung to ether, pass sequentially through AB-8 type large pore resin absorption column,
Sephadex LH-20 gel resin column obtains eluent, and eluent is concentrated under reduced pressure, and is freeze-dried to obtain 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) it measures, sugared content is not less than 82%.Measured through HPGPC method, the molecular weight of geranium wilfordii oligosaccharide 300-2000 it
Between.
Above-mentioned is only the several embodiments provided, in development, inventor through it is repeated multiple times experiment achieve it is identical or
It is similar as a result, preparation method it is scientific and reasonable effectively abundant raw material is cheap, and products application is extensive, there is the valence of applying
Value carries out repetition test to geranium wilfordii polysaccharide and oligosaccharide, achieves satisfied technical effect, and related testing data is as follows:
The immunocompetence of geranium wilfordii polysaccharide is tested
One, test material
1. experimental animal: the Kunming 18.5-23g kind detergent mouse, half male and half female.It is mentioned by Zhengzhou University's Experimental Animal Center
For.
2. testing reagent: the geranium wilfordii polyoses content of the method for the present invention preparation is 86.71%, Switzerland's dye liquor, lentinan
(Hubei Guangren Pharmaceutical Co., Ltd.), cyclophosphamide (Hengrui Medicine Co., Ltd., Jiangsu Prov.), sodium citrate, sodium chloride, wine
Stone acid potassium sodium, glucose, dipotassium hydrogen phosphate, potassium dihydrogen phosphate are phenol red etc..
Two, test methods
1. influence of the geranium wilfordii polysaccharide to normal mouse Peritoneal Macrophage Phagocytosis
Mouse 50 are taken, weight 18.5-23g, half male and half female is uniformly divided into 5 groups at random.Gavage respectively it is small, in, large dosage
Geranium wilfordii polysaccharide solution (10mg/ml, 20mg/ml, 40mg/ml；0.2ml/10g), lentinan suspension (5mg/ml；
0.2ml/10g) and the physiological saline of same volume (0.2ml/10g).It is administered once daily, successive administration 7 days.It is early in administration the 7th day
5% chicken red blood cell normal saline suspension 0.5ml is injected intraperitoneally in upper each group mouse.5% was injected after gastric infusion 2h in the 7th day
Chicken red blood cell, cervical dislocation is put to death after 4h, and Intraperitoneal injection Han Shi liquid 2.5ml gently rubs mouse web portion, then abdominal cut skin,
An aperture is cut on peritonaeum, is drawn peritoneal fluid 2ml with suction pipe and is placed in test tube, mixes；A little abdominal cavity drop is drawn on glass slide,
Liquid point size is about 1.5cm × 2cm.Glass 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, Wright's stain dyeing, and tap water flushing is dried；Microscopically observation Turnover of Mouse Peritoneal Macrophages gulps down
Situation is bitten, and calculates phagocytic percentage and phagocytic index.
1 Peritoneal Macrophage Phagocytosis result of table
* is indicated and blank group ratio P < 0.01, * are indicated and blank group ratio P < 0.05
As it can be seen from table 1 it is red to chicken that small dose group is remarkably improved Turnover of Mouse Peritoneal Macrophages with blank control group ratio
The phagocytic index (P < 0.05) of peritoneal macrophage can be improved in the phagocytic percentage of cell；Greatly, middle dose group and lentinan
The group extremely significant raising phagocytic percentage of energy and phagocytic index (P < 0.01), wherein especially with the effect of middle dosage geranium wilfordii polysaccharide group
It is most strong.
2. the influence that geranium wilfordii polysaccharide forms normal mouse hemolysin
Mouse 50 are taken, weight 18.5-23g, half male and half female is randomly divided into 5 groups.Gavage respectively it is small, in, large dosage it is old
Stork grass polysaccharide aqueous solution (10mg/ml, 20mg/ml, 40mg/ml；0.2ml/10g), lentinan suspension (5mg/ml；
0.2ml/10g) and the physiological saline of same volume (0.2ml/10g).It is administered once daily, successive administration 7 days.Meanwhile administration the 1st
5% chicken red blood cell normal saline suspension 0.2ml/ is injected intraperitoneally only in its each group mouse, is immunized, after administration in the 7th day
2h, mouse orbit take blood, and centrifugation separates serum.After being diluted with physiological saline 1: 100,1ml dilution and 5% chicken red blood cell are taken
Suspension 0.5ml, 10% complement 0.5ml (guinea pig serum is saturated 6h with chicken red blood cell in advance) are mixed, 37 DEG C of incubation 30min, ice
Reaction is terminated in water.It separately sets and the blank tube of complement is not added compares, draw each pipe supernatant in UV-T1810 type spectrophotometer
Colorimetric at 540nm measures each group hemolysin formational situation.
3. the influence that geranium wilfordii polysaccharide forms normal mouse hemolysis plaque
Mouse 50 are taken, weight 18.5-23g, half male and half female is uniformly divided into 5 groups at random.Gavage respectively it is small, in, large dosage
Geranium wilfordii polysaccharide solution (10mg/ml, 20mg/ml, 40mg/ml；0.2ml/10g), lentinan suspension (5mg/ml；
0.2ml/10g) and the physiological saline of same volume (0.2ml/10g).It is administered once daily, successive administration 7 days.Meanwhile administration the 1st
5% chicken red blood cell normal saline suspension 0.2ml/ is injected intraperitoneally only in its each group mouse, is immunized, after administration in the 7th day
Mouse is put to death and dissected to 2h, cervical dislocation, takes out spleen, as an example of two mouse spleens of group；Homogenate, and adjust splenocyte suspension
Spleens cell number is 5 × 10 in liquid6A/ml.Extracting spleen cell suspension 1.0ml, and 0.2% chicken red blood cell suspension 0.5ml and 1:
10 guinea pig serum 0.5ml is mixed.The blank tube that complement is not added, 37 DEG C of incubation 1h are separately set, centrifugation takes supernatant in UV-T1810
Colorimetric at type spectrophotometer 413nm surveys each group hemolysis plaque formational situation.
2 hemolysin of table, hemolysis plaque form result
* is indicated and blank group ratio P < 0.01, * are indicated and blank group ratio P < 0.05
From table 2 it can be seen that small dose group hemolysis plaque, which has, significantly increases (P < 0.05) with blank control group ratio, big,
Middle dose group and the extremely significant raising (P < 0.01) of lentinan group hemolysin and hemolysis plaque.Wherein especially with the old stork of middle dosage
The effect of grass polysaccharide group is optimal.
4. the influence of the immunosuppressed mice Peritoneal Macrophage Phagocytosis of geranium wilfordii Polysaccharides On Cyclophosphamide induction
Mouse 60 are taken, weight 18.5-23g, half male and half female is uniformly divided into 6 groups at random.In addition to blank control group, remaining
Each group establishes cyclophosphamide and causes immunosuppression model totally 5 groups of (dosage 8mg/ml；Continuous intraperitoneal injection in 1st, 2,3 day), mould
After the completion of type group is established, geranium wilfordii polysaccharide solution (5mg/ml, 10mg/ml, 20mg/ml are gavaged respectively；0.2ml/10g), 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.5% chicken red blood cell normal saline suspension 0.5ml is injected intraperitoneally in administration each group mouse in morning last day, fills
2h after stomach administration, to 4h after chicken red blood cell, cervical dislocation puts to death mouse.Intraperitoneal injection Han Shi liquid 2.5ml, gently rubs 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；It draws
For a little abdominal cavity drop on glass slide, liquid point size is about 1.5cm × 2cm.Glass slide is placed on to 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, tap water flushing is dried, under microscope
The phagocytosis situation of Turnover of Mouse Peritoneal Macrophages is observed, and calculates phagocytic percentage and phagocytic index.
3 Peritoneal Macrophage Phagocytosis result of table
* is indicated and model group ratio P < 0.01
From table 3 it can be seen that compared to the 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, illustrates modeling success.Compared with model group, large, medium and small dosage polysaccharide group and
Lentinan group is remarkably improved Turnover of Mouse Peritoneal Macrophages to phagocytic index and phagocytic percentage (the P < of chicken red blood cell
0.01), 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 are taken, weight 18.5-23g, half male and half female is uniformly divided into 6 groups at random.In addition to blank control group, remaining
Each group establishes cyclophosphamide and causes immunosuppression model totally 5 groups of (dosage 8mg/ml；Continuous intraperitoneal injection in 1st, 2,3 day)；Mould
After the completion of type group is established, 5% chicken red blood cell normal saline suspension 0.2ml/ is injected intraperitoneally only in each group mouse (containing blank group),
It is immunized；And start to gavage geranium wilfordii polysaccharide solution (5mg/ml, 10mg/ml, 20mg/ml respectively；0.2ml/10g), 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 take blood, are centrifuged, and separate serum；After being diluted with physiological saline 1: 100, take
(guinea pig serum is saturated in advance with chicken red blood cell by 1ml dilution and 5% chicken red blood cell suspension 0.5ml, 10% complement 0.5ml
6h) mix；37 DEG C of incubation 30min terminate reaction in ice water, separately set and the blank tube of complement is not added compares；Draw each pipe supernatant
Liquid colorimetric at UV-T1810 type spectrophotometer 540nm measures 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 are taken, weight 18.5-23g, half male and half female is uniformly divided into 6 groups at random.In addition to blank control group, remaining
Each group establishes cyclophosphamide and causes immunosuppression model totally 5 groups of (dosage 8mg/ml；Continuous intraperitoneal injection in 1st, 2,3 day)；Mould
After the completion of type group is established, 5% chicken red blood cell normal saline suspension 0.2ml/ is injected intraperitoneally only in each group mouse (containing blank group),
It is immunized；And start to gavage geranium wilfordii polysaccharide solution (5mg/ml, 10mg/ml, 20mg/ml respectively；0.2ml/10g), 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.Mouse is put to death and dissected to the 2h after last 1 time administration, cervical dislocation, takes out spleen, is with two mouse spleens in group
An example, homogenate, and adjusting spleens cell number in splenocyte suspension is 5 × 106/ml.Extracting spleen cell suspension 1.0ml, with
The guinea pig serum 0.5ml of 0.2% chicken red blood cell suspension 0.5ml and 1: 10 is mixed.The blank tube that complement is not added separately is set, 37 DEG C incubate
1h is educated, is centrifuged, supernatant colorimetric at UV-T1810 type spectrophotometer 413nm is taken, surveys each group hemolysis plaque formational situation, knot
Fruit is shown in Table 4.
4 hemolysin of table, hemolysis plaque form result
* is indicated and model group ratio P < 0.01
It 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 group
It is best.
The experiment of geranium wilfordii oligosaccharide antifatigue effect:
One, test material
1. experimental animal: the Kunming 18.5-23g kind detergent mouse, half male and half female.It is mentioned by Zhengzhou University's Experimental Animal Center
For.
2. testing reagent: the geranium wilfordii oligosaccharide content of the method for the present invention preparation is 89.35%, superoxide dismutase
(SOD) assay kit, malonaldehyde (MDA) assay kit etc..
Two, test methods
Kunming mouse 40 of weight 18.5-23g are taken, are randomly divided into 4 groups by weight: Normal group, low (200mg/
Kg), (500mg/kg), high dose (800mg/kg) administration group in；Daily gastric infusion is primary, and Normal group is with the life of equivalent
Salt water stomach-filling is managed, successive administration 30 days, in 30min after the last administration, mouse is put in swimming trunk, water temperature is 25 ± 5 DEG C, mouse
The sheet lead of 5% weight of root of the tail portion weight bearing, record mouse submerges the time that 8s in water does not go out the water surface to head since swimming, i.e., small
The walking weight load of mouse.Mouse is after swimming terminates to restore for 24 hours, and eye socket takes blood to every group of mouse respectively, separates serum, uses super oxygen
Compound mutase (SOD) assay kit, malonaldehyde (MDA) assay kit measure SOD in mice serum respectively, and MDA value is shown in
Table 5.
5 geranium wilfordii oligosaccharide of table is to the mice burden swimming time, the influence of SOD and MDA in serum
* P < 0.05 compared to the blank group is indicated；* indicates P < 0.01 compared to the blank group
As can be seen from Table 5, geranium wilfordii oligosaccharide high and low dose is remarkably improved body SOD vigor, reduces MDA and contains
Measure (P < 0.05), middle dose group can extremely significant raisings body SOD vigor, reduction MDA content (P < 0.01), to show old stork
Careless oligosaccharide has preferable antifatigue effect.
Embodiment described above is only presently preferred embodiments of the present invention, not does limit in any form to the present invention
System, although the present invention has been disclosed as a preferred embodiment, is not to limit the invention, any to be familiar with this profession
Technical staff, without departing from the scope of the present invention, when making few modifications using the technology contents of the disclosure above
Or it is modified to the equivalent embodiment of equivalent variations, but anything that does not depart from the technical scheme of the invention content, skill according to the present invention
Art any simple modification substantially made to the above embodiment, equivalent variations and modification, belong to the range of technical solution of the present invention
It is interior.
Claims (5)
1. a kind of application of geranium wilfordii polysaccharide E in the drug and health care product of preparation strengthen immunity, geranium wilfordii oligosaccharide F are making
Application in standby antifatigue drug and health care product, the geranium wilfordii polysaccharide E and geranium wilfordii oligosaccharide F are to make by the following method
It is standby to obtain: geranium wilfordii being taken to be dried, crushed into coarse powder；Geranium wilfordii coarse powder is taken, distilled water is added into geranium wilfordii coarse powder, geranium wilfordii is thick
The weight ratio of powder and distilled water is 1:11-22, impregnates 23-46min, 84 DEG C of -98 DEG C of ultrasonic extraction 41-97min, filtering, filtrate
It is concentrated into the medicinal extract that specific gravity is 1.11-1.20, ethyl alcohol, which is added, makes ethanol content reach volumetric concentration 66%-79%, 4 DEG C of standings
8-15h, 6000r/min are centrifuged 45min, obtain supernatant A and centrifugation B, and supernatant A is spare, centrifugation B freeze-drying,
Geranium wilfordii Thick many candies powder C, take geranium wilfordii Thick many candies powder C be added 10 times of weight distilled water make to dissolve geranium wilfordii is slightly more
Sugar juice D the yeast of its 1% weight is added into geranium wilfordii Thick many candies solution D, at room temperature revolving speed 150-195r/min shaking table
Ca (OH) is added in filtrate in cultivation and fermentation, fermentation time 26-74h, filtering2It is 8.1,88 DEG C of heating water baths to filtrate pH value
66min is passed through CO simultaneously2Gas is put to room temperature, filtering, and filtrate is concentrated into the medicinal extract that specific gravity is 1.11-1.20, and ethyl alcohol, which is added, to be made
It is 80% that ethanol content, which reaches volumetric concentration, and 4 DEG C of standings 12h, 6000r/min are centrifuged 35min, takes lower layer's pellet frozen dry i.e.
Obtain geranium wilfordii polysaccharide E；It takes supernatant A that ethyl alcohol is recovered under reduced pressure and is concentrated into the medicinal extract that specific gravity is 1.11-1.21, ether extraction is collected
Lower layer's extract liquor, 50 DEG C are flung to ether, pass sequentially through large pore resin absorption column, gel resin column, obtain eluent, eluent decompression
Concentration, is freeze-dried to obtain geranium wilfordii oligosaccharide F；The macroporous absorbent resin is AB-8 type macroporous absorbent resin, gel resin
For Sephadex LH-20.
2. application of the geranium wilfordii polysaccharide E described in claim 1 in the drug and health care product of preparation strengthen immunity, geranium wilfordii
Oligosaccharide F is preparing the application in antifatigue drug and health care product, which is characterized in that the geranium wilfordii polysaccharide E and geranium wilfordii
Oligosaccharide F is to be prepared by the following method to obtain: geranium wilfordii being taken to be dried, crushed into coarse powder；Geranium wilfordii coarse powder is taken, it is thick to geranium wilfordii
It is added distilled water in powder, the weight ratio of geranium wilfordii coarse powder and distilled water is 1:22,46min, 98 DEG C of ultrasonic extraction 97min are impregnated,
Filtering, filtrate are concentrated into the medicinal extract that specific gravity is 1.20, and ethyl alcohol, which is added, makes ethanol content reach volumetric concentration 79%, 4 DEG C of standings
15h, 6000r/min are centrifuged 45min, obtain supernatant A and centrifugation B, and supernatant A is spare, and centrifugation B freeze-drying obtains
Geranium wilfordii Thick many candies powder C, the distilled water for taking geranium wilfordii Thick many candies powder C that 10 times of weight are added make to dissolve to obtain geranium wilfordii Thick many candies
The yeast of its 1% weight is added into geranium wilfordii Thick many candies solution D for solution D, and revolving speed 195r/min shaking table culture is sent out at room temperature
Ca (OH) is added in filtrate in ferment, fermentation time 74h, filtering2To filtrate pH value be 8.1,88 DEG C of heating water bath 66min simultaneously
It is passed through CO2Gas is put to room temperature, filtering, and filtrate is concentrated into the medicinal extract that specific gravity is 1.20, and ethyl alcohol, which is added, makes ethanol content reach body
Product concentration is 80%, and 4 DEG C of standing 12h, 6000r/min centrifugation 35min take lower layer's pellet frozen to be drying to obtain geranium wilfordii polysaccharide E；
It takes supernatant A that ethyl alcohol is recovered under reduced pressure and is concentrated into the medicinal extract that specific gravity is 1.21, ether extraction collects lower layer's extract liquor, 50 DEG C are flung to
Ether passes sequentially through large pore resin absorption column, gel resin column, obtains eluent, and eluent is concentrated under reduced pressure, and is freeze-dried to obtain old stork
Careless oligosaccharide F；The macroporous absorbent resin is AB-8 type macroporous absorbent resin, and gel resin is Sephadex LH-20.
3. application of the geranium wilfordii polysaccharide E described in claim 1 in the drug and health care product of preparation strengthen immunity, geranium wilfordii
Oligosaccharide F is preparing the application in antifatigue drug and health care product, which is characterized in that the geranium wilfordii polysaccharide E and geranium wilfordii
Oligosaccharide F is to be prepared by the following method to obtain: geranium wilfordii being taken to be dried, crushed into coarse powder；Geranium wilfordii coarse powder is taken, it is thick to geranium wilfordii
It is added distilled water in powder, the weight ratio of geranium wilfordii coarse powder and distilled water is 1:11,23min, 84 DEG C of ultrasonic extraction 41min are impregnated,
Filtering, filtrate are concentrated into the medicinal extract that specific gravity is 1.11, and ethyl alcohol, which is added, makes ethanol content reach volumetric concentration 66%, 4 DEG C of standings
8h, 6000r/min are centrifuged 45min, obtain supernatant A and centrifugation B, and supernatant A is spare, and centrifugation B freeze-drying obtains always
Stork grass Thick many candies powder C, take geranium wilfordii Thick many candies powder C be added 10 times of weight distilled water make to dissolve geranium wilfordii Thick many candies are molten
The yeast of its 1% weight is added into geranium wilfordii Thick many candies solution D by liquid D, and revolving speed 150r/min shaking table culture is sent out at room temperature
Ca (OH) is added in filtrate in ferment, fermentation time 26h, filtering2To filtrate pH value be 8.1,88 DEG C of heating water bath 66min simultaneously
It is passed through CO2Gas is put to room temperature, filtering, and filtrate is concentrated into the medicinal extract that specific gravity is 1.11, and ethyl alcohol, which is added, makes ethanol content reach body
Product concentration is 80%, and 4 DEG C of standing 12h, 6000r/min centrifugation 35min take lower layer's pellet frozen to be drying to obtain geranium wilfordii polysaccharide E；
It takes supernatant A that ethyl alcohol is recovered under reduced pressure and is concentrated into the medicinal extract that specific gravity is 1.11, ether extraction collects lower layer's extract liquor, 50 DEG C are flung to
Ether passes sequentially through large pore resin absorption column, gel resin column, obtains eluent, and eluent is concentrated under reduced pressure, and is freeze-dried to obtain old stork
Careless oligosaccharide F；The macroporous absorbent resin is AB-8 type macroporous absorbent resin, and gel resin is Sephadex LH-20.
4. application of the geranium wilfordii polysaccharide E described in claim 1 in the drug and health care product of preparation strengthen immunity, geranium wilfordii
Oligosaccharide F is preparing the application in antifatigue drug and health care product, which is characterized in that the geranium wilfordii polysaccharide E and geranium wilfordii
Oligosaccharide F is to be prepared by the following method to obtain: geranium wilfordii being taken to be dried, crushed into coarse powder；Geranium wilfordii coarse powder is taken, it is thick to geranium wilfordii
It is added distilled water in powder, the weight ratio of geranium wilfordii coarse powder and distilled water is 1:16,35min, 91 DEG C of ultrasonic extraction 69min are impregnated,
Filtering, filtrate are concentrated into the medicinal extract that specific gravity is 1.15, and ethyl alcohol, which is added, makes ethanol content reach volumetric concentration 72.5%, and 4 DEG C quiet
11h is set, 6000r/min is centrifuged 45min, obtains supernatant A and centrifugation B, and supernatant A is spare, centrifugation B freeze-drying,
Geranium wilfordii Thick many candies powder C, take geranium wilfordii Thick many candies powder C be added 10 times of weight distilled water make to dissolve geranium wilfordii is slightly more
Sugar juice D the yeast of its 1% weight is added into geranium wilfordii Thick many candies solution D, at room temperature revolving speed 173r/min shaking table culture
Ca (OH) is added in filtrate in fermentation, fermentation time 50h, filtering2It is that 8.1,88 DEG C of heating water bath 66min are same to filtrate pH value
When be passed through CO2Gas is put to room temperature, filtering, and filtrate is concentrated into the medicinal extract that specific gravity is 1.15, and ethyl alcohol, which is added, reaches ethanol content
Volumetric concentration is 80%, and 4 DEG C of standing 12h, 6000r/min centrifugation 35min take lower layer's pellet frozen to be drying to obtain geranium wilfordii polysaccharide
E；It takes supernatant A that ethyl alcohol is recovered under reduced pressure and is concentrated into the medicinal extract that specific gravity is 1.16, ether extraction collects lower layer's extract liquor, 50 DEG C are waved
Ether is removed, large pore resin absorption column, gel resin column are passed sequentially through, obtains eluent, eluent is concentrated under reduced pressure, and is freeze-dried always
Stork grass oligosaccharide F；The macroporous absorbent resin is AB-8 type macroporous absorbent resin, and gel resin is Sephadex LH-20.
5. application of the geranium wilfordii polysaccharide E described in claim 1 in the drug and health care product of preparation strengthen immunity, geranium wilfordii
Oligosaccharide F is preparing the application in antifatigue drug and health care product, which is characterized in that the geranium wilfordii polysaccharide E and geranium wilfordii
Oligosaccharide F is to be prepared by the following method to obtain: geranium wilfordii being taken to be dried, crushed into coarse powder；Geranium wilfordii coarse powder 11kg is taken, to old stork
Distilled water 121kg is added in careless coarse powder, impregnates 46min, 91 DEG C of ultrasonic extraction 97min, filtering, it is 1.11 that filtrate, which is concentrated into specific gravity,
Medicinal extract, ethyl alcohol, which is added, makes ethanol content reach volumetric concentration 66%, and 4 DEG C of standing 11h, 6000r/min centrifugation 45min are obtained
Supernatant A and centrifugation B, supernatant A is spare, and centrifugation B freeze-drying obtains geranium wilfordii Thick many candies powder C, takes geranium wilfordii
The distilled water that 10 times of weight are added in Thick many candies powder C makes to dissolve to obtain geranium wilfordii Thick many candies solution D, to geranium wilfordii Thick many candies solution D
The middle yeast that its 1% weight is added, revolving speed 173r/min shaking table culture is fermented at room temperature, fermentation time 26h, filtering, in filter
Ca (OH) is added in liquid2It is that 8.1,88 DEG C of heating water bath 66min are passed through CO simultaneously to filtrate pH value2Gas is put to room temperature, filtering,
Filtrate is concentrated into the medicinal extract that specific gravity is 1.16, and ethyl alcohol, which is added, makes ethanol content reach volumetric concentration 80%, 4 DEG C of standing 12h,
6000r/min is centrifuged 35min, and lower layer's pellet frozen is taken to be drying to obtain geranium wilfordii polysaccharide E；Take supernatant A that ethyl alcohol is recovered under reduced pressure dense
It is reduced to the medicinal extract that specific gravity is 1.21, ether extraction collects lower layer's extract liquor, 50 DEG C are flung to ether, pass sequentially through macroporous absorption tree
Rouge column, gel resin column obtain eluent, and eluent is concentrated under reduced pressure, and are freeze-dried to obtain geranium wilfordii oligosaccharide F；The macropore is inhaled
Attached resin is AB-8 type macroporous absorbent resin, and gel resin is Sephadex LH-20.
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