CN106501422A - A kind of chromatographic fingerprinting method for differentiating three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) - Google Patents
A kind of chromatographic fingerprinting method for differentiating three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) Download PDFInfo
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Abstract
The invention discloses a kind of chromatographic fingerprinting method for differentiating three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae), comprises the following steps：Pretreatment, Polyose extraction, polysaccharide purification, the foundation of chromatographic fingerprinting, the identification of unknown species Pseudobulbus Bletillae (Rhizoma Bletillae).It is an advantage of the current invention that：Can effectively be distinguished by the molecular weight distribution of Pseudobulbus Bletillae (Rhizoma Bletillae) main pharmacodynamics composition bletilla polysaccharide and differentiate three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae), can overcome the disadvantages that the deficiency of the Pseudobulbus Bletillae (Rhizoma Bletillae) authentication method of 2015 editions settings of Chinese Pharmacopoeia, because the latter is the appearance purpose speckle chromatographed using silica gel thin-layer point sample is used as its quality evaluation and criterion, it is a kind of single feedback of the information, counterfeiter can be faked using the leak of detection method, and this method is closer to the essence of chemical constituent, it is finer information, counterfeiter cannot almost copy or counterfeit cost is expensive.
Description
Technical field
The present invention relates to orchid family Pseudobulbus Bletillae (Rhizoma Bletillae) belongs to the discriminating of medicinal plants product and method of quality control technical field, more particularly to
A kind of chromatographic fingerprinting method for differentiating three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae).
Background technology
Drying of the medical material Pseudobulbus Bletillae (Rhizoma Bletillae) for orchid family Pseudobulbus Bletillae (Rhizoma Bletillae) platymiscium Pseudobulbus Bletillae (Rhizoma Bletillae) (Bletilla striata (Thunb.) Reichb.f.)
Tuber, is China's tradition hemostasia and promoting granulation medicine, and tcm clinical practice is used for spitting blood, spits blood, traumatic hemorrhage, sore swollen toxin, chapped skin.In vain
And category (Bletilla Rchb.) the plant whole world has 6 kinds, is distributed in the Upper Myanmar in Asia through China to Japan.China produces 4
Kind：Magnificent Pseudobulbus Bletillae (Rhizoma Bletillae) Bletilla sinensis (Rolfe) Schltr.Schltr., little Pseudobulbus Bletillae (Rhizoma Bletillae) Btetilla formosana
(Hayata) Schltr., Hemerocallis citrina Baroni Pseudobulbus Bletillae (Rhizoma Bletillae) Bletilla ochracea Schltr, pale reddish brown trident Pseudobulbus Bletillae (Rhizoma Bletillae) Bletilla striate
(Thunb.ex A.Murray)Rchb.f..And traction and medical value highest kind are pale reddish brown trident Pseudobulbus Bletillae (Rhizoma Bletillae), and absolutely
Most cultivar is also pale reddish brown trident Pseudobulbus Bletillae (Rhizoma Bletillae), and its introduces a collection 90% is from " Jiangxi group " and " Guizhou group ", plantation
Area is about Guizhou and accounts for 20% (about 1500 mu), and Yunnan accounts for 18%, and four CHUANZHAN 15%, Anhui account for 15%, and Hubei accounts for 12%, river
Soviet Union, Guangxi, Hunan, Shaanxi respectively account for 5%.
Modern pharmacological research shows that water soluble polysaccharide is then its main medicine containing multiple effective medicinal ingredients in Pseudobulbus Bletillae (Rhizoma Bletillae) tuber
Use composition.Used as natural macromolecular material, bletilla polysaccharide has functional slow-release, local retention, auto-degradation, nothing simultaneously
Zest, the characteristic of the adjuvant such as have no toxic side effect, the effect in prepared by modern medicines are more and more important.
Main active one of of the polysaccharide compound as natural drug, the space structure of its activity and its polysaccharide and
Molecular weight distribution is closely related, is the dependency for exploring its component and drug effect, and this programme will be using 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
The ratio of amount polysaccharide and distribution are set up after HPSEC chromatographic fingerprintings are characterizing the construction featuress of different plant species bletilla polysaccharide
The formulation of continuous quality standard provides feasible solution.And so far, have no with regard to setting up medicinal bletilla polysaccharide using HPSEC chromatographs
The open report of chromatographic fingerprinting.
Content of the invention
The technical problem to be solved is to provide a kind of color for more accurately and reliably differentiating three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae)
Spectrum fingerprint spectrum method, the atlas calculation are the HPSEC colors that is set up according to three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) main pharmacodynamics basic substance polysaccharide
Spectrogram is composed.The polysaccharide molecular weight scattergram Prepenem that the present invention sets up effectively distinguishes different Pseudobulbus Bletillae (Rhizoma Bletillae) species, can both make up profit in pharmacopeia
The deficiency to judge Pseudobulbus Bletillae (Rhizoma Bletillae) quality and the true and false of the appearance purpose speckle chromatographed with silica gel thin-layer point sample, it is also possible to differentiate three kinds
Medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) (little Pseudobulbus Bletillae (Rhizoma Bletillae) Btetilla formosana (Hayata) Schltr.；Hemerocallis citrina Baroni Pseudobulbus Bletillae (Rhizoma Bletillae) Bletilla ochracea
Schltr；Pale reddish brown trident Pseudobulbus Bletillae (Rhizoma Bletillae) Bletilla striate (Thunb.ex A.Murray) Rchb.f.) belong to other kind of Pseudobulbus Bletillae (Rhizoma Bletillae)
Plant.
For solving above-mentioned technical problem, the present invention provides a kind of chromatographic fingerprinting method for differentiating three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae),
Comprise the steps：
(1)：Pretreatment
Mix after the medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) material of the known different cultivars obtained according to statistical method sampling is thinly sliced, in
Dry to constant weight in 60-70 DEG C of baking oven and be ground into powder；
(2)：Polyose extraction
1. colourless to backflow using gained powder in 80 DEG C -90 DEG C of dehydrated alcohol surname extraction step (1), volatilize molten
Agent, by gained Pseudobulbus Bletillae (Rhizoma Bletillae) medicinal residues according to solid-liquid ratio (1-2)：(4-6) add in distilled water, and in 75 DEG C -85 DEG C water-bath condensing refluxes
Extract 3-5 time, each 2-3h；
2. merging filtrate and in filtrate add mass fraction for 0.3%-0.5% activated carbon mix decolourize, in 60 DEG C-
Sucking filtration after 1-2h is stood under 65 DEG C of environment；
3., after sucking filtration, above-mentioned solution is carried out 30%- of the negative pressure concentration for original volume in 60 DEG C -65 DEG C in Rotary Evaporators
35%, add dehydrated alcohol so as to which final concentration reaches 80%-85% (v/v), 2-3h is stood in 3-5 DEG C of refrigerator, connect
, under 3000-4000r/min rotating speeds, after centrifugation 10-20min, leave and take precipitation；
4. precipitation distillation water dissolution is obtained final product medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) crude polysaccharides solution；
(3)：Polysaccharide purification
Further polysaccharide purification is carried out to the crude polysaccharides solution of step (2) gained；
(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, further according to each corresponding chromatogram calculation polysaccharide molecular weight calibration curve equation；
2. the medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) sample difference sample introduction after polysaccharide purification obtains corresponding color in Efficient numerical method HPSEC
Spectrogram, calculates the relative molecular weight of the chromatographic curve each several part of each sample, most further according to polysaccharide molecular weight calibration curve equation
The HPSEC for setting up corresponding known medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) sample eventually represents mode chromatographic collection of illustrative plates；
(5)：The identification of unknown species Pseudobulbus Bletillae (Rhizoma Bletillae)
The Pseudobulbus Bletillae (Rhizoma Bletillae) of unknown species is processed according to above step (1)-(4), to obtain its corresponding chromatographic fingerprinting,
Its chromatographic fingerprinting with the known Pseudobulbus Bletillae (Rhizoma Bletillae) sample that sets up is carried out similarity-rough set again, when its similarity is more than 90%
When above, you can be judged to same class Pseudobulbus Bletillae (Rhizoma Bletillae) platymiscium, otherwise it is not same kind.
Preferably, in step (1), the kind of the medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) of different cultivars is respectively：Pale reddish brown trident Pseudobulbus Bletillae (Rhizoma Bletillae), Hemerocallis citrina Baroni are white
And with little Pseudobulbus Bletillae (Rhizoma Bletillae).
Preferably, in step (1), medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) material is medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) fresh goods tuber or dry tuber.
Preferably, in step (1), statistical method is：Press in Guizhou, Yunnan, Sichuan, four, Anhui area respectively
Medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) sample is collected according to five point sampling.
Preferably, in step (3), polysaccharide purification adopts Sevage methods：According to sample and extractant volume ratio (3-5)：
(0.5-2) Sevage reagents are added, is mixed, vibrate 25-35min, 5-10min is centrifuged with 3000-4000r/min, is discarded and is removed down
Layer chloroform and medial degeneration albumen；Take upper strata aqueous solution again and repeat Deproteinization operation, to without obvious middle level；Finally, collect
Upper strata aqueous solution after Deproteinization, by ultrafiltration, sloughs small-molecule substance, that is, the polysaccharide concentration for obtaining after purification is molten
Liquid.
Preferably, the Sevage reagents are respectively (3-5) for volume ratio: chloroform (0.5-1.5) is mixed with n-butyl alcohol
Close liquid.
Preferably, the ultrafiltration apparatus are the ultrafiltration apparatus that membrane retention molecular weight is 1K.
Preferably, in step (4), weight average molecular weight Mw of the dextran standard of different molecular weight is respectively：
2000KDa、580KDa、70KDa、10KDa、5KDa.
Preferably, in step (4), the computational methods of polysaccharide molecular weight calibration curve equation are：According to each glucosan mark
The chromatographic peak retention time of quasi- product and molecular weight logarithm, as vertical coordinate, chromatographic peak retains the molecular weight logarithm lgMw with standard substance
Time Rt is abscissa, is fitted to obtain polysaccharide molecular weight calibration curve equation using linear equation.
Preferably, carrying out corresponding chromatographic condition when Efficient numerical method HPSEC is detected is：Mobile phase is 0.1M acetic acid
Sodium, flow velocity are 0.5mL/min, and column temperature is 45 DEG C, and sample size is 20uL.
It is an advantage of the current invention that：The HPSEC chromatographic fingerprint figures of three kinds of bletilla polysaccharide molecular weight distribution that the present invention is provided
Compose as representing pattern collection of illustrative plates, produced using Chinese Pharmacopoeia committee《Similarity evaluation》Software
Judged, can effectively be distinguished by the molecular weight distribution of Pseudobulbus Bletillae (Rhizoma Bletillae) main pharmacodynamics composition-bletilla polysaccharide and be differentiated three kinds medicinal
Pseudobulbus Bletillae (Rhizoma Bletillae), its method can overcome the disadvantages that the deficiency of the Pseudobulbus Bletillae (Rhizoma Bletillae) authentication method of 2015 editions settings of Chinese Pharmacopoeia, because the latter is to utilize silica gel
The appearance purpose speckle of thin-layer sample application chromatography is used as its quality evaluation and criterion, is a kind of single feedback of the information,
Counterfeiter can be faked using the leak of detection method, and this method is closer to the essence of chemical constituent, is finer information,
Counterfeiter cannot almost copy or counterfeit cost is expensive.
Description of the drawings
Fig. 1 is a kind of stream of the chromatographic fingerprinting method of three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) of discriminating that embodiment of the present invention 1-2 is provided
Cheng Tu.
Fig. 2 is a kind of the pale reddish brown of the chromatographic fingerprinting method of three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) of discriminating that the embodiment of the present invention 1 is provided
The graph of molecular weight distribution of trident bletilla polysaccharide；
Fig. 3 is a kind of Hemerocallis citrina Baroni of the chromatographic fingerprinting method of three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) of discriminating that the embodiment of the present invention 1 is provided
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 Pseudobulbus Bletillae (Rhizoma Bletillae) of discriminating that the embodiment of the present invention 1 is provided
And the graph of molecular weight distribution of polysaccharide.
Specific embodiment
Below embodiments of the invention are elaborated, the present embodiment is carried out under premised on technical solution of the present invention
Implement, give detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following enforcements
Example.
Embodiment 1
A kind of chromatographic fingerprinting method for differentiating three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae), as shown in figure 1, comprise the steps：
(1)：Pretreatment
The three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) that will be collected according to five point sampling in Guizhou, Yunnan, Sichuan, four, Anhui area respectively
After the fresh tuber of (pale reddish brown trident Pseudobulbus Bletillae (Rhizoma Bletillae), Hemerocallis citrina Baroni Pseudobulbus Bletillae (Rhizoma Bletillae), little Pseudobulbus Bletillae (Rhizoma Bletillae)) is thinly sliced, mix by same species respectively, in 60 DEG C
Dry in baking oven to constant weight and be ground into powder；
(2)：Polyose extraction
1. colourless to backflow using gained powder in 85 DEG C of dehydrated alcohol surname extraction step (1), solvent is volatilized, will
Gained Pseudobulbus Bletillae (Rhizoma Bletillae) medicinal residues are according to solid-liquid ratio 1：5 add in distilled water, and extract 3 times in 80 DEG C of water-bath condensing refluxes, each 3h；
2. merging filtrate and in filtrate add mass fraction be 0.5% activated carbon mix decolourize, under 60 DEG C of environment
Stand sucking filtration after 1h；
3., after sucking filtration, it is the 33% of original volume to carry out negative pressure concentration in 60 DEG C in Rotary Evaporators, adds dehydrated alcohol,
Make its final concentration reach 85% (v/v), 3h is stood in 4 DEG C of refrigerators, then, stayed under 4000r/min rotating speeds after centrifugation 15min
Take precipitation；
4. precipitation distillation water dissolution is obtained final product medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) crude polysaccharides solution；
(3)：Polysaccharide purification
Further polysaccharide purification is carried out to the crude polysaccharides solution of step (2) gained, using Sevage methods：According to sample with
Extractant volume ratio 4：1 addition Sevage reagents (volume ratio is respectively 4: 1 chloroform and n-butyl alcohol mixed liquor), mix, vibration
30min, is centrifuged 15min with 4000r/min, discards except lower floor's chloroform and medial degeneration albumen；Take upper strata aqueous solution again to repeat
Albumen is operated, to without obvious middle level；Finally, the upper strata aqueous solution after Deproteinization is collected, by ultrafiltration, filter membrane
Molecular cut off is 1K, sloughs small-molecule substance, that is, obtain polysaccharide concentrate solution after purification；
(4)：The foundation of chromatographic fingerprinting
1. weight average molecular weight Mw is respectively：Five kinds of glucosan 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, the molecular weight logarithm lgMw with standard substance as vertical coordinate, chromatographic peak retention time
Rt is abscissa, is fitted to obtain polysaccharide molecular weight calibration curve equation using linear equation：LgMw=y=-0.3486x+11.133,
R2=0.9968；Wherein, carrying out corresponding chromatographic condition when Efficient numerical method HPSEC is detected is：Chromatographic column：G6000PWXL 7.8mm ID×300mm；Detector：Differential refraction detector (RID)；Mobile phase：0.1M vinegar
Sour sodium；Flow velocity：0.5mL/min；Column temperature：45 DEG C, 20 μ L of sample size；
2. the medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) sample difference sample introduction after polysaccharide purification obtains corresponding color in Efficient numerical method HPSEC
Spectrogram, calculates the relative molecular weight of the chromatographic curve each several part of each sample, most further according to polysaccharide molecular weight calibration curve equation
The HPSEC for setting up corresponding known Pseudobulbus Bletillae (Rhizoma Bletillae) sample eventually represents mode chromatographic collection of illustrative plates；Fig. 2-4 is respectively three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) in height
The peak spectrogram obtained in effect size exclusion chromatograph HPSEC, i.e., the (unit with abscissa as appearance time：Minute), vertical coordinate is light
Response value (the unit of the signal of telecommunication：Millivolt) polysaccharide graph of molecular weight distribution, 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 collection of illustrative plates of three kinds of Pseudobulbus Bletillae (Rhizoma Bletillae) samples
Peak cut into 6 parts by fluctuating, molecular weight is respectively：More than 500KDa, 500KDa-200KDa, 200KDa-100KDa,
100KDa-50KDa, 50KDa-10Kda, be less than 10KDa；Collection of illustrative plates is tested and reappearance test through stability test, elaboration
Deng methodological study, the RSD of relative peak area change is respectively less than 3%, meets chromatographic process requirement；
From Fig. 2-4, the molecular weight distribution of bletilla polysaccharide is more concentrated, three kinds of Pseudobulbus Bletillae (Rhizoma Bletillae) platymiscium sample (pale reddish brown tridents
Pseudobulbus Bletillae (Rhizoma Bletillae), Hemerocallis citrina Baroni Pseudobulbus Bletillae (Rhizoma Bletillae), little Pseudobulbus Bletillae (Rhizoma Bletillae)) weight average molecular weight (Mw) be respectively：590KDa, 98KDa, 72KDa, i.e. different cultivars Pseudobulbus Bletillae (Rhizoma Bletillae)
Weight average molecular weight have notable difference, numerically Pseudobulbus Bletillae (Rhizoma Bletillae) is much larger than Hemerocallis citrina Baroni Pseudobulbus Bletillae (Rhizoma Bletillae) and little Pseudobulbus Bletillae (Rhizoma Bletillae)；
Therefore, three collection of illustrative plates of Fig. 2-4 are the generation of the HPSEC chromatographic fingerprintings of three kinds of bletilla polysaccharide molecular weight distribution
Table schema collection of illustrative plates；
(5)：The identification of unknown species Pseudobulbus Bletillae (Rhizoma Bletillae)
Two kinds of unknown Pseudobulbus Bletillae (Rhizoma Bletillae) platymiscium fresh tubers are processed according to above step (1)-(4), right to obtain which
Chromatographic fingerprinting is answered, is produced further according to Chinese Pharmacopoeia committee《Similarity evaluation》Software
Judged, its HPSEC chromatographic fingerprinting with the known Pseudobulbus Bletillae (Rhizoma Bletillae) sample that sets up is carried out similarity-rough set, as a result table
Up to 94%, the similarity of a kind of collection of illustrative plates of bright Pseudobulbus Bletillae (Rhizoma Bletillae) platymiscium and the pale reddish brown trident Pseudobulbus Bletillae (Rhizoma Bletillae) of species is judged to that the pale reddish brown trident of species is white
And, and the similarity that the collection of illustrative plates of another kind of Pseudobulbus Bletillae (Rhizoma Bletillae) platymiscium represents pattern finger printing with three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) is not reached
90%, then judge the Pseudobulbus Bletillae (Rhizoma Bletillae) platymiscium this three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) non-.
Embodiment 2
A kind of chromatographic fingerprinting method for differentiating three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae), as shown in figure 1, comprise the steps：
(1)：Pretreatment
The three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) that will be collected according to five point sampling in Guizhou, Yunnan, Sichuan, four, Anhui area respectively
After the dry tuber of (pale reddish brown trident Pseudobulbus Bletillae (Rhizoma Bletillae), Hemerocallis citrina Baroni Pseudobulbus Bletillae (Rhizoma Bletillae), little Pseudobulbus Bletillae (Rhizoma Bletillae)) is thinly sliced, mix by same species respectively, in 60 DEG C
Dry in baking oven to constant weight and be ground into powder；
(2)：Polyose extraction
1. colourless to backflow using gained powder in 80 DEG C of dehydrated alcohol surname extraction step (1), solvent is volatilized, will
Gained Pseudobulbus Bletillae (Rhizoma Bletillae) medicinal residues are according to solid-liquid ratio 1：6 add in distilled water, and extract 4 times in 85 DEG C of water-bath condensing refluxes, each 3h；
2. merging filtrate and in filtrate add mass fraction be 0.5% activated carbon mix decolourize, under 65 DEG C of environment
Stand sucking filtration after 2h；
3., after sucking filtration, it is the 34% of original volume that above-mentioned solution is carried out negative pressure concentration in 65 DEG C in Rotary Evaporators, then plus
Enter dehydrated alcohol so as to which final concentration reaches 85% (v/v), in 5 DEG C of refrigerators stand 2h, then, under 3000r/min rotating speeds from
Precipitation is left and taken after heart 20min；
4. precipitation distillation water dissolution is obtained final product Pseudobulbus Bletillae (Rhizoma Bletillae) crude polysaccharides solution；
(3)：Polysaccharide purification
Further polysaccharide purification is carried out to the crude polysaccharides solution of step (2) gained, using Sevage methods：According to sample with
Extractant volume ratio 4：1 addition Sevage reagents (volume ratio is respectively 4: 1 chloroform and n-butyl alcohol mixed liquor), mix, vibration
35min, is centrifuged 10min with 4000r/min, discards except lower floor's chloroform and medial degeneration albumen；Take upper strata aqueous solution again to repeat
Albumen is operated, to without obvious middle level；Finally, the upper strata aqueous solution after Deproteinization is collected, by ultrafiltration, filter membrane
Molecular cut off is 1K, sloughs small-molecule substance, that is, obtain polysaccharide concentrate solution after purification；
(4)：The foundation of chromatographic fingerprinting
1. weight average molecular weight Mw is respectively：Five kinds of glucosan 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, the molecular weight logarithm lgMw with standard substance as vertical coordinate, chromatographic peak retention time
Rt is abscissa, is fitted to obtain polysaccharide molecular weight calibration curve equation using linear equation：LgMw=y=-0.3486x+11.133,
R2=0.9968；Wherein, carrying out corresponding chromatographic condition when Efficient numerical method HPSEC is detected is：Chromatographic column：G6000PWXL 7.8mm ID×300mm；Detector：Differential refraction detector (RID)；Mobile phase：0.1M vinegar
Sour sodium；Flow velocity：0.5mL/min；Column temperature：45 DEG C, 20 μ L of sample size；
2. the medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) sample difference sample introduction after polysaccharide purification obtains corresponding color in Efficient numerical method HPSEC
Spectrogram, calculates the relative molecular weight of the chromatographic curve each several part of each sample, most further according to polysaccharide molecular weight calibration curve equation
The chromatographic fingerprinting of corresponding known medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) sample is set up eventually；
(5)：The identification of unknown species Pseudobulbus Bletillae (Rhizoma Bletillae)
Three kinds of unknown Pseudobulbus Bletillae (Rhizoma Bletillae) platymiscium dry tubers are processed according to above step (1)-(4), right to obtain which
Chromatographic fingerprinting is answered, is produced further according to Chinese Pharmacopoeia committee《Similarity evaluation》Software
Judged, by its with set up known to the chromatographic fingerprinting of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) sample carry out similarity-rough set, as a result table
The similarity of the collection of illustrative plates and species Hemerocallis citrina Baroni Pseudobulbus Bletillae (Rhizoma Bletillae) of a kind of bright Pseudobulbus Bletillae (Rhizoma Bletillae) platymiscium is judged to Hemerocallis citrina Baroni Pseudobulbus Bletillae (Rhizoma Bletillae), a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) up to 90%
The similarity of the little Pseudobulbus Bletillae (Rhizoma Bletillae) of the collection of illustrative plates and species of platymiscium is judged to the little Pseudobulbus Bletillae (Rhizoma Bletillae) of species up to 91%, and the third Pseudobulbus Bletillae (Rhizoma Bletillae) platymiscium
The collection of illustrative plates similarity that represents pattern finger printing with above-mentioned three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) do not reach 90%, then judge that Pseudobulbus Bletillae (Rhizoma Bletillae) category is planted
Thing this three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) non-.
Presently preferred embodiments of the present invention is the foregoing is only, not in order to limit the present invention, all in essence of the invention
Any modification, equivalent and improvement that is made within god and principle etc., should be included within the scope of the present invention.
Claims (10)
1. a kind of differentiate three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) chromatographic fingerprinting method, it is characterised in that comprise the steps：
(1)：Pretreatment
Mix after the medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) material of the known different cultivars obtained according to statistical method sampling is thinly sliced, in 60-
Dry to constant weight in 70 DEG C of baking oven and be ground into powder；
(2)：Polyose extraction
1. colourless to backflow using gained powder in 80 DEG C -90 DEG C of dehydrated alcohol surname extraction step (1), solvent is volatilized,
By gained Pseudobulbus Bletillae (Rhizoma Bletillae) medicinal residues according to solid-liquid ratio (1-2)：(4-6) add in distilled water, and extract in 75 DEG C -85 DEG C water-bath condensing refluxes
3-5 time, each 2-3h；
2. merging filtrate activated carbon mixing decolouring of the interpolation mass fraction for 0.3%-0.5% in filtrate, in 60 DEG C -65 DEG C
Sucking filtration after 1-2h is stood under environment；
3., after sucking filtration, above-mentioned solution is carried out 30%- of the negative pressure concentration for original volume in 60 DEG C -65 DEG C in Rotary Evaporators
35%, add dehydrated alcohol so as to which final concentration reaches 80%-85% (v/v), 2-3h is stood in 3-5 DEG C of refrigerator, connect
, under 3000-4000r/min rotating speeds, after centrifugation 10-20min, leave and take precipitation；
4. precipitation distillation water dissolution is obtained final product medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) crude polysaccharides solution；
(3)：Polysaccharide purification
Further polysaccharide purification is carried out to the crude polysaccharides solution of step (2) gained；
(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 chromatograph HPSEC, further according to each corresponding chromatogram calculation polysaccharide molecular weight calibration curve equation；
2. the medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) sample difference sample introduction after polysaccharide purification obtains corresponding chromatogram in Efficient numerical method HPSEC,
The relative molecular weight of the chromatographic curve each several part of each sample is calculated further according to polysaccharide molecular weight calibration curve equation, final foundation
The HPSEC of corresponding known medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) sample represents mode chromatographic collection of illustrative plates；
(5)：The identification of unknown species Pseudobulbus Bletillae (Rhizoma Bletillae)
The Pseudobulbus Bletillae (Rhizoma Bletillae) of unknown species is processed according to above step (1)-(4), to obtain its corresponding chromatographic fingerprinting, then will
Which carries out similarity-rough set with the chromatographic fingerprinting of the known Pseudobulbus Bletillae (Rhizoma Bletillae) sample that sets up, when its similarity is more than more than 90%
When, you can it is judged to same class Pseudobulbus Bletillae (Rhizoma Bletillae) platymiscium, is not otherwise same kind.
2. according to claim 1 a kind of differentiate three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) chromatographic fingerprinting method, it is characterised in that institute
The kind for stating the medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) of different cultivars in step (1) is respectively：Pale reddish brown trident Pseudobulbus Bletillae (Rhizoma Bletillae), Hemerocallis citrina Baroni Pseudobulbus Bletillae (Rhizoma Bletillae) and little Pseudobulbus Bletillae (Rhizoma Bletillae).
3. according to claim 1 a kind of differentiate three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) chromatographic fingerprinting method, it is characterised in that institute
It is medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) fresh goods tuber or dry tuber to state medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) material in step (1).
4. according to claim 1 a kind of differentiate three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) chromatographic fingerprinting method, it is characterised in that institute
Stating statistical method in step (1) is：Medicine is collected respectively in Guizhou, Yunnan, Sichuan, four, Anhui area according to five point sampling
Use Pseudobulbus Bletillae (Rhizoma Bletillae) sample.
5. according to claim 1 a kind of differentiate three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) chromatographic fingerprinting method, it is characterised in that institute
State polysaccharide purification in step (3) and adopt Sevage methods：According to sample and extractant volume ratio (3-5)：(0.5-2) Sevage is added
Reagent, mixes, and vibrates 25-35min, is centrifuged 5-10min with 3000-4000r/min, discards except lower floor's chloroform and medial degeneration egg
In vain；Take upper strata aqueous solution again and repeat Deproteinization operation, to without obvious middle level；Finally, the upper strata that collects after Deproteinization is water-soluble
Liquid, by ultrafiltration, sloughs small-molecule substance, that is, obtains polysaccharide concentrate solution after purification.
6. according to claim 5 a kind of differentiate three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) chromatographic fingerprinting method, it is characterised in that institute
State Sevage reagents to be respectively (3-5) for volume ratio: chloroform (0.5-1.5) and the mixed liquor of n-butyl alcohol.
7. according to claim 5 a kind of differentiate three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) chromatographic fingerprinting method, it is characterised in that institute
State the ultrafiltration apparatus that ultrafiltration apparatus are that membrane retention molecular weight is 1K.
8. according to claim 1 a kind of differentiate three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) chromatographic fingerprinting method, it is characterised in that institute
Weight average molecular weight Mw for stating the dextran standard of different molecular weight in step (4) is respectively：2000KDa、580KDa、70KDa、
10KDa、5KDa.
9. according to claim 1 a kind of differentiate three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) chromatographic fingerprinting method, it is characterised in that institute
The computational methods for stating polysaccharide molecular weight calibration curve equation in step (4) are：Retained according to the chromatographic peak of each dextran standard
Time and molecular weight logarithm, as vertical coordinate, chromatographic peak retention time Rt is abscissa to the molecular weight logarithm lgMw with standard substance, makes
Polysaccharide molecular weight calibration curve equation is fitted to obtain with linear equation.
10. according to a kind of chromatographic fingerprinting method of the arbitrary described three kinds of medicinal Pseudobulbus Bletillae (Rhizoma Bletillae) of discriminating of claim 1-9, its feature
It is, carrying out corresponding chromatographic condition when Efficient numerical method HPSEC is detected is：Mobile phase is 0.1M sodium acetates, and flow velocity is
0.5mL/min, column temperature are 45 DEG C, and sample size is 20uL.
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| CN107340348A (en) * | 2017-06-07 | 2017-11-10 | 贵阳中医学院 | A kind of method for building up of Bletilla striata medicinal materials HPLC finger-prints |
| CN107340348B (en) * | 2017-06-07 | 2020-05-12 | 贵阳中医学院 | Method for establishing HPLC (high Performance liquid chromatography) fingerprint spectrum of rhizoma bletillae medicinal material |
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