CN106990153A - A kind of method for differentiating astragalus mongolicus and Astragalus membranacus - Google Patents
A kind of method for differentiating astragalus mongolicus and Astragalus membranacus Download PDFInfo
- Publication number
- CN106990153A CN106990153A CN201710400065.9A CN201710400065A CN106990153A CN 106990153 A CN106990153 A CN 106990153A CN 201710400065 A CN201710400065 A CN 201710400065A CN 106990153 A CN106990153 A CN 106990153A
- Authority
- CN
- China
- Prior art keywords
- astragalus
- polysaccharide
- derivatization
- saccharides
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
- G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Electrochemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Saccharide Compounds (AREA)
Abstract
The present invention relates to a kind of method for differentiating astragalus mongolicus and Astragalus membranacus, present invention aim to address existing astragalus mongolicus and Astragalus membranacus discrimination method specificity it is low, be difficult to the technical problem that differentiates Radix Astragali kind；The technical scheme is that：Milkvetch Root is dried, crushed；The method of water extract-alcohol precipitation extracts astragalus polyose；Prepare astragalus polyose Partial acid hydrolysis product；The derivatization of hydrolysate；Electrophoretic analysis is carried out to astragalus polyose hydrolysate；Through Quantity One to electrophoretic image processing, the content of trisaccharide and disaccharides in astragalus polyose is calculated, and astragalus mongolicus and Astragalus membranacus are differentiated according to following standard：As two sugared content ＜ 1.0mg/mL, and it is astragalus mongolicus during two sugared contents/tri- sugared content ＜ 0.3；As two sugared content ＞ 1.0mg/mL, and it is Astragalus membranacus during two sugared contents/tri- sugared content ＞ 0.3；Not only easy to operate, experimental cost of the invention is low, and the saccharic composition difference formed after the astragalus polyose Partial acid hydrolysis of different genera is substantially, the discriminating available for Milkvetch Root kind.
Description
Technical field
The present invention relates to a kind of method for differentiating astragalus mongolicus and Astragalus membranacus, fluoroscopic assist gel electrophoresis skill is particularly belonged to
Art, using the content of otherness bglii fragment after astragalus polyose Partial acid hydrolysis as the astragalus mongolicus of appraisal basis and the mirror of Astragalus membranacus
Other method.
Background technology
The Radix Astragali, ancient name astragalus have the good reputation of " length of help ", records in going through version《Chinese Pharmacopoeia》, it is that beans plant is covered
The dry root of the ancient Radix Astragali or Astragalus membranacus, with invigorating qi for strengthening superficies, diuretic and maintain drug, the effects such as expelling pus and promoting granulation, have " stilbene of ten medicine nine " it
Say, enjoy the favor of the successive dynasties traditional Chinese medical science.And the Chinese patent drug up to more than 200 using the Radix Astragali as raw material plants demand pole of the domestic market to the Radix Astragali
Greatly, wherein there are about 50% is used to produce Radix Astragali medicine materical crude slice, nearly 50% is used for Chinese patent drug and extract and preparation.
Astragalus mongolicus and Astragalus membranacus cause its drug effect different because its chemical composition is variant, although in plant
Also there is significant difference in terms of upper portion forms feature and biological property, but its medicinal effects astragalus root profile is more close, meat
Eye is difficult to distinguish.Therefore, occur in that two kinds of Radixs Astragali mix phenomenon in medicinal material market in recent years, upset Chinese Medicinal Materials Markets.
Clinical research shows that carbohydrate content is the main matter that the Radix Astragali plays immunoregulation effect.Using carbohydrate content as
Radix Astragali quality control and the standard of quality evaluation, with good Research Prospects and meaning.Domestic and foreign scholars are also attempted with total many
Sugared content is compared to the Radix Astragali of different genera, however, the index determined lacks the specificity feature of Radix Astragali kind, it is difficult to
Discriminating for Radix Astragali kind.
The content of the invention
Present invention aim to address existing astragalus mongolicus and Astragalus membranacus discrimination method specificity it is low, be difficult to differentiate yellow
There is provided a kind of method for differentiating astragalus mongolicus and Astragalus membranacus for the technical problem of stilbene kind.
In order to solve the above technical problems, the technical solution adopted by the present invention is：One kind differentiates astragalus mongolicus and Astragalus membranacus
Method, comprise the following steps：
1) pretreatment of raw material：The dry Radix Astragali is ground into after powder 100 mesh sieves excessively and obtains Radix Astragali fine powder, it is standby；
2) Thick many candies are extracted：Take above-mentioned Radix Astragali fine powder add water after at a temperature of 100 DEG C refluxing extraction 1h, be down to after room temperature
Centrifuging and taking supernatant；Residue adds water repetition and extracted 1 time, takes supernatant, merges supernatant；Concentration, the supernatant of merging is used
It is that 80%, 3000r/min centrifuges 10min that 95% ethanol, which is adjusted to alcohol content, merges precipitation, is freeze-dried to obtain polysaccharide coarse powder；
3) purified polysaccharide：By step 2) obtained polysaccharide coarse powder is soluble in water, obtains polysaccharide meal solution, the polysaccharide is thick
The mass ratio of powder and water is 1:200, potassium ferrocyanide solution and concentration of the concentration for 10.6% are added into polysaccharide meal solution
For 21.9% acetic acid zinc solution, the zinc acetate that the potassium ferrocyanide solution and concentration that the concentration is 10.6% are 21.9% is molten
The addition of liquid is the 10% of polysaccharide meal solution volume, and 30min is stood after shaking, and centrifugation obtains purified polysaccharide, freezes, standby
With；
4) polysaccharide sour water solution：Take step 3) obtained purified polysaccharide 10mg, the μ L of 0.5mol/L trifluoroacetic acids 500 are added, are mixed
It is even, mixed liquor is then placed in 90 DEG C of insulation 1h, after reaction terminates, drying instrument is blown with nitrogen and dries polysaccharide acid hydrolysis products, then
Proper amount of methanol cleaning hydrolysate is added, continues nitrogen drying；
5) derivatization of polysaccharide acid hydrolysis products, three saccharides and two saccharides：Take three saccharide 6mg, disaccharides
Standard items 2mg and step 4) in obtained polysaccharide acid hydrolysis products, be separately added into 200 μ L NaCNBH3 solution, then respectively plus
Enter 200 μ L ANTS derivatization reagents, be vortexed, mix；After 37 DEG C of isothermal reaction 15h, three kinds of products are dried up with nitrogen, are dissolved in
1mL6mol/L urea liquid, is made the derivatization sample of three saccharides, two saccharides and polysaccharide acid hydrolysis products, its
In the derivatization samples of three saccharides and two saccharides be diluted to no less than 6 concentration respectively with 6mol/L urea liquid
The derivatization sample of gradient；
6) derivatization sample electrophoresis are analyzed：Take step 5) in polysaccharide acid hydrolysis products derivatization sample, three saccharides spread out
Biological sample and two saccharide derivatization samples, each derivatization sample is separated using vertical slab gel electrophoresis, and separation gel is
Concentration is 30% polyacrylamide gel, and concentration glue is the polyacrylamide gel that concentration is 8%, and electrophoretic buffer is
PH8.0-9.0, concentration 0.1-0.2mol/L Tris-boric, applied sample amount are 1-3 μ L, and gel is imaged under the conditions of UV300nm,
Obtain the electrophoresis of polysaccharide acid hydrolysis products derivatization sample, three saccharide derivatization samples and two saccharide derivatization samples
Figure；
7) standard curve of three saccharides and two saccharides is drawn：By polysaccharide acid hydrolysis products derivatization sample, three
The electrophoretogram of saccharide derivatization sample and two saccharide derivatization samples imports Quantity One softwares, by processing
Afterwards, the OD value of polysaccharide acid hydrolysis products and three saccharides and two saccharides is obtained, with three saccharides and disaccharides mark
The OD value of quasi- product is that ordinate, concentration are abscissa, draws standard curve；
8) discriminating of astragalus mongolicus and Astragalus membranacus：Bring the OD value of polysaccharide acid hydrolysis products into step 7) in mark
Directrix curve, calculates the content of trisaccharide and disaccharides in astragalus polyose, and differentiate astragalus mongolicus and Astragalus membranacus according to following standard：
As two sugared content ＜ 1.0mg/mL, and it is astragalus mongolicus during two sugared contents/tri- sugared content ＜ 0.3；
As two sugared content ＞ 1.0mg/mL, and it is Astragalus membranacus during two sugared contents/tri- sugared content ＞ 0.3.
Further, step 2) described in Radix Astragali fine powder and water mass ratio be 1:100.
Further, step 2) described in residue and water mass ratio be 1:80.
Further, step 6) in separation gel and concentration glue preparation solvent for pH8.0~9.0, concentration 0.1~
0.2mol/L Tris-boric buffer solutions.
Further, step 6) in electrophoretic separation process be carried out in two steps, first using 60-80V separation voltage electricity
Swimming separation 40-60min, then using 100-200V separation voltage electrophoretic separation 100-120min.
The beneficial effects of the invention are as follows：
1) the saccharide portion sour water solution in the Radix Astragali is analyzed, finds out illiteracy by the present invention using fluoroscopic assist gel electrophoresis technology
The difference of the sugared composition of the ancient Radix Astragali and Astragalus membranacus, the discriminating for Radix Astragali kind.This method is not only easy to operate, experimental cost
It is low, and the saccharic composition difference formed after different genera astragalus polyose Partial acid hydrolysis is substantially, available for Milkvetch Root kind
Differentiate.
2) fluoroscopic assist gel electrophoresis (PACE) technology for using of the present invention, is a kind of very easily polysaccharide hydrolysate
Isolation technics, with high resolution, reproducible, stability it is high, can multiple samples analyze simultaneously the features such as；Due to carbohydrate chemical combination
Thing uses derivatization reagent 8- amino naphthalenes -1,3,6- tri- without ultraviolet or fluorophor, the present invention before PACE analyses are carried out
Sodium sulfonate (ANTS) does derivatization treatment to polysaccharide hydrolysate, polysaccharide hydrolysate is carried ultraviolet group and fluorophor,
Greatly improve the sensitivity of detection.
Brief description of the drawings
Fig. 1 is the polyacrylamide gel electrophoresis figure of different genera astragalus polyose Partial acid hydrolysis product；
Fig. 2 is the finger-print of different genera astragalus polyose Partial acid hydrolysis product；
Fig. 3 is the T check analysis figures of otherness bglii fragment；
Fig. 4 schemes for the PLS-DA of the astragalus polyose Partial acid hydrolysis product of different genera；
Fig. 5 is PLS-DA analysis model proof diagrams；
Fig. 6 is the load diagram of otherness bglii fragment；
Fig. 7 is the otherness bglii fragment number figure of the astragalus polyose Partial acid hydrolysis product of different genera；
Fig. 8 is the canonical plotting of two saccharides；
Fig. 9 is the canonical plotting of three saccharides.
Embodiment
The invention will be further described with reference to the accompanying drawings and examples.
Embodiment 1
A kind of method of discriminating astragalus mongolicus and Astragalus membranacus in the present embodiment, comprises the following steps：
1) pretreatment of raw material：The dry Radix Astragali is ground into after powder 100 mesh sieves excessively and obtains Radix Astragali fine powder, it is standby；
2) Thick many candies are extracted：Take above-mentioned Radix Astragali fine powder about 5g, it is accurately weighed after be placed in 1000mL round-bottomed flasks, add water
500mL, the refluxing extraction 1h at a temperature of 100 DEG C, is down to centrifuging and taking supernatant after room temperature；Residue adds water 400mL, and repetition is carried
Take 1 time, take supernatant, merge supernatant；Concentration, it is 80% that the supernatant of merging, which is adjusted with 95% ethanol to alcohol content,
3000r/min centrifuges 10min, merges precipitation, is freeze-dried to obtain polysaccharide coarse powder；
3) purified polysaccharide：By step 2) obtained polysaccharide coarse powder is soluble in water, obtains polysaccharide meal solution, the polysaccharide is thick
The mass ratio of powder and water is 1:200, to polysaccharide meal solution add concentration be 10.6% potassium ferrocyanide solution and concentration be
21.9% acetic acid zinc solution, the acetic acid zinc solution that the potassium ferrocyanide solution and concentration that the concentration is 10.6% are 21.9%
Addition be the 10% of polysaccharide meal solution volume, stand 30min after shaking, centrifugation obtains purified polysaccharide, freeze, it is standby
With；
4) polysaccharide sour water solution：Take step 3) obtained purified polysaccharide 10mg, the μ L of 0.5mol/L trifluoroacetic acids 500 are added, are mixed
It is even, mixed liquor is then placed in 90 DEG C of insulation 1h, after reaction terminates, drying instrument is blown with nitrogen and dries polysaccharide acid hydrolysis products, to go
Except the trifluoroacetic acid wherein remained, proper amount of methanol cleaning hydrolysate is then added, continues nitrogen drying；
5) derivatization of polysaccharide acid hydrolysis products, three saccharides and two saccharides：Take three saccharide 6mg, disaccharides
Standard items 2mg and step 4) in obtained polysaccharide acid hydrolysis products, be separately added into 200 μ L NaCNBH3 solution, then respectively plus
Enter 200 μ L ANTS derivatization reagents, be vortexed, mix；After 37 DEG C of isothermal reaction 15h, three kinds of products are dried up with nitrogen, are dissolved in
1mL6mol/L urea liquid, is made the derivatization sample of three saccharides, two saccharides and polysaccharide acid hydrolysis products, its
In the derivatization samples of three saccharides and two saccharides be diluted to no less than 6 concentration respectively with 6mol/L urea liquid
The derivatization sample of gradient；
6) derivatization sample electrophoresis are analyzed：Take step 5) in polysaccharide acid hydrolysis products derivatization sample, three saccharides
Derivatization sample and two saccharide derivatization samples, each derivatization sample is separated using vertical slab gel electrophoresis；Separation gel
The polyacrylamide gel for being 30% for concentration, concentration glue is polyacrylamide gel that concentration is 8%, the separation gel and dense
The preparation solvent of contracting glue is pH8.2, concentration 0.1mol/L Tris-boric buffer solutions；Electrophoretic buffer is pH8.2, concentration
0.1mol/L Tris-boric, applied sample amount is 1 μ L；First using 60V separation voltage electrophoretic separation 60min, then use
200V separation voltage electrophoretic separation 120min；Gel is imaged under the conditions of UV300nm, obtains polysaccharide acid hydrolysis products derivatization
The electrophoretogram of sample, three saccharide derivatization samples and two saccharide derivatization samples；
7) standard curve of three saccharides and two saccharides is drawn：By polysaccharide acid hydrolysis products derivatization sample, three
The electrophoretogram of saccharide derivatization sample and two saccharide derivatization samples imports Quantity One softwares, by processing
Afterwards, the OD value of polysaccharide acid hydrolysis products and three saccharides and two saccharides is obtained, with three saccharides and disaccharides mark
The OD value of quasi- product is that ordinate, concentration are abscissa, draws standard curve；
8) discriminating of astragalus mongolicus and Astragalus membranacus：Bring the OD value of polysaccharide acid hydrolysis products into step 7) in mark
Directrix curve, calculates the content of trisaccharide and disaccharides in astragalus polyose, and differentiate astragalus mongolicus and Astragalus membranacus according to following standard：
As two sugared content ＜ 1.0mg/mL, and it is astragalus mongolicus during two sugared contents/tri- sugared content ＜ 0.3；
As two sugared content ＞ 1.0mg/mL, and it is Astragalus membranacus during two sugared contents/tri- sugared content ＞ 0.3.
Gained two sugared contents=0.945mg/mL is calculated in the present embodiment, sugared content=0.289 of two sugared contents/tri- differentiates
As a result it is astragalus mongolicus.
Embodiment 2
A kind of method of discriminating astragalus mongolicus and Astragalus membranacus in the present embodiment, comprises the following steps：
1) pretreatment of raw material：The dry Radix Astragali is ground into after powder 100 mesh sieves excessively and obtains Radix Astragali fine powder, it is standby；
2) Thick many candies are extracted：Take above-mentioned Radix Astragali fine powder about 5g, it is accurately weighed after be placed in 1000mL round-bottomed flasks, add water
500mL, the refluxing extraction 1h at a temperature of 100 DEG C, is down to centrifuging and taking supernatant after room temperature；Residue adds water 400mL, and repetition is carried
Take 1 time, take supernatant, merge supernatant；Concentration, it is 80% that the supernatant of merging, which is adjusted with 95% ethanol to alcohol content,
3000r/min centrifuges 10min, merges precipitation, is freeze-dried to obtain polysaccharide coarse powder；
3) purified polysaccharide：By step 2) obtained polysaccharide coarse powder is soluble in water, obtains polysaccharide meal solution, the polysaccharide is thick
The mass ratio of powder and water is 1:200, to polysaccharide meal solution add concentration be 10.6% potassium ferrocyanide solution and concentration be
21.9% acetic acid zinc solution, the acetic acid zinc solution that the potassium ferrocyanide solution and concentration that the concentration is 10.6% are 21.9%
Addition be the 10% of polysaccharide meal solution volume, stand 30min after shaking, centrifugation obtains purified polysaccharide, freeze, it is standby
With；
4) polysaccharide sour water solution：Take step 3) obtained purified polysaccharide 10mg, the μ L of 0.5mol/L trifluoroacetic acids 500 are added, are mixed
It is even, mixed liquor is then placed in 90 DEG C of insulation 1h, after reaction terminates, drying instrument is blown with nitrogen and dries polysaccharide acid hydrolysis products, to go
Except the trifluoroacetic acid wherein remained, proper amount of methanol cleaning hydrolysate is then added, continues nitrogen drying；
5) derivatization of polysaccharide acid hydrolysis products, three saccharides and two saccharides：Take three saccharide 6mg, disaccharides
Standard items 2mg and step 4) in obtained polysaccharide acid hydrolysis products, be separately added into 200 μ L NaCNBH3 solution, then respectively plus
Enter 200 μ L ANTS derivatization reagents, be vortexed, mix；After 37 DEG C of isothermal reaction 15h, three kinds of products are dried up with nitrogen, are dissolved in
1mL6mol/L urea liquid, is made the derivatization sample of three saccharides, two saccharides and polysaccharide acid hydrolysis products, its
In the derivatization samples of three saccharides and two saccharides be diluted to no less than 6 concentration respectively with 6mol/L urea liquid
The derivatization sample of gradient；
6) derivatization sample electrophoresis are analyzed：Take step 5) in polysaccharide acid hydrolysis products derivatization sample, three saccharides
Derivatization sample and two saccharide derivatization samples, each derivatization sample is separated using vertical slab gel electrophoresis；Separation gel
The polyacrylamide gel for being 30% for concentration, concentration glue is polyacrylamide gel that concentration is 8%, the separation gel and dense
The preparation solvent of contracting glue is pH8.5, concentration 0.2mol/L Tris-boric buffer solutions；Electrophoretic buffer is pH8.5, concentration
0.2mol/L Tris-boric, applied sample amount is 1 μ L；First using 70V separation voltage electrophoretic separation 50min, then use
150V separation voltage electrophoretic separation 110min；Gel is imaged under the conditions of UV300nm, obtains polysaccharide acid hydrolysis products derivatization
The electrophoretogram of sample, three saccharide derivatization samples and two saccharide derivatization samples；
7) standard curve of three saccharides and two saccharides is drawn：By polysaccharide acid hydrolysis products derivatization sample, three
The electrophoretogram of saccharide derivatization sample and two saccharide derivatization samples imports Quantity One softwares, by processing
Afterwards, the OD value of polysaccharide acid hydrolysis products and three saccharides and two saccharides is obtained, with three saccharides and disaccharides mark
The OD value of quasi- product is that ordinate, concentration are abscissa, draws standard curve；
8) discriminating of astragalus mongolicus and Astragalus membranacus：Bring the OD value of polysaccharide acid hydrolysis products into step 7) in mark
Directrix curve, calculates the content of trisaccharide and disaccharides in astragalus polyose, and differentiate astragalus mongolicus and Astragalus membranacus according to following standard：
As two sugared content ＜ 1.0mg/mL, and it is astragalus mongolicus during two sugared contents/tri- sugared content ＜ 0.3；
As two sugared content ＞ 1.0mg/mL, and it is Astragalus membranacus during two sugared contents/tri- sugared content ＞ 0.3.
Gained two sugared contents=1.06mg/mL is calculated in the present embodiment, sugared content=0.35 of two sugared contents/tri- differentiates knot
Fruit is Astragalus membranacus.
Embodiment 3
A kind of method of discriminating astragalus mongolicus and Astragalus membranacus in the present embodiment, comprises the following steps：
1) pretreatment of raw material：The dry Radix Astragali is ground into after powder 100 mesh sieves excessively and obtains Radix Astragali fine powder, it is standby；
2) Thick many candies are extracted：Take above-mentioned Radix Astragali fine powder about 5g, it is accurately weighed after be placed in 1000mL round-bottomed flasks, add water
500mL, the refluxing extraction 1h at a temperature of 100 DEG C, is down to centrifuging and taking supernatant after room temperature；Residue adds water 400mL, and repetition is carried
Take 1 time, take supernatant, merge supernatant；Concentration, it is 80% that the supernatant of merging, which is adjusted with 95% ethanol to alcohol content,
3000r/min centrifuges 10min, merges precipitation, is freeze-dried to obtain polysaccharide coarse powder；
3) purified polysaccharide：By step 2) obtained polysaccharide coarse powder is soluble in water, obtains polysaccharide meal solution, the polysaccharide is thick
The mass ratio of powder and water is 1:200, to polysaccharide meal solution add concentration be 10.6% potassium ferrocyanide solution and concentration be
21.9% acetic acid zinc solution, the acetic acid zinc solution that the potassium ferrocyanide solution and concentration that the concentration is 10.6% are 21.9%
Addition be the 10% of polysaccharide meal solution volume, stand 30min after shaking, centrifugation obtains purified polysaccharide, freeze, it is standby
With；
4) polysaccharide sour water solution：Take step 3) obtained purified polysaccharide 10mg, the μ L of 0.5mol/L trifluoroacetic acids 500 are added, are mixed
It is even, mixed liquor is then placed in 90 DEG C of insulation 1h, after reaction terminates, drying instrument is blown with nitrogen and dries polysaccharide acid hydrolysis products, to go
Except the trifluoroacetic acid wherein remained, proper amount of methanol cleaning hydrolysate is then added, continues nitrogen drying；
5) derivatization of polysaccharide acid hydrolysis products, three saccharides and two saccharides：Take three saccharide 6mg, disaccharides
Standard items 2mg and step 4) in obtained polysaccharide acid hydrolysis products, be separately added into 200 μ L NaCNBH3 solution, then respectively plus
Enter 200 μ L ANTS derivatization reagents, be vortexed, mix；After 37 DEG C of isothermal reaction 15h, three kinds of products are dried up with nitrogen, are dissolved in
1mL6mol/L urea liquid, is made the derivatization sample of three saccharides, two saccharides and polysaccharide acid hydrolysis products, its
In the derivatization samples of three saccharides and two saccharides be diluted to no less than 6 concentration respectively with 6mol/L urea liquid
The derivatization sample of gradient；
6) derivatization sample electrophoresis are analyzed：Take step 5) in polysaccharide acid hydrolysis products derivatization sample, three saccharides
Derivatization sample and two saccharide derivatization samples, each derivatization sample is separated using vertical slab gel electrophoresis；Separation gel
The polyacrylamide gel for being 30% for concentration, concentration glue is polyacrylamide gel that concentration is 8%, the separation gel and dense
The preparation solvent of contracting glue is pH9.0, concentration 0.15mol/L Tris-boric buffer solutions；Electrophoretic buffer is pH9.0, dense
0.15mol/L Tris-boric is spent, applied sample amount is 1 μ L；First using 80V separation voltage electrophoretic separation 40min, then adopt
100min is separated by electrophoresis with 100V separation voltage；Gel is imaged under the conditions of UV300nm, is obtained polysaccharide acid hydrolysis products and is derived
Change the electrophoretogram of sample, three saccharide derivatization samples and two saccharide derivatization samples；
7) standard curve of three saccharides and two saccharides is drawn：By polysaccharide acid hydrolysis products derivatization sample, three
The electrophoretogram of saccharide derivatization sample and two saccharide derivatization samples imports Quantity One softwares, by processing
Afterwards, the OD value of polysaccharide acid hydrolysis products and three saccharides and two saccharides is obtained, with three saccharides and disaccharides mark
The OD value of quasi- product is that ordinate, concentration are abscissa, draws standard curve；
8) discriminating of astragalus mongolicus and Astragalus membranacus：Bring the OD value of polysaccharide acid hydrolysis products into step 7) in mark
Directrix curve, calculates the content of trisaccharide and disaccharides in astragalus polyose, and differentiate astragalus mongolicus and Astragalus membranacus according to following standard：
As two sugared content ＜ 1.0mg/mL, and it is astragalus mongolicus during two sugared contents/tri- sugared content ＜ 0.3；
As two sugared content ＞ 1.0mg/mL, and it is Astragalus membranacus during two sugared contents/tri- sugared content ＞ 0.3.
Gained two sugared contents=1.225mg/mL is calculated in the present embodiment, sugared content=0.287 of two sugared contents/tri- differentiates
As a result it is astragalus mongolicus.
Two saccharides and three saccharides in above-described embodiment come from Nat'l Pharmaceutical ＆ Biological Products Control Institute, lot number point
Wei not 100287-201102,100274.
The present invention differentiates that the standard of Radix Astragali kind is obtained by following experiment.
The astragalus mongolicus used in experiment is derived from the ground such as the Gansu west of Gansu Province and Shandong Wendeng City, Astragalus membranacus be derived from Shanxi Huiyuan and
The ground such as Heilungkiang, the 2 class Radixs Astragali respectively take 12 samples.
Method according to the present invention is handled the Radix Astragali sample of selection, is then separated using gel electrophoresis,
The electrophoretogram of each Radix Astragali sample is obtained, as shown in figure 1, in figure, S is the saccharide containing 6 degree of polymerization, No. 1-12 is illiteracy
Ancient Radix Astragali sample, No. 13-24 is Astragalus membranacus sample, and DP represents the monose degree of polymerization, and DP1-DP6 is respectively a sugar, disaccharides, three
Sugar, disaccharides, pentasaccharides and six sugar.Electrophoretogram in Fig. 1 is imported into Quantity One softwares, after treatment, obtained and electrophoresis
The corresponding finger-print of figure, as shown in Fig. 2 in figure, No. 1-12 is astragalus mongolicus sample, and No. 13-24 is Astragalus membranacus sample.
Finger-print data in Fig. 2 are imported into SPSS16.0 and carry out T check analyses, Fig. 3 is obtained, from the figure 3, it may be seen that disaccharides has significantly
Sex differernce, i.e. disaccharides are to discriminate between the polysaccharide fragment that * in the Main Differences fragment of Radix Astragali kind, figure represents significant difference.
Finger-print data in Fig. 2 are imported into SMICA-P13.0 to be analyzed, analysis result such as Fig. 4, Fig. 5 and Fig. 6,
Fig. 4 schemes for the PLS-DA of the astragalus polyose Partial acid hydrolysis product of different genera, as shown in Figure 4, astragalus mongolicus and Astragalus membranacus
Apparent separation can be obtained；Fig. 5 is R in the proof diagram of PLS-DA analysis models, figure2For interpretation model value, Q2For prediction mould
Offset, as shown in Figure 5 PLS-DA models checking is set up, and the model can be used for otherness bglii fragment in Radix Astragali species discrimination to seek
Look for；Fig. 6 be otherness bglii fragment load diagram, as shown in Figure 6 scatterplot from center farther out, between the Radix Astragali of different genera difference compared with
Greatly.Otherness sugar-tablet segment number is counted, Fig. 7 is obtained, as shown in Figure 7, trisaccharide and disaccharides are to discriminate between the master of Radix Astragali kind
Want otherness fragment.
OD value in Fig. 2 finger-prints is brought into the standard curve of two saccharides and three saccharides respectively, counted
The disaccharides and three sugared contents in Radix Astragali sample are calculated, Fig. 8 and Fig. 9 are respectively the standard curve of two saccharides and three saccharides
In figure, figure, y is OD value, and x is concentration value, R2For coefficient correlation, result of calculation is as shown in table 1；
| Radix Astragali sample number into spectrum | Two sugared contents (mg/mL) | Three sugared contents (mg/mL) | The sugared content of two sugared contents/tri- |
| 1 | 0.873 | 3.310 | 0.264 |
| 2 | 0.809 | 3.088 | 0.262 |
| 3 | 0.985 | 3.384 | 0.291 |
| 4 | 0.945 | 3.270 | 0.289 |
| 5 | 0.681 | 3.013 | 0.226 |
| 6 | 0.783 | 3.058 | 0.256 |
| 7 | 0.864 | 2.958 | 0.292 |
| 8 | 0.748 | 2.662 | 0.281 |
| 9 | 0.857 | 2.866 | 0.299 |
| 10 | 0.685 | 2.991 | 0.229 |
| 11 | 0.795 | 2.978 | 0.267 |
| 12 | 0.774 | 2.660 | 0.291 |
| 13 | 1.026 | 3.167 | 0.324 |
| 14 | 1.102 | 3.158 | 0.349 |
| 15 | 1.298 | 3.197 | 0.406 |
| 16 | 1.096 | 3.036 | 0.361 |
| 17 | 1.112 | 3.038 | 0.366 |
| 18 | 1.372 | 3.330 | 0.412 |
| 19 | 1.016 | 2.509 | 0.405 |
| 20 | 1.134 | 2.961 | 0.383 |
| 21 | 1.028 | 2.763 | 0.372 |
| 22 | 1.123 | 2.894 | 0.388 |
| 23 | 1.231 | 2.959 | 0.416 |
| 24 | 1.035 | 2.974 | 0.348 |
Table 1
Table 1 is the disaccharides of 24 Radix Astragali samples and the content and its ratio of trisaccharide；As shown in Table 1,1-12 astragalus mongolicus
Two sugared contents in sample are respectively less than 1.0mg/mL, and the ratio of two sugared contents and three sugared contents is respectively less than 0.3；13-24 films
Two sugared contents in pod Radix Astragali sample are all higher than 1.0mg/mL, and the ratio of two sugared contents and three sugared contents is all higher than 0.3.
Therefore, the present invention can using disaccharides, trisaccharide content as differentiate Radix Astragali kind standard, i.e., as two sugared content ＜
1.0mg/mL, and be astragalus mongolicus during two sugared contents/tri- sugared content ＜ 0.3；As two sugared content ＞ 1.0mg/mL, and disaccharides contains
It is Astragalus membranacus when measuring/tri- sugared content ＞ 0.3.
Claims (5)
1. a kind of method for differentiating astragalus mongolicus and Astragalus membranacus, it is characterised in that comprise the following steps：
1) pretreatment of raw material：The dry Radix Astragali is ground into after powder 100 mesh sieves excessively and obtains Radix Astragali fine powder, it is standby；
2) Thick many candies are extracted：Take above-mentioned Radix Astragali fine powder add water after at a temperature of 100 DEG C refluxing extraction 1h, be down to after room temperature centrifuge
Take supernatant；Residue adds water repetition and extracted 1 time, takes supernatant, merges supernatant；Concentration, by the supernatant of merging with 95%
It is that 80%, 3000r/min centrifuges 10min that ethanol, which is adjusted to alcohol content, merges precipitation, is freeze-dried to obtain polysaccharide coarse powder；
3) purified polysaccharide：By step 2) obtained polysaccharide coarse powder is soluble in water, obtains polysaccharide meal solution, the polysaccharide coarse powder with
The mass ratio of water is 1:200, into polysaccharide meal solution add concentration be 10.6% potassium ferrocyanide solution and concentration be
21.9% acetic acid zinc solution, the acetic acid zinc solution that the potassium ferrocyanide solution and concentration that the concentration is 10.6% are 21.9%
Addition be the 10% of polysaccharide meal solution volume, stand 30min after shaking, centrifugation obtains purified polysaccharide, freeze, it is standby
With；
4) polysaccharide sour water solution：Take step 3) obtained purified polysaccharide 10mg, the μ L of 0.5mol/L trifluoroacetic acids 500 are added, are mixed, with
Mixed liquor is placed in 90 DEG C of insulation 1h afterwards, after reaction terminates, drying instrument is blown with nitrogen and dries polysaccharide acid hydrolysis products, then add suitable
Methanol cleaning hydrolysate is measured, continues nitrogen drying；
5) derivatization of polysaccharide acid hydrolysis products, three saccharides and two saccharides：Take three saccharide 6mg, two Standard for Sugars
Product 2mg and step 4) in obtained polysaccharide acid hydrolysis products, be separately added into 200 μ L NaCNBH3 solution, then be separately added into
200 μ L ANTS derivatization reagents, are vortexed, and mix；After 37 DEG C of isothermal reaction 15h, three kinds of products are dried up with nitrogen, are dissolved in
1mL6mol/L urea liquid, is made the derivatization sample of three saccharides, two saccharides and polysaccharide acid hydrolysis products, its
In the derivatization samples of three saccharides and two saccharides be diluted to no less than 6 concentration respectively with 6mol/L urea liquid
The derivatization sample of gradient；
6) derivatization sample electrophoresis are analyzed：Take step 5) in polysaccharide acid hydrolysis products derivatization sample, three saccharide derivatizations
Sample and two saccharide derivatization samples, separate each derivatization sample, separation gel is concentration using vertical slab gel electrophoresis
For 30% polyacrylamide gel, concentration glue is the polyacrylamide gel that concentration is 8%, and electrophoretic buffer is pH8.0-
9.0th, concentration 0.1-0.2mol/L Tris-boric, applied sample amount is 1-3 μ L, and gel is imaged under the conditions of UV300nm, is obtained many
The electrophoretogram of saccharic acid hydrolysate derivatization sample, three saccharide derivatization samples and two saccharide derivatization samples；
7) standard curve of three saccharides and two saccharides is drawn：By polysaccharide acid hydrolysis products derivatization sample, trisaccharide mark
The electrophoretogram of quasi- product derivatization sample and two saccharide derivatization samples imports Quantity One softwares, after treatment,
The OD value of polysaccharide acid hydrolysis products and three saccharides and two saccharides is obtained, with three saccharides and two saccharides
OD value be that ordinate, concentration are abscissa, draw standard curve；
8) discriminating of astragalus mongolicus and Astragalus membranacus：Bring the OD value of polysaccharide acid hydrolysis products into step 7) in standard it is bent
Line, calculates the content of trisaccharide and disaccharides in astragalus polyose, and differentiate astragalus mongolicus and Astragalus membranacus according to following standard：
As two sugared content ＜ 1.0mg/mL, and it is astragalus mongolicus during two sugared contents/tri- sugared content ＜ 0.3；
As two sugared content ＞ 1.0mg/mL, and it is Astragalus membranacus during two sugared contents/tri- sugared content ＞ 0.3.
2. a kind of method for differentiating astragalus mongolicus and Astragalus membranacus according to claim 1, it is characterised in that：Step 2) in
The mass ratio of the Radix Astragali fine powder and water is 1:100.
3. a kind of method for differentiating astragalus mongolicus and Astragalus membranacus according to claim 1, it is characterised in that：Step 2) in
The mass ratio of the residue and water is 1:80.
4. a kind of method for differentiating astragalus mongolicus and Astragalus membranacus according to claim 1, it is characterised in that：Step 6) in
The solvent of preparing of separation gel and concentration glue is pH8.0~9.0,0.1~0.2mol/L of concentration Tris-boric buffer solutions.
5. a kind of method for differentiating astragalus mongolicus and Astragalus membranacus according to claim 1, it is characterised in that：Step 6) in
Electrophoretic separation process be carried out in two steps, first using 60-80V separation voltage electrophoretic separation 40-60min, then use
100-200V separation voltage electrophoretic separation 100-120min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710400065.9A CN106990153A (en) | 2017-05-31 | 2017-05-31 | A kind of method for differentiating astragalus mongolicus and Astragalus membranacus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710400065.9A CN106990153A (en) | 2017-05-31 | 2017-05-31 | A kind of method for differentiating astragalus mongolicus and Astragalus membranacus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106990153A true CN106990153A (en) | 2017-07-28 |
Family
ID=59421432
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710400065.9A Pending CN106990153A (en) | 2017-05-31 | 2017-05-31 | A kind of method for differentiating astragalus mongolicus and Astragalus membranacus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106990153A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110632223A (en) * | 2019-07-16 | 2019-12-31 | 夏永刚 | Traditional Chinese medicine polysaccharide structural feature fingerprint spectrum and construction method and application thereof |
| CN110632162A (en) * | 2019-09-11 | 2019-12-31 | 山西大学 | Method for identifying wild astragalus and cultivated astragalus in ground |
| CN112986408A (en) * | 2019-12-13 | 2021-06-18 | 中国科学院大连化学物理研究所 | Method for identifying and analyzing astragalus and hedysarum polybotrys |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1965886A (en) * | 2005-11-14 | 2007-05-23 | 兰州大学 | Method for distinguishing astragalus root from its counterfeit drug by using SSR molecular marking process |
| CN103923909A (en) * | 2013-01-15 | 2014-07-16 | 复旦大学 | Specific molecular marker for panax ginseng and panax quinquefolius and identification method thereof |
-
2017
- 2017-05-31 CN CN201710400065.9A patent/CN106990153A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1965886A (en) * | 2005-11-14 | 2007-05-23 | 兰州大学 | Method for distinguishing astragalus root from its counterfeit drug by using SSR molecular marking process |
| CN103923909A (en) * | 2013-01-15 | 2014-07-16 | 复旦大学 | Specific molecular marker for panax ginseng and panax quinquefolius and identification method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 高凡茸: "黄芪药材甜味与品质关联性研究及黄芪糖谱分析方法的建立", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110632223A (en) * | 2019-07-16 | 2019-12-31 | 夏永刚 | Traditional Chinese medicine polysaccharide structural feature fingerprint spectrum and construction method and application thereof |
| CN110632162A (en) * | 2019-09-11 | 2019-12-31 | 山西大学 | Method for identifying wild astragalus and cultivated astragalus in ground |
| CN110632162B (en) * | 2019-09-11 | 2021-07-27 | 山西大学 | Method for identifying wild astragalus and cultivated astragalus in ground |
| CN112986408A (en) * | 2019-12-13 | 2021-06-18 | 中国科学院大连化学物理研究所 | Method for identifying and analyzing astragalus and hedysarum polybotrys |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103142669B (en) | Enzyme method for extracting ginsenoside and obtained ginsenoside extractive | |
| CN106990153A (en) | A kind of method for differentiating astragalus mongolicus and Astragalus membranacus | |
| CN107024524A (en) | It is a kind of to differentiate the method for transplanting the Radix Astragali and the wild Radix Astragali | |
| CN105560320A (en) | Ginseng, glossy ganoderma and cordyceps militaris powder formula and preparation method | |
| WO2020093510A1 (en) | Separation and purification method for polysaccharide in ganoderma lucidum spores | |
| CN102445514B (en) | Detection method of traditional Chinese medicine preparation jinshuibao capsule | |
| CN103788038B (en) | Red sesame lactone compound and pharmaceutical composition thereof and its preparation method and application | |
| CN101028460B (en) | Method for inspecting throat-clearing Chinese medicinal pills | |
| CN113354610A (en) | Method for extracting flavonoid components in yin-nourishing qi-tonifying blood-activating prescription medicine by using natural eutectic solvent and process optimization method thereof | |
| CN113968916B (en) | Extraction method and application of phlebopus portentosus polysaccharide | |
| CN103156920B (en) | A kind of drying means of Schisandra chinens P.E | |
| CN104407092A (en) | Quality detection method of traditional Chinese medicinal composition with efficacy of appetizing and invigorating spleen | |
| CN112485354A (en) | Ganoderma lucidum index detection method and authenticity identification method | |
| CN101195646A (en) | Production method of stilbene glycoside extract | |
| CN103183740B (en) | Production method of fructus schisandrae polysaccharide | |
| CN108424469B (en) | Gorgon fruit kernel polysaccharide and separation and extraction method and application thereof | |
| CN112076151B (en) | A Chinese medicinal oral liquid for treating diabetes due to deficiency of both qi and yin, and its preparation method and quality control method | |
| CN103623039A (en) | Astragaloside extract product, preparing method therefor and quality standard control method therefor | |
| CN110123946A (en) | A kind of process using saccharomyces cerevisiae solid state fermentation rhizoma polygonati | |
| CN104697950A (en) | Method for detecting pseudo-ginseng content and optimally extracting and purifying | |
| CN101411836A (en) | Quality standard of Chinese medicament preparation for treating cough after common cold and inspection method thereof | |
| WO2009155756A1 (en) | Method for determining the contents of oligosaccharides in morinda officinalis chinese medicine or extraction thereof | |
| CN113176366A (en) | Evaluation method of secondary refined polysaccharide and flavonoid components based on spectral efficiency relationship | |
| CN110483658A (en) | A kind of preparation method of hypoglycemic corn silk polysaccharide | |
| CN111484567B (en) | Extraction and preparation method of polysaccharide in long red dates, polysaccharide and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170728 |
|
| WD01 | Invention patent application deemed withdrawn after publication |