CN106501421A - A kind of chromatographic fingerprinting method for differentiating three kinds of medicinal dendrobiums - Google Patents
A kind of chromatographic fingerprinting method for differentiating three kinds of medicinal dendrobiums Download PDFInfo
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- 241001523681 Dendrobium Species 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 60
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 89
- 239000005017 polysaccharide Substances 0.000 claims abstract description 85
- 150000004676 glycans Chemical class 0.000 claims abstract description 84
- 238000000746 purification Methods 0.000 claims abstract description 30
- 241000894007 species Species 0.000 claims abstract description 17
- 239000000126 substance Substances 0.000 claims abstract description 11
- 238000000605 extraction Methods 0.000 claims abstract description 9
- 239000012141 concentrate Substances 0.000 claims abstract description 7
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 26
- 238000011993 High Performance Size Exclusion Chromatography Methods 0.000 claims description 20
- 238000011088 calibration curve Methods 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 12
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 12
- 239000007864 aqueous solution Substances 0.000 claims description 12
- 230000003544 deproteinization Effects 0.000 claims description 12
- 230000014759 maintenance of location Effects 0.000 claims description 10
- 229920002307 Dextran Polymers 0.000 claims description 9
- 238000001556 precipitation Methods 0.000 claims description 8
- 238000005070 sampling Methods 0.000 claims description 8
- 238000000108 ultra-filtration Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 7
- 239000012153 distilled water Substances 0.000 claims description 7
- 238000001228 spectrum Methods 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 5
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical class [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
- 239000012491 analyte Substances 0.000 claims description 4
- 238000004090 dissolution Methods 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- 230000003252 repetitive effect Effects 0.000 claims description 4
- 235000011091 sodium acetates Nutrition 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 238000007619 statistical method Methods 0.000 claims description 4
- 238000004364 calculation method Methods 0.000 claims description 3
- 230000035613 defoliation Effects 0.000 claims description 3
- 230000002786 root growth Effects 0.000 claims description 3
- 238000000205 computational method Methods 0.000 claims description 2
- 230000007850 degeneration Effects 0.000 claims description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 2
- 239000010931 gold Substances 0.000 claims description 2
- 229910052737 gold Inorganic materials 0.000 claims description 2
- 150000003384 small molecules Chemical class 0.000 claims description 2
- 241000208140 Acer Species 0.000 claims 1
- 230000000717 retained effect Effects 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 6
- 238000001514 detection method Methods 0.000 abstract description 5
- 230000007812 deficiency Effects 0.000 abstract description 3
- 230000003285 pharmacodynamic effect Effects 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 239000000470 constituent Substances 0.000 abstract description 2
- 238000007639 printing Methods 0.000 description 4
- 241000026010 Dendrobium candidum Species 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 229930013930 alkaloid Natural products 0.000 description 3
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 2
- 150000003797 alkaloid derivatives Chemical class 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 239000000460 chlorine Substances 0.000 description 2
- 229910052801 chlorine Inorganic materials 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000857 drug effect Effects 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- QWUWMCYKGHVNAV-UHFFFAOYSA-N 1,2-dihydrostilbene Chemical group C=1C=CC=CC=1CCC1=CC=CC=C1 QWUWMCYKGHVNAV-UHFFFAOYSA-N 0.000 description 1
- 241001678082 Dendrobium huoshanense Species 0.000 description 1
- 241001076416 Dendrobium tosaense Species 0.000 description 1
- 229920001503 Glucan Polymers 0.000 description 1
- 241000594394 Hedyotis Species 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- -1 Polysaccharide compound Chemical class 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- TWNIBLMWSKIRAT-VFUOTHLCSA-N levoglucosan Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@H]2CO[C@@H]1O2 TWNIBLMWSKIRAT-VFUOTHLCSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a kind of chromatographic fingerprinting method for differentiating three kinds of medicinal dendrobiums, comprises the following steps：Polyose extraction, obtains crude polysaccharides solution；Polysaccharide purification, concentration, obtain polysaccharide concentrate solution after purification；Chromatography, sets up chromatographic fingerprinting；Unknown species Herba Dendrobii identification.It is an advantage of the current invention that：The present invention can effectively distinguish and differentiate three kinds of Herba Dendrobiis that Dabie Mts, anhui is produced by the molecular weight distribution of Herba Dendrobii main pharmacodynamics composition dendrobium polysaccharide, can overcome the disadvantages that the deficiency of the authentication method in 2016 editions Anhui Native standards of 2015 editions Herba Dendrobii authentication methods for setting of Chinese Pharmacopoeia and Herba Dendrobii, because being a kind of rough overlapped information both rear, 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 the discriminating of orchid family Dendrobium 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 dendrobiums.
Background technology
3 kinds of medicinal dendrobiums are mainly produced in Dabie Mountains, Anhui Province area, plant name and are respectively：Herba Dendrobii (Dendrobium
Huoshanense C.Z.Tang et S.J.Cheng) (also known as Huoshan rice dry measure used in former times), Herba Dendrobii (Dendrobium candidum
Wall.exLindl.) (also known as Herba hedyotis costatae) and dendrobium moniliformeSweet (Dendrobium moniliforme (Linn.) Sw.) (also known as
Dendrobium Moniliforme).Three is orchid family Herba Dendrobii, belongs to herbaceos perennial, mainly contains polysaccharide, alkaloid, bibenzyl and luxuriant and rich with fragrance class
Isoreactivity composition, with multiple efficacies such as antitumor, immunomodulating, antioxidation, expansion of blood vessels, blood sugar lowering.Three is pacified jointly
Emblem provincial government declares as geography symbol product " Herba Dendrobii " that (species name " Herba Dendrobii " only refers to one kind, geographical sign " Huoshan
Herba Dendrobii " refers to three kinds).
Polysaccharide compound is one of main active of natural drug.Its activity and the space structure of its polysaccharide and point
Son amount distribution is closely related.For exploring the dependency of its component and drug effect, this programme adopts Efficient numerical method method
(HPSEC) analysis is compared to the distribution of the relative molecular mass of same place of production variety classes dendrobium 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 dendrobium polysaccharide
The formulation of continuous quality standard provides feasible solution.So far, have no with regard to setting up many fried sugars of medicinal dendrobium using HPSEC chromatographs
The open report of spectrum finger printing.
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 dendrobiums
Spectrum fingerprint spectrum method, the atlas calculation are the HPSEC chromatograms that is set up according to medicinal dendrobium main pharmacodynamics basic substance polysaccharide
Spectrum, the polysaccharide molecular weight scattergram Prepenem that the present invention sets up effectively distinguish different Herba Dendrobii species, can both make up only according to total polysaccharidess, life
The content of alkaloids and polysaccharide acidolysis into monosaccharide ratio of components judging that Herba Dendrobii or Herba Dendrobii be good and bad and the deficiency of the true and false,
Can be used to the Herba Dendrobii kind for differentiating geography symbol product " Herba Dendrobii " and other producing regions.
For solving above-mentioned technical problem, the present invention provides a kind of chromatographic fingerprinting method for differentiating three kinds of medicinal dendrobiums,
Comprise the steps：
(1)：Polyose extraction
The medicinal dendrobium material of the known different cultivars obtained according to statistical method sampling is mixed according to same species, in
Dry to constant weight in 50-60 DEG C of baking oven, and be ground into powder, 80 DEG C -90 DEG C of dehydrated alcohol surname extraction is colourless to backflow,
Solvent is volatilized, by gained Herba Dendrobii medicinal residues according to solid-liquid ratio (0.5-1)：(5-7) add in distilled water, and in 75 DEG C of -85 DEG C of water-baths
Condensing reflux is extracted 4-5 time, each 3-4h；Then, merge said extracted filtrate and concentrating under reduced pressure obtains crude polysaccharides solution；
(2)：Polysaccharide purification and concentration
By gained crude polysaccharides solution according to volume ratio (0.5-1):(3-4) add in dehydrated alcohol, in 3-5 DEG C of refrigerator
20-30h is stood, and then, leaves and takes precipitation, go forward side by side after 2-5min, and repetitive operation 3-4 time being centrifuged under 3000-4000r/min rotating speeds
One step carries out polysaccharide purification to which, and wherein, polysaccharide purification adopts two-step purifying method：
1. first step purification is realized to the process of polysaccharide solution deproteinization using Sevage methods；
2. first step polysaccharide solution after purification is collected, by ultrafiltration, small-molecule substance while concentration is sloughed
Polysaccharide solution, realizes that second step is purified to the final polysaccharide concentrate solution for obtaining after purification；
(3)：Chromatography and the foundation of chromatographic fingerprinting
1. chromatographic-type
Efficient numerical method HPSEC；
2. chromatographic condition
Mobile phase：0.1-0.2M sodium acetates；Flow velocity：0.5-1.0mL/min；Column temperature：35-45℃；Sample size：20μL；
3. the acquisition of polysaccharide standard curve
The aqueous solution difference sample introduction that the dextran standard of different molecular weight is configured to 4-5mg/mL is arranged in efficient volume
In resistance chromatograph HPSEC, further according to each corresponding chromatogram calculation polysaccharide molecular weight calibration curve equation；
4. molecular weight analyte distributional analysiss and the foundation of chromatographic fingerprinting
Herba Dendrobii sample difference sample introduction after polysaccharide purification obtains corresponding chromatogram in Efficient numerical method HPSEC, then
The relative molecular weight of the chromatographic curve each several part of each sample is calculated according to polysaccharide molecular weight calibration curve equation, finally sets up phase
Answer the chromatographic fingerprinting of known Herba Dendrobii sample；
(4)：The identification of unknown species Herba Dendrobii
The Herba Dendrobii of unknown species is processed according to above step (1)-(3), to obtain its corresponding chromatographic fingerprinting,
Its chromatographic fingerprinting with the known Herba Dendrobii 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 Dendrobium Sw, otherwise it is not same kind.
Preferably, in step (1), the kind of medicinal dendrobium is respectively：Herba Dendrobii, Herba Dendrobii and dendrobium moniliformeSweet.
Preferably, in step (1), medicinal dendrobium material includes that medicinal dendrobium dry product and fresh goods, the medicinal dendrobium are done
Product further include Herba Dendrobii extract, very little gold, powder；The medicinal dendrobium fresh goods is specially root growth more than 3 years, then stripping
The fresh stem of Herba Dendrobii defoliation after surviving the winter, also, the medicinal dendrobium fresh goods need to be used in mixed way after thinly slicing.
Preferably, in step (1), statistical method is：Carried out using five point sampling in Land in Dabieshan Area of Anhui Province medicinal
The collection of Herba Dendrobii sample.
Preferably, concretely comprised the following steps using Sevage methods in step (2)：Gained precipitation is used distillation water dissolution simultaneously first
Mix, then, according to sample and extractant volume ratio (3-5)：(0.5-2) Sevage reagents are added, mix vibration 25-35min,
5-6min is centrifuged with 3000-4000r/min again, is discarded except lower floor's chloroform and medial degeneration albumen；Take upper strata aqueous solution again to repeat
Deproteinization is operated, to without obvious middle level；Finally, the upper strata aqueous solution after Deproteinization is collected.
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, in step (2), ultrafiltration apparatus are the ultrafiltration apparatus that membrane retention molecular weight is 1K.
Preferably, in step (3), weight average molecular weight Mw of the dextran standard of different molecular weight is respectively：
2000KDa、580KDa、70KDa、10KDa、5KDa.
Preferably, in step (3), 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, in step (3), chromatographic condition also includes：
Chromatograph column type number and dress post mode：UltrahydrogelTMLinear 300mm × 7.8mm, two series connection；Detection
Device type：Differential refraction detector RID.
It is an advantage of the current invention that：The Dabie Mts, anhui that the present invention is provided produces three kinds of dendrobium polysaccharide molecular weight distribution
HPSEC chromatographic fingerprintings for representing pattern collection of illustrative plates, can by the molecular weight distribution of Herba Dendrobii main pharmacodynamics composition-dendrobium polysaccharide come
Effectively distinguish and differentiate three kinds of Herba Dendrobiis that Dabie Mts, anhui is produced；The method can overcome the disadvantages that the Herba Dendrobii of 2015 editions settings of Chinese Pharmacopoeia
The deficiency of the authentication method in 2016 editions Anhui Native standards (DB34/T 486-2016) of authentication method and Herba Dendrobii,
Because rear both be ratio of components using total polysaccharidess content, total alkaloid content and polysaccharide acidolysis into monosaccharide as its quality evaluation and
Criterion, is a kind of rough overlapped information, and counterfeiter can be faked using the leak of detection method, and this method closer to
The essence of chemical constituent, is finer information, and 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 dendrobiums of discriminating that embodiment of the present invention 1-2 is provided
Cheng Tu；
Fig. 2 is a kind of Anhui of the chromatographic fingerprinting method of three kinds of medicinal dendrobiums of discriminating that the embodiment of the present invention 1 is provided
Produce the graph of molecular weight distribution of Herba Dendrobii polysaccharide in Dabie Mountain；
Fig. 3 is a kind of Anhui of the chromatographic fingerprinting method of three kinds of medicinal dendrobiums of discriminating that the embodiment of the present invention 1 is provided
Produce the graph of molecular weight distribution of dendrobium moniliformeSweet polysaccharide in Dabie Mountain；
Fig. 4 is a kind of Anhui of the chromatographic fingerprinting method of three kinds of medicinal dendrobiums of discriminating that the embodiment of the present invention 1 is provided
Produce the graph of molecular weight distribution of Dendrobium officinale polysaccharide in Dabie Mountain.
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 dendrobiums, as shown in figure 1, comprise the steps：
(1)：Polyose extraction
The known three kinds of root growths medicine of more than 3 years that will be obtained in Land in Dabieshan Area of Anhui Province sampling according to five point sampling
Processed with the fresh stem defoliation after stripping is survived the winter then of Herba Dendrobii (Herba Dendrobii, Herba Dendrobii, dendrobium moniliformeSweet), cut by species respectively
Flakiness mixes, and dries to constant weight, and be ground into powder in 60 DEG C of baking oven, and 85 DEG C of dehydrated alcohol surname extraction is to backflow
Liquid is colourless, volatilizes solvent, by gained Herba Dendrobii medicinal residues according to solid-liquid ratio 1：5 add in distilled water, and in 80 DEG C of water-bath condensing refluxes
Extract 4 times, each 4h；Then, merge said extracted filtrate and concentrating under reduced pressure obtains crude polysaccharides solution；
(2)：Polysaccharide purification and concentration
By gained crude polysaccharides solution according to volume ratio 1:4 add in dehydrated alcohol, stand 24h, connect in 4 DEG C of refrigerators
, 2-5min, and repetitive operation 3 times is centrifuged under 4000r/min rotating speeds, is merged precipitation, and polysaccharide purification is carried out to which further,
Wherein, polysaccharide purification adopts two-step purifying method：
1. first step purification is realized to the process of polysaccharide solution deproteinization using Sevage methods：First gained precipitation is distilled
Water dissolution is simultaneously mixed, then, according to sample and extractant volume ratio 4：1 adds Sevage reagents, and (volume ratio is respectively 4: 1 chlorine
Imitative and n-butyl alcohol mixed liquor), vibration 30min is mixed, then 5min is centrifuged with 4000r/min, discarded and become except lower floor's chloroform and middle level
Property albumen；Take upper strata aqueous solution again and repeat Deproteinization operation, to without obvious middle level；Finally, the upper strata after Deproteinization is collected
Aqueous solution；
2. first step polysaccharide solution after purification is collected, and by ultrafiltration, membrane retention molecular weight is 1K, sloughs little
Molecular substance simultaneously concentrates polysaccharide solution simultaneously, realizes that second step is purified to the final polysaccharide concentrate solution for obtaining after purification；
(3)：Chromatography and the foundation of chromatographic fingerprinting
1. chromatographic-type
Efficient numerical method HPSEC；
2. chromatographic condition
Chromatograph column type number and dress post mode：UltrahydrogelTMLinear 300mm × 7.8mm, two series connection；Detection
Device type：Differential refraction detector RID；Mobile phase：0.1M sodium acetates；Flow velocity：0.8mL/min；Column temperature：45℃；Sample size：20
μL；
3. the acquisition of polysaccharide standard curve
Weight average molecular weight Mw is respectively：Five kinds of dextran standards of 2000KDa, 580KDa, 70KDa, 10KDa, 5KDa
Product are configured to the aqueous solution difference sample introduction of 5mg/mL in Efficient numerical method HPSEC, further 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=-0.3488x+11.134
R2=0.9948；
4. molecular weight analyte distributional analysiss and the foundation of chromatographic fingerprinting
Herba Dendrobii sample difference sample introduction after polysaccharide purification obtains corresponding chromatogram in Efficient numerical method HPSEC, then
The relative molecular weight of the chromatographic curve each several part of each sample is calculated according to polysaccharide molecular weight calibration curve equation, finally sets up phase
Answer the chromatographic fingerprinting of known Herba Dendrobii sample；Fig. 2-4 is respectively three kinds of Huoshan and produces Herba Dendrobii in Efficient numerical method
The peak spectrogram obtained in HPSEC, i.e., the (unit with abscissa as appearance time：Minute), response value of the vertical coordinate for photosignal
(unit：Millivolt) polysaccharide graph of molecular weight distribution, wherein, the data on chromatographic peak are the relative molecular weight Mw of each cut point,
It is to be calculated by above-mentioned polysaccharide molecular weight calibration curve equation；Press molecular weight in the peak of the HPSEC collection of illustrative plates of three kinds of Herba Dendrobii samples
Scope cuts into 6 parts, and molecular weight is respectively：It is more than 500KDa, 500KDa-200KDa, 200KDa-100KDa, 100KDa-
50KDa、50KDa-10Kda、<10KDa；Collection of illustrative plates is examined through methodologies such as stability test, elaboration test and reappearance tests
Examine, the RSD of relative peak area change is respectively less than 3%, meet chromatographic process requirement；
The polysaccharide of general part of the molecular weight between 50KDa-200KDa may have significant anti-tumor activity, will divide
Son amount accounts for the percent data of total peak area in the peak area of 50KDa-200KDa and is listed and obtains table 1, i.e. different molecular weight
Dendrobium polysaccharide accounts for the ratio table of total polysaccharidess；
The dendrobium polysaccharide of 1 different molecular weight of table accounts for the ratio of total polysaccharidess
As shown in Table 1, Dabie Mts, anhui produces the polyoses content of three kinds of Herba Dendrobiis and molecular weight distribution notable difference, different
The relative scale of the polysaccharide of molecular weight is different, and particularly polysaccharide proportion of the molecular weight between 50KDa～200KDa has bright
Significant difference is different, respectively：Herba Dendrobii 16.38 ± 1.4%, dendrobium moniliformeSweet 21.61 ± 1.3%, Herba Dendrobii 14.19 ± 1.5%.
This shows that the polysaccharide molecular weight distribution of three kinds of Herba Dendrobiis under the same place of production, same condition of culture has notable difference, this species diversity
May be closely related with its species and physiologically active, also point out the drug effect difference of three kinds of Herba Dendrobiis come from active polysaccharide phase simultaneously
The difference of comparative example；
Therefore, three collection of illustrative plates of Fig. 2-4 are the HPSEC chromatographs that Dabie Mts, anhui produces three kinds of dendrobium polysaccharide molecular weight distribution
The representative pattern collection of illustrative plates of finger printing；
(4)：The identification of unknown species Herba Dendrobii
The Herba Dendrobii of two kinds of unknown species is processed according to above step (1)-(3), its corresponding chromatographic fingerprint figure is obtained
Spectrum, produces further according to Chinese Pharmacopoeia committee《Similarity evaluation》Software is judged, is tied
Fruit shows that a kind of collection of illustrative plates of Herba Dendrobii and Dabie Mts, anhui produce the similarity of Herba Dendrobii up to 94%, that is, be judged to that Dabie Mts, anhui is produced
Herba Dendrobii, and the similarity that the collection of illustrative plates of another kind of Herba Dendrobii represents pattern finger printing with Dabie Mts, anhui three kinds of Herba Dendrobiis of product reaches
Less than 90%, then judge that not Dabie Mts, anhui produces Herba Dendrobii to the Herba Dendrobii.
Embodiment 2
A kind of chromatographic fingerprinting method for differentiating three kinds of medicinal dendrobiums, as shown in figure 1, comprise the steps：
(1)：Polyose extraction
Known three kinds of medicinal dendrobiums (Herba Dendrobii, the ferrum that will be obtained in Land in Dabieshan Area of Anhui Province sampling according to five point sampling
Skin Herba Dendrobii, dendrobium moniliformeSweet) Herba Dendrobii extract by species mix, dry to constant weight in 55 DEG C of baking oven, and be ground into powder, 80 DEG C of nothing
Water-ethanol surname extraction is colourless to backflow, volatilizes solvent, by gained Herba Dendrobii medicinal residues according to solid-liquid ratio 1：6 add in distilled water,
And extract 5 times in 80 DEG C of water-bath condensing refluxes, each 3h；Then, merge said extracted filtrate and concentrating under reduced pressure obtains crude polysaccharides
Solution；
(2)：Polysaccharide purification and concentration
By gained crude polysaccharides solution according to volume ratio 1:4 add in dehydrated alcohol, stand 30h, connect in 5 DEG C of refrigerators
, 4min, and repetitive operation 4 times is centrifuged under 3500r/min rotating speeds, is merged precipitation, and polysaccharide purification is carried out to which further, its
In, polysaccharide purification adopts two-step purifying method：
1. first step purification is realized to the process of polysaccharide solution deproteinization using Sevage methods：First gained precipitation is distilled
Water dissolution is simultaneously mixed, then, according to sample and extractant volume ratio 4：1 adds Sevage reagents, and (volume ratio is respectively 4: 1 chlorine
Imitative and n-butyl alcohol mixed liquor), vibration 25min is mixed, then 6min is centrifuged with 4000r/min, discarded and become except lower floor's chloroform and middle level
Property albumen；Take upper strata aqueous solution again and repeat Deproteinization operation, to without obvious middle level；Finally, the upper strata after Deproteinization is collected
Aqueous solution；
2. first step polysaccharide solution after purification is collected, and by ultrafiltration, membrane retention molecular weight is 1K, sloughs little
Molecular substance simultaneously concentrates polysaccharide solution simultaneously, realizes that second step is purified to the final polysaccharide concentrate solution for obtaining after purification；
(3)：Chromatography and the foundation of chromatographic fingerprinting
1. chromatographic-type
Efficient numerical method HPSEC；
2. chromatographic condition
Chromatograph column type number and dress post mode：UltrahydrogelTMLinear 300mm × 7.8mm, two series connection；Detection
Device type：Differential refraction detector RID；Mobile phase：0.1M sodium acetates；Flow velocity：1.0mL/min；Column temperature：45℃；Sample size：20
μL；
3. the acquisition of polysaccharide standard curve
Weight average molecular weight Mw is respectively：Five kinds of dextran standards of 2000KDa, 580KDa, 70KDa, 10KDa, 5KDa
Product are configured to the aqueous solution difference sample introduction of 5mg/mL in Efficient numerical method HPSEC, further 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=-0.3488x+
11.134R2=0.9948；
4. molecular weight analyte distributional analysiss and the foundation of chromatographic fingerprinting
Herba Dendrobii sample difference sample introduction after polysaccharide purification obtains corresponding chromatogram in Efficient numerical method HPSEC, then
The relative molecular weight of the chromatographic curve each several part of each sample is calculated according to polysaccharide molecular weight calibration curve equation, finally sets up phase
Answer the chromatographic fingerprinting of known Herba Dendrobii sample；
(4)：The identification of unknown species Herba Dendrobii
The Herba Dendrobii of three kinds of unknown species is processed according to above step (1)-(3), its corresponding chromatographic fingerprint figure is obtained
Spectrum, produces further according to Chinese Pharmacopoeia committee《Similarity evaluation》Software is judged, is tied
Fruit shows that a kind of collection of illustrative plates of Herba Dendrobii produces the similarity of Herba Dendrobii up to 92% with Dabie Mts, anhui, that is, be judged to Land in Dabieshan Area of Anhui Province
Herba Dendrobii, a kind of collection of illustrative plates of Herba Dendrobii and Dabie Mts, anhui produce the similarity of dendrobium moniliformeSweet up to 90%, that is, be judged to that Anhui is other greatly
Mountain products dendrobium moniliformeSweet, and the collection of illustrative plates of the third Herba Dendrobii represents the similar of pattern finger printing to three kinds of Herba Dendrobiis that Dabie Mts, anhui is produced
Degree does not reach 90%, then judge that not Herba Dendrobii is produced in Land in Dabieshan Area of Anhui Province to the Herba Dendrobii.
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 dendrobiums chromatographic fingerprinting method, it is characterised in that comprise the steps：
(1)：Polyose extraction
The medicinal dendrobium material of the known different cultivars obtained according to statistical method sampling is mixed according to same species, in 50-
Dry to constant weight in 60 DEG C of baking oven, and be ground into powder, 80 DEG C -90 DEG C of dehydrated alcohol surname extraction is colourless to backflow, waves
Dry solvent, by gained Herba Dendrobii medicinal residues according to solid-liquid ratio (0.5-1)：(5-7) add in distilled water, and cold in 75 DEG C of -85 DEG C of water-baths
Solidifying reflux, extract, 4-5 time, each 3-4h；Then, merge said extracted filtrate and concentrating under reduced pressure obtains crude polysaccharides solution；
(2)：Polysaccharide purification and concentration
By gained crude polysaccharides solution according to volume ratio (0.5-1):(3-4) add in dehydrated alcohol, stand in 3-5 DEG C of refrigerator
20-30h, then, leaves and takes precipitation under 3000-4000r/min rotating speeds after being centrifuged 2-5min, and repetitive operation 3-4 time, and further
Polysaccharide purification is carried out to which, wherein, polysaccharide purification adopts two-step purifying method：
1. first step purification is realized to the process of polysaccharide solution deproteinization using Sevage methods；
2. first step polysaccharide solution after purification is collected, by ultrafiltration, small-molecule substance is sloughed and while is concentrated polysaccharide
Solution, realizes that second step is purified to the final polysaccharide concentrate solution for obtaining after purification；
(3)：Chromatography and the foundation of chromatographic fingerprinting
1. chromatographic-type
Efficient numerical method HPSEC；
2. chromatographic condition
Mobile phase：0.1-0.2M sodium acetates；Flow velocity：0.5-1.0mL/min；Column temperature：35-45℃；Sample size：20μL；
3. the acquisition of polysaccharide standard curve
The dextran standard of different molecular weight is configured to the aqueous solution difference sample introduction of 4-5mg/mL in efficient volume-exclusion color
In spectrum HPSEC, further according to each corresponding chromatogram calculation polysaccharide molecular weight calibration curve equation；
4. molecular weight analyte distributional analysiss and the foundation of chromatographic fingerprinting
Herba Dendrobii sample difference sample introduction after polysaccharide purification obtains corresponding chromatogram in Efficient numerical method HPSEC, further according to
Polysaccharide molecular weight calibration curve equation calculates the relative molecular weight of the chromatographic curve each several part of each sample, and final foundation is corresponding
The chromatographic fingerprinting of the Herba Dendrobii sample that knows；
(4)：The identification of unknown species Herba Dendrobii
The Herba Dendrobii of unknown species is processed according to above step (1)-(3), to obtain its corresponding chromatographic fingerprinting, then will
Which carries out similarity-rough set with the chromatographic fingerprinting of the known Herba Dendrobii sample that sets up, when its similarity is more than more than 90%
When, you can it is judged to same class Dendrobium Sw, is not otherwise same kind.
2. according to claim 1 a kind of differentiate three kinds of medicinal dendrobiums chromatographic fingerprinting method, it is characterised in that institute
The kind for stating medicinal dendrobium in step (1) is respectively：Herba Dendrobii, Herba Dendrobii and dendrobium moniliformeSweet.
3. according to claim 1 a kind of differentiate three kinds of medicinal dendrobiums chromatographic fingerprinting method, it is characterised in that institute
Stating medicinal dendrobium material in step (1) includes that medicinal dendrobium dry product and fresh goods, the medicinal dendrobium dry product further include maple
Bucket, very little gold, powder；The medicinal dendrobium fresh goods is specially root growth more than 3 years, the Herba Dendrobii defoliation after stripping is survived the winter then
Fresh stem, also, the medicinal dendrobium fresh goods need to be used in mixed way after thinly slicing.
4. according to claim 1 a kind of differentiate three kinds of medicinal dendrobiums chromatographic fingerprinting method, it is characterised in that institute
Stating statistical method in step (1) is：The collection of medicinal dendrobium sample is carried out using five point sampling in Land in Dabieshan Area of Anhui Province.
5. according to claim 1 a kind of differentiate three kinds of medicinal dendrobiums chromatographic fingerprinting method, it is characterised in that institute
State and concretely comprised the following steps using Sevage methods in step (2)：Gained precipitation is distilled water dissolution first and is mixed, then, according to sample
Product and extractant volume ratio (3-5)：(0.5-2) Sevage reagents are added, mixes vibration 25-35min, then with 3000-4000r/
Min is centrifuged 5-6min, discards except lower floor's chloroform and medial degeneration albumen；Take upper strata aqueous solution again and repeat Deproteinization operation, to nothing
Substantially till middle level；Finally, the upper strata aqueous solution after Deproteinization is collected.
6. according to claim 5 a kind of differentiate three kinds of medicinal dendrobiums 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 1 a kind of differentiate three kinds of medicinal dendrobiums chromatographic fingerprinting method, it is characterised in that institute
State the ultrafiltration apparatus that ultrafiltration apparatus in step (2) are that membrane retention molecular weight is 1K.
8. according to claim 1 a kind of differentiate three kinds of medicinal dendrobiums 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 (3) is respectively：2000KDa、580KDa、70KDa、
10KDa、5KDa.
9. according to claim 1 a kind of differentiate three kinds of medicinal dendrobiums chromatographic fingerprinting method, it is characterised in that institute
The computational methods for stating polysaccharide molecular weight calibration curve equation in step (3) 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 dendrobiums of discriminating of claim 1-9, its feature
It is, in step (3), chromatographic condition also includes：
Chromatograph column type number and dress post mode：UltrahydrogelTMLinear 300mm × 7.8mm, two series connection；Detector class
Type：Differential refraction detector RID.
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