CN106501422B - A kind of chromatographic fingerprinting method identifying three kinds of medicinal bletillas - Google Patents
A kind of chromatographic fingerprinting method identifying three kinds of medicinal bletillas Download PDFInfo
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Abstract
The invention discloses a kind of chromatographic fingerprinting methods for identifying three kinds of medicinal bletillas, comprising the following steps: pretreatment, Polyose extraction, polysaccharide purification, the foundation of chromatographic fingerprinting, the identification of unknown type bletilla.The present invention has the advantages that three kinds of medicinal bletillas effectively can be distinguished and be identified by bletilla main pharmacodynamics ingredient-bletilla polysaccharide molecular weight distribution, it can overcome the disadvantages that the deficiency of the bletilla identification method of Chinese Pharmacopoeia 2015 editions settings, because the latter is the appearance purpose spot chromatographed using silica gel thin-layer point sample as its quality evaluation and judgment criteria, it is a kind of single information feedback, counterfeiter can be faked using the loophole of detection method, and this method is closer to the essence of chemical constituent, it is finer information, counterfeiter can not almost copy or counterfeit cost is expensive.
Description
Technical field
The present invention relates to the identifications and method of quality control technical field of orchid family bletilla category medicinal plant product, more particularly to
A kind of chromatographic fingerprinting method identifying three kinds of medicinal bletillas.
Background technique
Medicinal material bletilla is the drying of orchid family bletilla platymiscium bletilla (Bletilla striata (Thunb.) Reichb.f.)
Stem tuber is China's tradition hemostasia and promoting granulation medicine, and tcm clinical practice is for spitting blood, haematemesis, traumatic hemorrhage, sore swollen toxin, chapped skin.It is white
And the category plant whole world (Bletilla Rchb.) has 6 kinds, is distributed in the Upper Myanmar in Asia through China to Japan.China produces 4
Kind: magnificent bletilla Bletilla sinensis (Rolfe) Schltr.Schltr., small bletilla Btetilla formosana
(Hayata) Schltr., chrysanthemum bletilla Bletilla ochracea Schltr, pale reddish brown trident bletilla Bletilla striate
(Thunb.exA.Murray)Rchb.f..And traction and highest kind of medical value are pale reddish brown trident bletillas, and big absolutely
Most cultivars is also pale reddish brown trident bletilla, and provenance 90% derives from " Jiangxi group " and " Guizhou group ", growing surface
Product is about Guizhou and accounts for 20% (about 1500 mu), and Yunnan accounts for 18%, and Sichuan accounts for 15%, and Anhui accounts for 15%, and Hubei accounts for 12%, river
Soviet Union, Guangxi, Hunan, Shaanxi respectively account for 5%.
Effective medicinal ingredient there are many containing in modern pharmacological research display bletilla stem tuber, water-soluble polysaccharide is then its main medicine
Use ingredient.As natural macromolecular material, bletilla polysaccharide has functional slow-release, local retention, auto-degradation, nothing simultaneously
The characteristic of irritation, the auxiliary materials such as have no toxic side effect, the effect in modern medicines preparation are more and more important.
Main active one of of the polysaccharide compound as natural drug, activity and the space structure of its polysaccharide and
Molecular weight distribution is closely related, and for the correlation for exploring its component and drug effect, this programme will use Efficient numerical method method
(HPSEC) analysis is compared to the distribution of the relative molecular mass of same place of production variety classes bletilla polysaccharide, from different molecular
Ratio and the distribution of polysaccharide are measured to characterize the design feature of different plant species bletilla polysaccharide, is established after HPSEC chromatographic fingerprinting is
The formulation of continuous quality standard provides feasible solution.And so far, it has no and establishes medicinal bletilla polysaccharide about using HPSEC chromatography
The open report of chromatographic fingerprinting.
Summary of the invention
Technical problem to be solved by the present invention lies in provide a kind of more acurrate reliable color for identifying three kinds of medicinal bletillas
Fingerprint spectrum method is composed, which is the HPSEC color established according to three kinds of medicinal bletilla main pharmacodynamics basic substance polysaccharide
Spectrogram spectrum.The polysaccharide molecular weight distribution map that the present invention establishes can effectively distinguish different bletilla types, can both make up sharp in pharmacopeia
The appearance purpose spot chromatographed with silica gel thin-layer point sample judges the deficiency of bletilla superiority and inferiority and the true and false, it is also possible to identify three kinds
Medicinal bletilla (small bletilla Btetilla formosana (Hayata) Schltr.；Chrysanthemum bletilla Bletilla ochracea
Schltr；Pale reddish brown trident bletilla Bletilla striate (Thunb.exA.Murray) Rchb.f.) belong to other kind of bletilla and planting
Object.
In order to solve the above technical problems, the present invention provides a kind of chromatographic fingerprinting method for identifying three kinds of medicinal bletillas,
Include the following steps:
(1): pretreatment
It is mixed after the medicinal bletilla material of the known different cultivars sampled according to statistical method is thinly sliced, in
Drying to constant weight in 60-70 DEG C of baking oven and is ground into powder；
(2): Polyose extraction
1. it is colourless to phegma using gained powder in 80 DEG C -90 DEG C of dehydrated alcohol Soxhlet extraction step (1), it volatilizes molten
Agent, by the gained bletilla dregs of a decoction according to solid-liquid ratio (1-2): (4-6) is added in distilled water, and is condensed back in 75 DEG C of -85 DEG C of water-baths
It extracts 3-5 times, each 2-3h；
2. merging filtrate simultaneously into filtrate add mass fraction be 0.3%-0.5% active carbon mix decoloration, in 60 DEG C-
It is filtered after standing 1-2h under 65 DEG C of environment；
3. after filtering, above-mentioned solution being carried out negative pressure in Rotary Evaporators in 60 DEG C -65 DEG C, the 30%- for original volume being concentrated
35%, dehydrated alcohol is added, its final concentration is made to reach 80%-85%v/v, 2-3h is stood in 3-5 DEG C of refrigerator, then,
Precipitating is left and taken after being centrifuged 10-20min under 3000-4000r/min revolving speed；
4. being dissolved precipitating with distilled water up to medicinal bletilla Thick many candies solution；
(3): polysaccharide purification
Further polysaccharide purification is carried out to the resulting Thick many candies solution of step (2)；
(4): the foundation of chromatographic fingerprinting
1. the dextran standard of different molecular weight is configured to the aqueous solution difference sample introduction of 4-5mg/mL in efficient volume
In exclusion chromatography HPSEC, polysaccharide molecular weight calibration curve equation is calculated further according to each corresponding chromatogram；
2. the medicinal bletilla sample difference sample introduction after polysaccharide purification obtains corresponding color in Efficient numerical method HPSEC
Spectrogram calculates the relative molecular weight of chromatographic curve each section of each sample further according to polysaccharide molecular weight calibration curve equation, most
The HPSEC for establishing corresponding known medicinal bletilla sample eventually represents mode chromatographic map；
(5): the identification of unknown type bletilla
The bletilla of unknown type is handled according to above step (1)-(4), corresponds to chromatographic fingerprinting to obtain it,
The chromatographic fingerprinting of itself and established known bletilla sample is subjected to similarity-rough set again, when its similarity is greater than 90%
It when above, can be determined as same class bletilla platymiscium, otherwise not be same kind.
Preferably, to be respectively as follows: pale reddish brown trident bletilla, chrysanthemum white for the kind of the medicinal bletilla of different cultivars in the step (1)
And with small bletilla.
Preferably, medicinal bletilla material is medicinal bletilla fresh goods stem tuber or dry tuber in the step (1).
Preferably, statistical method in the step (1) are as follows: pressed respectively in Guizhou, Yunnan, Sichuan, four, Anhui area
Medicinal bletilla sample is collected according to five point sampling.
Preferably, polysaccharide purification uses Sevage method in the step (3): according to sample and extractant volume ratio (3-5):
Sevage reagent is added in (0.5-2), mixes, and vibrates 25-35min, is centrifuged 5-10min with 3000-4000r/min, discards and remove down
Layer chloroform and medial degeneration albumen；Upper layer aqueous solution is taken to repeat deproteinized operation again, until without obvious middle layer；Finally, collecting
Upper layer aqueous solution after deproteinized sloughs small-molecule substance by ultrafiltration to get molten to polysaccharide concentration after purification
Liquid.
Preferably, it is respectively (3-5) that the Sevage reagent, which is volume ratio: the chloroform of (0.5-1.5) and mixing for n-butanol
Close liquid.
Preferably, the ultrafiltration apparatus is the ultrafiltration apparatus that membrane retention molecular weight is 1K.
Preferably, the weight average molecular weight Mw of the dextran standard of different molecular weight is respectively as follows: in the step (4)
2000KDa、580KDa、70KDa、10KDa、5KDa。
Preferably, in the step (4) polysaccharide molecular weight calibration curve equation calculation method are as follows: according to each glucan mark
The chromatographic peak retention time and molecular weight logarithm of quasi- product, using the molecular weight logarithm lgMw of standard items as ordinate, chromatographic peak retains
Time Rt is abscissa, is fitted to obtain polysaccharide molecular weight calibration curve equation using linear equation.
Preferably, corresponding chromatographic condition when Efficient numerical method HPSEC detection is carried out are as follows: mobile phase is 0.1M acetic acid
Sodium, flow velocity 0.5mL/min, column temperature are 45 DEG C, and sample volume is 20 μ L.
The present invention has the advantages that the HPSEC chromatographic fingerprint figure of three kinds of bletilla polysaccharide molecular weight distributions provided by the invention
Spectrum utilizes " similarity evaluation " software of Chinese Pharmacopoeia committee product to represent mode map
Determined, can by bletilla main pharmacodynamics ingredient-bletilla polysaccharide molecular weight distribution come effectively distinguish and identify three kinds it is medicinal
Bletilla, method can overcome the disadvantages that the deficiency of the bletilla identification method of Chinese Pharmacopoeia 2015 editions settings, because the latter is to utilize silica gel
The appearances purpose spot of thin-layer sample application chromatography as its quality evaluation and judgment criteria, be that a kind of single information is fed back,
Counterfeiter can be faked using the loophole of detection method, and this method is closer to the essence of chemical constituent, is finer information,
Counterfeiter can not almost copy or counterfeit cost is expensive.
Detailed description of the invention
Fig. 1 is a kind of stream of the chromatographic fingerprinting method of three kinds of medicinal bletillas of identification that 1-2 of the embodiment of the present invention is provided
Cheng Tu.
Fig. 2 is a kind of the pale reddish brown of the chromatographic fingerprinting method of three kinds of medicinal bletillas of identification that the embodiment of the present invention 1 provides
The graph of molecular weight distribution of trident bletilla polysaccharide；
Fig. 3 is a kind of chrysanthemum of the chromatographic fingerprinting method of three kinds of medicinal bletillas of identification that the embodiment of the present invention 1 provides
The graph of molecular weight distribution of bletilla polysaccharide；
Fig. 4 is a kind of little Bai of the chromatographic fingerprinting method of three kinds of medicinal bletillas of identification that the embodiment of the present invention 1 provides
And the graph of molecular weight distribution of polysaccharide.
Specific embodiment
It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention
Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation
Example.
Embodiment 1
A kind of chromatographic fingerprinting method identifying three kinds of medicinal bletillas, as shown in Figure 1, including the following steps:
(1): pretreatment
The three kinds of medicinal bletillas that will be collected into respectively in Guizhou, Yunnan, Sichuan, four, Anhui area according to five point sampling
After the fresh tuber of (pale reddish brown trident bletilla, chrysanthemum bletilla, small bletilla) is thinly sliced, mixed respectively by same type, in 60 DEG C
Drying to constant weight in baking oven and is ground into powder；
(2): Polyose extraction
1. it is colourless to phegma using gained powder in 85 DEG C of dehydrated alcohol Soxhlet extraction step (1), solvent is volatilized, it will
The gained bletilla dregs of a decoction are added in distilled water according to solid-liquid ratio 1:5, and are condensed back and extract 3 times in 80 DEG C of water-baths, each 3h；
2. merging filtrate simultaneously adds the active carbon mixing decoloration that mass fraction is 0.5% into filtrate, under 60 DEG C of environment
It is filtered after standing 1h；
3. after filtering, carrying out negative pressure concentration in Rotary Evaporators in 60 DEG C is the 33% of original volume, dehydrated alcohol is added,
So that its final concentration is reached 85%v/v, stand 3h in 4 DEG C of refrigerators, then, is left and taken after being centrifuged 15min under 4000r/min revolving speed
Precipitating；
4. being dissolved precipitating with distilled water up to medicinal bletilla Thick many candies solution；
(3): polysaccharide purification
Further polysaccharide purification is carried out to the resulting Thick many candies solution of step (2), using Sevage method: according to sample with
Sevage reagent (volume ratio is respectively 4: 1 chloroform and n-butanol mixed liquor) is added in extractant volume ratio 4:1, mixes, oscillation
30min is centrifuged 15min with 4000r/min, discards except lower layer's chloroform and medial degeneration albumen；Upper layer aqueous solution is taken to repeat to go again
Albumen operation, until without obvious middle layer；Finally, collecting the upper layer aqueous solution after deproteinized, pass through ultrafiltration, filter membrane
Molecular cut off is 1K, sloughs small-molecule substance to get the polysaccharide concentrate solution arrived after purification；
(4): the foundation of chromatographic fingerprinting
1. weight average molecular weight Mw to be respectively as follows: to five kinds of glucan marks of 2000KDa, 580KDa, 70KDa, 10KDa, 5KDa
Quasi- product are configured to the aqueous solution difference sample introduction of 5mg/mL in Efficient numerical method HPSEC, according to each dextran standard
Chromatographic peak retention time and molecular weight logarithm, using the molecular weight logarithm lgMw of standard items as ordinate, chromatographic peak retention time
Rt is abscissa, is fitted to obtain polysaccharide molecular weight calibration curve equation: lgMw=y=-0.3486x+11.133 using linear equation,
R2=0.9968；Wherein, corresponding chromatographic condition when Efficient numerical method HPSEC detection is carried out are as follows: chromatographic column:G6000PWXL 7.8mm ID×300mm；Detector: differential refraction detector (RID)；Mobile phase: 0.1M
Sodium acetate；Flow velocity: 0.5mL/min；Column temperature: 45 DEG C, 20 μ L of sample volume；
2. the medicinal bletilla sample difference sample introduction after polysaccharide purification obtains corresponding color in Efficient numerical method HPSEC
Spectrogram calculates the relative molecular weight of chromatographic curve each section of each sample further according to polysaccharide molecular weight calibration curve equation, most
The HPSEC for establishing corresponding known bletilla sample eventually represents mode chromatographic map；Fig. 2-4 are respectively three kinds of medicinal bletillas in height
Peak spectrogram obtained in size exclusion chromatograph HPSEC is imitated, i.e., using abscissa as appearance time (unit: minute), ordinate is light
The graph of molecular weight distribution of the polysaccharide of the response (unit: millivolt) of electric signal, wherein the data on chromatographic peak are each cut point
Relative molecular weight Mw is calculated by above-mentioned polysaccharide molecular weight calibration curve equation；The HPSEC map of three kinds of bletilla samples
Peak be cut into 6 parts by fluctuating, molecular weight be respectively as follows: greater than 500KDa, 500KDa-200KDa, 200KDa-100KDa,
100KDa-50KDa, 50KDa-10Kda, it is less than 10KDa；Map is by stability test, accuracy test and reappearance test
The methods of learn investigate, relative peak area variation RSD be respectively less than 3%, meet chromatographic process require；
By Fig. 2-4 it is found that the molecular weight distribution of bletilla polysaccharide is more concentrated, three kinds of bletilla platymiscium sample (pale reddish brown tridents
Bletilla, chrysanthemum bletilla, small bletilla) weight average molecular weight (Mw) be respectively as follows: 590KDa, 98KDa, 72KDa, i.e. different cultivars bletilla
Weight average molecular weight have notable difference, numerically bletilla be much larger than chrysanthemum bletilla and small bletilla；
Therefore, three maps of Fig. 2-4 are the generation of the HPSEC chromatographic fingerprinting of three kinds of bletilla polysaccharide molecular weight distributions
Table schema map；
(5): the identification of unknown type bletilla
Two kinds of unknown bletilla platymiscium fresh tubers are handled according to above step (1)-(4), it is right to obtain its
Chromatographic fingerprinting is answered, " similarity evaluation " software produced further according to the Chinese Pharmacopoeia committee
Determined, the HPSEC chromatographic fingerprinting of itself and established known bletilla sample is subjected to similarity-rough set, as a result table
A kind of map of bright bletilla platymiscium and the similarity of the pale reddish brown trident bletilla of species are determined as that the pale reddish brown trident of species is white up to 94%
And and similarity that the map of another bletilla platymiscium represents mode finger-print with three kinds of medicinal bletillas is not achieved
90%, then determine this non-three kinds of medicinal bletillas of the bletilla platymiscium.
Embodiment 2
A kind of chromatographic fingerprinting method identifying three kinds of medicinal bletillas, as shown in Figure 1, including the following steps:
(1): pretreatment
The three kinds of medicinal bletillas that will be collected into respectively in Guizhou, Yunnan, Sichuan, four, Anhui area according to five point sampling
After the dry tuber of (pale reddish brown trident bletilla, chrysanthemum bletilla, small bletilla) is thinly sliced, mixed respectively by same type, in 60 DEG C
Drying to constant weight in baking oven and is ground into powder；
(2): Polyose extraction
1. it is colourless to phegma using gained powder in 80 DEG C of dehydrated alcohol Soxhlet extraction step (1), solvent is volatilized, it will
The gained bletilla dregs of a decoction are added in distilled water according to solid-liquid ratio 1:6, and are condensed back and extract 4 times in 85 DEG C of water-baths, each 3h；
2. merging filtrate simultaneously adds the active carbon mixing decoloration that mass fraction is 0.5% into filtrate, under 65 DEG C of environment
It is filtered after standing 2h；
3. after filtering, above-mentioned solution is carried out negative pressure to be concentrated being the 34% of original volume in 65 DEG C in Rotary Evaporators, then plus
Enter dehydrated alcohol, its final concentration is made to reach 85%v/v, stand 2h in 5 DEG C of refrigerators, then, is centrifuged under 3000r/min revolving speed
Precipitating is left and taken after 20min；
4. being dissolved precipitating with distilled water up to bletilla Thick many candies solution；
(3): polysaccharide purification
Further polysaccharide purification is carried out to the resulting Thick many candies solution of step (2), using Sevage method: according to sample with
Sevage reagent (volume ratio is respectively 4: 1 chloroform and n-butanol mixed liquor) is added in extractant volume ratio 4:1, mixes, oscillation
35min is centrifuged 10min with 4000r/min, discards except lower layer's chloroform and medial degeneration albumen；Upper layer aqueous solution is taken to repeat to go again
Albumen operation, until without obvious middle layer；Finally, collecting the upper layer aqueous solution after deproteinized, pass through ultrafiltration, filter membrane
Molecular cut off is 1K, sloughs small-molecule substance to get the polysaccharide concentrate solution arrived after purification；
(4): the foundation of chromatographic fingerprinting
1. weight average molecular weight Mw to be respectively as follows: to five kinds of glucan marks of 2000KDa, 580KDa, 70KDa, 10KDa, 5KDa
Quasi- product are configured to the aqueous solution difference sample introduction of 5mg/mL in Efficient numerical method HPSEC, according to each dextran standard
Chromatographic peak retention time and molecular weight logarithm, using the molecular weight logarithm lgMw of standard items as ordinate, chromatographic peak retention time
Rt is abscissa, is fitted to obtain polysaccharide molecular weight calibration curve equation: lgMw=y=-0.3486x+11.133 using linear equation,
R2=0.9968；Wherein, corresponding chromatographic condition when Efficient numerical method HPSEC detection is carried out are as follows: chromatographic column:G6000PWXL 7.8mm ID×300mm；Detector: differential refraction detector (RID)；Mobile phase: 0.1M
Sodium acetate；Flow velocity: 0.5mL/min；Column temperature: 45 DEG C, 20 μ L of sample volume；
2. the medicinal bletilla sample difference sample introduction after polysaccharide purification obtains corresponding color in Efficient numerical method HPSEC
Spectrogram calculates the relative molecular weight of chromatographic curve each section of each sample further according to polysaccharide molecular weight calibration curve equation, most
The chromatographic fingerprinting of corresponding known medicinal bletilla sample is established eventually；
(5): the identification of unknown type bletilla
Three kinds of unknown bletilla platymiscium dry tubers are handled according to above step (1)-(4), it is right to obtain its
Chromatographic fingerprinting is answered, " similarity evaluation " software produced further according to the Chinese Pharmacopoeia committee
Determined, the chromatographic fingerprinting of itself and established known medicinal bletilla sample is subjected to similarity-rough set, as a result table
A kind of map of bright bletilla platymiscium and the similarity of species chrysanthemum bletilla are determined as chrysanthemum bletilla, a kind of bletilla up to 90%
The similarity of the small bletilla of map and species of platymiscium is determined as the small bletilla of species up to 91%, and the third bletilla platymiscium
Map and above-mentioned three kinds of medicinal bletillas similarity for representing mode finger-print be not achieved 90%, then determine that the bletilla belongs to and plant
This non-three kinds of medicinal bletillas of object.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of chromatographic fingerprinting method for identifying three kinds of medicinal bletillas, which comprises the steps of:
(1): pretreatment
It is mixed after the medicinal bletilla material of the known different cultivars sampled according to statistical method is thinly sliced, in 60-
Drying to constant weight in 70 DEG C of baking oven and is ground into powder；
(2): Polyose extraction
1. it is colourless to phegma using gained powder in 80 DEG C -90 DEG C of dehydrated alcohol Soxhlet extraction step (1), solvent is volatilized,
By the gained bletilla dregs of a decoction according to solid-liquid ratio (1-2): (4-6) is added in distilled water, and is condensed back and extracts in 75 DEG C of -85 DEG C of water-baths
3-5 times, each 2-3h；
2. merging filtrate simultaneously adds the active carbon mixing decoloration that mass fraction is 0.3%-0.5% into filtrate, in 60 DEG C -65 DEG C
It is filtered after standing 1-2h under environment；
3. after filtering, above-mentioned solution being carried out negative pressure in Rotary Evaporators in 60 DEG C -65 DEG C, the 30%- for original volume being concentrated
35%, dehydrated alcohol is added, its final concentration is made to reach 80%-85%v/v, 2-3h is stood in 3-5 DEG C of refrigerator, then,
Precipitating is left and taken after being centrifuged 10-20min under 3000-4000r/min revolving speed；
4. being dissolved precipitating with distilled water up to medicinal bletilla Thick many candies solution；
(3): polysaccharide purification
Further polysaccharide purification is carried out to the resulting Thick many candies solution of step (2)；
(4): the foundation of chromatographic fingerprinting
1. the dextran standard of different molecular weight is configured to the aqueous solution difference sample introduction of 4-5mg/mL in efficient volume exclusion
In chromatography HPSEC, polysaccharide molecular weight calibration curve equation is calculated further according to each corresponding chromatogram；
2. the medicinal bletilla sample difference sample introduction after polysaccharide purification obtains corresponding chromatogram in Efficient numerical method HPSEC,
The relative molecular weight of chromatographic curve each section of each sample is calculated further according to polysaccharide molecular weight calibration curve equation, it is final to establish
The HPSEC of corresponding known medicinal bletilla sample represents mode chromatographic map；
(5): the identification of unknown type bletilla
The bletilla of unknown type is handled according to above step (1)-(4), corresponds to chromatographic fingerprinting to obtain it, then will
The chromatographic fingerprinting of itself and established known bletilla sample carries out similarity-rough set, when its similarity is greater than 90% or more
When, it can be determined as same class bletilla platymiscium, otherwise not be same kind.
2. a kind of chromatographic fingerprinting method for identifying three kinds of medicinal bletillas according to claim 1, which is characterized in that institute
The kind for stating the medicinal bletilla of different cultivars in step (1) is respectively as follows: pale reddish brown trident bletilla, chrysanthemum bletilla and small bletilla.
3. a kind of chromatographic fingerprinting method for identifying three kinds of medicinal bletillas according to claim 1, which is characterized in that institute
Stating medicinal bletilla material in step (1) is medicinal bletilla fresh goods stem tuber or dry tuber.
4. a kind of chromatographic fingerprinting method for identifying three kinds of medicinal bletillas according to claim 1, which is characterized in that institute
State statistical method in step (1) are as follows: collect medicine according to five point sampling in Guizhou, Yunnan, Sichuan, four, Anhui area respectively
With bletilla sample.
5. a kind of chromatographic fingerprinting method for identifying three kinds of medicinal bletillas according to claim 1, which is characterized in that institute
State polysaccharide purification in step (3) and use Sevage method: according to sample and extractant volume ratio (3-5): Sevage is added in (0.5-2)
Reagent mixes, and vibrates 25-35min, is centrifuged 5-10min with 3000-4000r/min, discards except lower layer's chloroform and medial degeneration egg
It is white；Upper layer aqueous solution is taken to repeat deproteinized operation again, until without obvious middle layer；Finally, the upper layer collected after deproteinized is water-soluble
Liquid sloughs small-molecule substance by ultrafiltration to get the polysaccharide concentrate solution arrived after purification.
6. a kind of chromatographic fingerprinting method for identifying three kinds of medicinal bletillas according to claim 5, which is characterized in that institute
It is respectively (3-5) that Sevage reagent, which is stated, as volume ratio: the chloroform of (0.5-1.5) and the mixed liquor of n-butanol.
7. a kind of chromatographic fingerprinting method for identifying three kinds of medicinal bletillas according to claim 5, which is characterized in that institute
Stating ultrafiltration apparatus is the ultrafiltration apparatus that membrane retention molecular weight is 1K.
8. a kind of chromatographic fingerprinting method for identifying three kinds of medicinal bletillas according to claim 1, which is characterized in that institute
State the dextran standard of different molecular weight in step (4) weight average molecular weight Mw be respectively as follows: 2000KDa, 580KDa, 70KDa,
10KDa、5KDa。
9. a kind of chromatographic fingerprinting method for identifying three kinds of medicinal bletillas according to claim 1, which is characterized in that institute
State the calculation method of polysaccharide molecular weight calibration curve equation in step (4) are as follows: retain according to the chromatographic peak of each dextran standard
Time and molecular weight logarithm, using the molecular weight logarithm lgMw of standard items as ordinate, chromatographic peak retention time Rt is abscissa, is made
Polysaccharide molecular weight calibration curve equation is fitted to obtain with linear equation.
10. the chromatographic fingerprinting method of -9 any described three kinds of medicinal bletillas of a kind of identification according to claim 1, feature
It is, carries out corresponding chromatographic condition when Efficient numerical method HPSEC detection are as follows: mobile phase is 0.1M sodium acetate, and flow velocity is
0.5mL/min, column temperature are 45 DEG C, and sample volume is 20 μ L.
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