CN105911081B - A kind of method for differentiating mannatide - Google Patents
A kind of method for differentiating mannatide Download PDFInfo
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Abstract
The invention discloses a kind of method for differentiating mannatide, belong to field of chemical detection.The present invention obtains the hydrogen spectrum of mannosan poly saccharide peptide standard product and test sample using nuclear magnetic resonance measuring, corresponded according to the spectral peak chemical displacement value in the Qing Puzhong anomer hydrogens area of mannatide, peak shape from the mannose residue of different connected modes in mannatide sugar chain structure repeat unit, it is compared with similarity, qualitative analysis is carried out, accurately mannatide can be identified.The inventive method has very strong specificity, can significantly distinguish the glucomannans and galactomannans similar with mannatide composition；And it is reproducible, day to day precision is high.Quality control and quality standard for mannatide provide new method.
Description
Technical field
The present invention relates to a kind of method for differentiating mannatide, belong to field of chemical detection.
Background technology
At present, it is differentiated using chemical colour reaction method in mannatide quality standard, main function mechanism is
Polysaccharide is hydrolyzed into monose, then rapid dehydration generation alditol derivative in the presence of sulfuric acid, is then generated with phenol coloured
Compound, as long as therefore have sugar structure can develop the color, specificity is not strong.But this discrimination method specificity is not strong, main cause
It is due to that mannatide structure is indefinite.Therefore a kind of strong, easy to operate, the reproducible discriminating side of specificity need to be established
Method, the quality control for mannatide.
Hydrogen nuclear magnetic resonance spectroscopy is the effective ways that polysaccharide composition and repeat unit structure are identified, need not during analysis
Polysaccharide is degraded or derived, the nuance between different polysaccharide structures can be analyzed, can also show a small amount of endogenous and
Exogenous pollutant.But use nuclear magnetic resonance to differentiate that mannatide has problems, include the phase of various saccharide residues
Measure, the ownership of dew glycan peptide proton nmr spectra anomer hydrogen area spectral peak and the tool of mannatide nuclear-magnetism detection to content
Concrete conditions in the establishment of a specific crime.
The content of the invention
The invention solves first technical problem be to provide the method for nuclear magnetic resonance identification mannatide a kind of.
Methods described is the proton nmr spectra by the hydrogen nuclear magnetic resonance spectrogram of testing sample and mannosan poly saccharide peptide standard product
The peak type and spectral peak shift value of figure are compared, and the spectral regions of specificity identification are used as in selected spectrogram, and calculate selected spectral regions
Similarity.
In one embodiment of the invention, the similarity ρ of mannatide sample and mannosan poly saccharide peptide standard product>
0.95.That is, testing sample and the n of the hydrogen nuclear magnetic resonance spectrogram of mannosan poly saccharide peptide standard product>0.95, it is judged to mannatide.
In one embodiment of the invention, the spectral regions as specificity identification, it is δ 4.89ppm~δ
5.90ppm anomer hydrogen area.
In one embodiment of the invention, δ 5.29 be 1,2- connections mannose residue anomer hydrogen signal, δ
5.14 be the anomer hydrogen signal of the mannose residue of 1,3- connections, and δ 5.12 and δ 5.08 are the mannose residue of 1,2,6- connections
Anomer hydrogen signal, the mannose residue that δ 5.04 is the anomer hydrogen signal of the mannose residue of 1- connections, δ 4.90 is 1,6- connections
Anomer hydrogen signal.
In one embodiment of the invention, the similarity ρ is calculated using correlation coefficient process.
In one embodiment of the invention, the hydrogen nuclear magnetic resonance spectrogram and mannatide standard of testing sample are obtained
The step of hydrogen nuclear magnetic resonance spectrogram of product, includes：
(1) preparation of sample：
Mannosan poly saccharide peptide standard product and each 30mg of testing sample accurately are weighed, is dissolved in 0.5mL D2In O solution, ultrasound is extremely
It is completely dissolved, adds 10 μ L 0.2% TSP heavy aqueous solutions, ultrasonic mixing is uniform, is transferred in 5mm nuclear magnetic tube；
(2) NMR is determined：
1H NMR resonant frequency is 400MHz, and sampling parameter is as follows：Pulse train is zgcppr, sampling number 64k,
Scanning times are 64, and it is 4 that sky, which sweeps number, spectrum width 8012.8Hz, pulse angle 45, sampling time 3.7s, reception gain
For 10, test temperature 303K, the relaxation delay time is 1.0s, and window function linewidth factor is 1Hz.
The present invention also provides a kind of method of magnetic resonance detection mannatide, the described method comprises the following steps：
(1) preparation of sample：
Mannosan poly saccharide peptide standard product and each 30mg of testing sample accurately are weighed, is dissolved in 0.5mL D2In O solution, ultrasound is extremely
It is completely dissolved, adds 10 μ L 0.2% TSP heavy aqueous solutions, ultrasonic mixing is uniform, is transferred in 5mm nuclear magnetic tube；
(2) NMR is determined：
1H NMR resonant frequency is 400MHz, and sampling parameter is as follows：Pulse train is zgcppr, sampling number 64k,
Scanning times are 64, and it is 4 that sky, which sweeps number, spectrum width 8012.8Hz, pulse angle 45, sampling time 3.7s, reception gain
For 10, test temperature 303K, the relaxation delay time is 1.0s, and window function linewidth factor is 1Hz.
The present invention obtains the hydrogen spectrum of mannosan poly saccharide peptide standard product and test sample using nuclear magnetic resonance measuring, is gathered according to sweet dew
The spectral peak chemical displacement value in the Qing Puzhong anomer hydrogens area of glycopeptide, peak shape are different from mannatide sugar chain structure repeat unit even
The mannose residue for connecing mode corresponds, and is compared with similarity, carries out qualitative analysis, accurately mannatide can be entered
Row identification.The inventive method has very strong specificity, can significantly distinguish the glucomannans similar with mannatide composition and
Galactomannans；And it is reproducible, day to day precision is high.The raising of quality control and quality standard for mannatide carries
For new method.
Brief description of the drawings
Fig. 1 mannosan poly saccharide peptide standard products1HNMR spectrograms
Fig. 2 exchanges pretreated sample through heavy water1HNMR spectrograms
Fig. 3 temperature pair1The influence of HNMR spectrograms
TSP's splits a point situation under Fig. 4 different temperatures
Fig. 5 glucomannans (2), galactomannans (3) and mannatide (1)1H-NMR spectrum stacking chart's (interceptions
4.85~6.00ppm areas)
Fig. 6 Radix Isatidis glycopeptide (2), polysaccharide-peptide (3), Flos Mume polysaccharide (4) and mannatide (1)1H-NMR is superimposed
Spectrogram (4.85~6.00ppm of interception areas)
The mannatide test sample of tri- parts of parallel preparations of Fig. 71H-NMR spectrum
Same date does not detect three times Fig. 8 mannatides test sample1H-NMR spectrum
Fig. 9 mannatide test samples1H-NMR superpositions spectrogram (4.85~66.00ppm of interception areas)；1. sweet glycan peptide
Standard items；2~10. mannatide test samples；Lot number：091202nd, 110901, G100102,131101 injection powder, 131102
Inject powder, 131101 oral powder, 140401 injection powder, 140501 injection powder, 140502 injection powder.
Embodiment
Reagent and instrument
Mannosan poly saccharide peptide standard product (140652-200401, Chinese pharmaceutical biological product inspection institute)；TSP
(trimethylsilylpropionic, tritium is for trimethyl silane propionic acid sodium salt), Sigma Co., USA；Heavy water, Beijing are wished triumphant
Creative Technology Ltd.；Mannatide sample, Chengdu rel Co., Ltd；(Community in Baiyunshan, Guangzhou and note are yellow for Radix Isatidis glycopeptide
Pu Chinese medicine Co., Ltd)；Polysaccharide-peptide (Nanjing Sen Beijia bio tech ltd)；Chitosan oligosaccharide (the big health biotechnology in Shanghai
Co., Ltd)；Flos Mume polysaccharide (laboratory extraction).The preparation process of Flos Mume polysaccharide includes：(1) pretreatment of Flos Mume：
Fresh Flos Mume is dried to constant weight with 50 DEG C of thermostatic drying chambers before preparing, is crushed with pulverizer, is put into hermetic bag, be placed in drying
It is sealed in device.(2) extraction of Flos Mume Thick many candies：The Flos Mume powder of 80.0g dryings is weighed, adds 1600mL deionizations
Water (solid-liquid ratio 1:20), 100 DEG C of reflux condensation modes 2 hours in 2L round-bottomed flask, are cooled to room temperature, first with filtered through gauze,
Filtered again with Buchner funnel, it is residue obtained to be extracted again once with same method.The filtrate for filtering to obtain twice is merged, with rotation
Turn evaporimeter and 30~40mL is concentrated at 50 DEG C, add 4 times of volume ethanols, be placed in overnight precipitation in 4 DEG C of refrigerators, centrifuge, it is cold
It is lyophilized dry, weigh.
DD2400-MR nuclear magnetic resonance spectrometers (Agilent Co., Ltd)；METTLER AE240 type analysis balances (Mei Te
Le-support benefit Co., Ltd).
The preparation of TSP heavy aqueous solutions
The deuterated TSP of 10mg [3- (trimethyl silicon substrate) sodium propionate] accurately are weighed, are dissolved in 5mL D2In O (W/V), it is made into
0.2% TSP heavy aqueous solutions.
The preparation of sample solution
Mannosan poly saccharide peptide standard product and each 30mg of test sample accurately are weighed, is dissolved in 0.5mL D2In O solution, ultrasound is extremely
It is completely dissolved, adds 10 μ L 0.2% TSP heavy aqueous solutions, ultrasonic mixing is uniform, is transferred in 5mm nuclear magnetic tube.
Nuclear-magnetism (NMR) test condition
1H NMR resonant frequency is 400MHz, and sampling parameter is as follows：Pulse train (PULPROG) zgcppr, sampled point
Number (TD) 64k, scanning times (NS) 64, sky sweep number (DS) 4, spectrum width (SWH) 8012.8Hz, pulse angle (PW) 45, sampling
Time (AQ) 3.7s, reception gain (RG) 10, test temperature (TE) 303K, relaxation delay time (D1)=1.0s, window function line
The wide factor (Lb) 1Hz.
The structural analysis of the mannatide of embodiment 1
Four kinds of homogeneous glycopeptides are isolated from the oral raw material of mannatide, are made up of molecular weight distribution, monose, methyl
Change the means researchs such as analysis, NMR spectrum, amino acid analysis to show, four kinds of homogeneous glycopeptides, relative molecular mass and amino
Acid content is different, but each homogeneous glycopeptide monose composition, saccharide residue connected mode, proton nmr spectra are nearly identical.Meanwhile
Isolate 2 kinds of homogeneous glycopeptides in different batches mannatide injection stage raw material, to its monose composition, saccharide residue connected mode and
Proton nmr spectra is determined, and has obtained identical conclusion.May thereby determine that mannatide for relative molecular mass,
The mixture for the homogeneous glycopeptide that amino acid content is different but sugar chain structure is essentially identical, can be carried out by hydrogen nuclear magnetic resonance spectroscopy
Identification.
Fine structure research is carried out to the homogeneous glycopeptide that is separated in mannatide, the results showed that its form using mannose as
It is main, and contain a small amount of glucose；Main chain is made up of 1,6- mannose residues, and side chain is by 1,2- mannose residues, 1,3- sweet dew
Saccharide residue and terminal mannose residues are formed, branch point 1,2, the O-2 positions of 6- mannose residues.Containing threonine, serine,
16 kinds of amino acid such as asparatate, alanine, lysine, proline.
The nuclear-magnetism of embodiment 2 (NMR) determines the condition of mannatide
The selection of 1 calibration thing
The selection of calibration thing will consider that calibrate the intermiscibility of thing and sample and calibration thing does not produce interference to the signal of spectrogram
Etc. factor.NMR experiments calibration thing used typically have TMS (tetramethylsilane), DSS [3- (trimethyl silicon substrate) propane sulfonic acid sodium salt],
TSP [3- (trimethyl silicon substrate) sodium propionate] etc..TMS is not soluble in water, and mannatide sample is soluble in because polarity is larger
Water, it is necessary to using heavy water as solvent, therefore DSS or TSP can be used as calibration thing.But there are 3 methylene in DSS structures
1There is strong signal to occur in H H NMR spectroscopies, and be multiplet, may be disturbed with the signal coherence in sample.Therefore, present invention choosing
Deuterated TSP is selected as calibration thing.
The selection of 2 test temperatures
Because the mannose residue content of 1,6- connections in mannatide is very low, at 25 DEG C, its anomer hydrogen signal (Fig. 2
Middle D peaks) it is very weak, additionally, due to its chemical shift and water peak very close to so appearing in water peak in the form of acromion at normal temperatures
Left side.
As shown in figure 3, with the rise of temperature, water peak shifts to High-Field, and the mannose residue anomer hydrogen of 1,6- connection is believed
Number displacement changes very little.But with the rise of temperature, internal standard compound TSP spectral peak appearance is a certain degree of to split point (Fig. 4), so that
Sample spectra peaking displacement study is difficult to Accurate Determining；In addition, temperature rise also has certain influence to the life-span of nuclear-magnetism probe.Work as temperature
30 DEG C are risen to, internal standard compound spectral peak does not occur substantially splitting point compared with 25 DEG C, and the saccharide residue anomer hydrogen signal of 1,6- connection is also basic
Separated with water peak.
The selection of 3 sample concentrations
Sampling 64 times in the case of, investigate various concentrations mannatide sample the peaks of δ 5.29 signal to noise ratio (table 1.1) and
The uniformity (range estimation) of sample solution.When concentration reaches 60mg/mL, signal to noise ratio is good, and sample can be caused by continuing increase concentration
Dissolubility and mobility it is poor, increase the difficulty of pre-treatment and shimming.
The influence of the sample concentration of table 1
The selection of 4 scanning times
Scanning 8,16,32,64,128 times, the change of spectral peak signal to noise ratio, the results are shown in Table 2 at δ 5.29.
The influence of the scanning times of table 2
Consider the factors such as time efficiency, the signal to noise ratio of instrument, the scanning times that sample determines are set to 64 times.
The selection of 5 relaxation delay times
Different relaxation delay times (1,2,5,10s) influence to peak signal to noise ratio at δ 5.29, the results are shown in Table 3.
The influence of the relaxation delay time of table 3
The relaxation delay time is little to spectral peak SNR influence at δ 5.29, the time efficiency of instrument test is considered, by relaxation
Time delay is set as 1s.
Select NMR testing conditions for：1H NMR resonant frequency is 400MHz, and sampling parameter is as follows：Pulse train
(PULPROG) zgcppr, sampling number (TD) 64k, scanning times (NS) 64, sky sweep number (DS) 4, spectrum width (SWH)
8012.8Hz, pulse angle (PW) 45, sampling time (AQ) 3.7s, reception gain (RG) 10, test temperature (TE) 303K, relaxation
Time delay (D1)=1.0s, window function linewidth factor (Lb) 1Hz.
The standard spectrogram of the mannosan poly saccharide peptide standard product of embodiment 3
The NMR testing conditions of reference embodiment 2, mannosan poly saccharide peptide standard product1HNMR is as shown in figure 1, Spectral Signal master
Concentrate between 3.20~5.80ppm of δ, be divided into two regions：1. anomer hydrogen area, 5.80~4.40ppm；②Huan protons area, δ
4.40~3.20ppm.Wherein, δ 5.29；δ5.14；δ 5.12, δ 5.08；δ5.04；δ 4.90 is respectively 1,2-；1,3-；1,2,6-；
1- connects the anomer hydrogen signal of mannose residue with 1,6-.Glucose and amino acid do not show obvious core because content is less
Magnetic signal.
The method of embodiment 4 specificity
For the specificity of verification method, the Portugal sweet dew similar with mannatide composition is determined under the same conditions and is gathered
The nuclear magnetic spectrogram (Fig. 5) of sugar and galactomannans.The monose composition of glucomannans is similar with mannatide, and by Portugal
Two kinds of monose compositions of grape sugar and mannose, but connected mode is presently considered to be and passes through β-Isosorbide-5-Nitrae-pyranose by glucose and mannose
The heteroglycan that glycosidic bond combines, there is the branched structure closed by β -1,3 bonds on main chain mannose C3 positions.Galactomannan gathers
Sugar is the heteroglycan being made up of galactolipin and mannose, similar with mannatide, and sugar chain main chain is also to be made up of mannose, but
Its structure is on the main chain being made up of β-(1-4)-D-MANNOSE, and single D- galactolipins are connected with by α-(1-6)-glycosidic bond
Branch.From fig. 5, it can be seen that due to the difference of connected mode, cause the signal and sweet dew in two kinds of mannosan anomer hydrogen regions
Glycan peptide signal is widely different, and both are (remote with the correlation coefficient ρ only -0.280 1, -0.157 9 of mannosan poly saccharide peptide standard product
Much smaller than 0.95), it is seen then that this method has very strong specificity.
In addition, under the conditions of same measured, nuclear-magnetism measure has been carried out to commercially available other several polysaccharide and glycopeptide class product
(Fig. 6).As a result show, several samples anomer hydrogen region peak type and peak shift value with mannatide reference substance spectrogram difference more
Greatly, Flos Mume Thick many candies, polysaccharide-peptide, the spectrogram of Radix Isatidis glycopeptide and the correlation coefficient ρ of mannatide reference substance spectrogram point
Not Zhi You 0.560 4,0.045 7, -0.039 8 (far smaller than 0.95), it is very high special to further illustrate that this method has
Property.
The method of embodiment 5 repeatability
Mannatide need testing solution spectrogram such as Fig. 7 of three parts of parallel preparations, it can be seen that three each spies of sample spectrogram
Sign chemical shift of proton is consistent with peak type, and the coefficient correlation between reference substance is respectively 0.990 9,0.991 3,0.997
6, it is seen that this method has preferably repeatability.
The day to day precision of embodiment 6
Same mannatide test sample determines under the not same date same time, as a result such as Fig. 8.Each chromatogram characteristic proton
Chemical shift is consistent with peak type, and the coefficient correlation between mannatide reference substance is respectively 0.991 1,0.994 8,
0.990 5, it is seen that this method has higher day to day precision.
The measure of the hydrogen nuclear magnetic resonance spectrogram similarity of embodiment 7
Using the 1H-NMR spectrums of mannosan poly saccharide peptide standard product as standard spectrum, δ (4.89~5.90) ppm anomer hydrogens area is selected to make
Identify that area is used as the spectral regions of CHARACTERISTICS IDENTIFICATION for " fingerprint ".
Circular is as follows：The 1H-NMR modal datas of reference substance and test sample are converted into the XY numbers of ASCII forms
According to file (wherein X=chemical shifts, Y=signal intensities), using Microsoft EXCEL softwares to analyze selected spectral regions
Data, by the Y-axis coordinate of spectrum file compared with the Y-axis coordinate of standard diagram, calculate correlation coefficient ρ.If calculate
The sample collection of illustrative plates of gained is consistent with standard diagram peak type and peak shift value, and coefficient correlation is more than 0.95 between the two, then can recognize
It is consistent with control sample structure for the sample of identification；Conversely, test sample and reference substance structure are inconsistent.Spectrogram correlation result
Such as table 4.
From fig. 9, it can be seen that the nuclear magnetic spectrogram of the mannatide test sample of 9 different batches and reference substance spectrogram peak type
And peak shift value is almost completely the same, coefficient correlation is all higher than 0.96, illustrates that this method can be with Qualitive test mannatide sample
Product.
Correlation coefficient process and co sinus vector included angle method are used equally for calculating spectrogram similarity, but result of calculation difference.
Calculated using Cosin method, the mannatide raw material spectrogram and mannosan poly saccharide peptide standard product spectrogram similarity of different batches are equal
More than 0.95, the similarity of chitosan oligosaccharide, Flos Mume polysaccharide, Radix Isatidis glycopeptide and polysaccharide-peptide and mannatide standard spectrogram is then
Respectively：0.7578th, 0.8398,0.6761 and 0.6641.Calculated using correlation coefficient process, the mannosan peptide former of different batches
Material spectrogram and mannosan poly saccharide peptide standard product spectrogram similarity are all higher than 0.95, and chitosan oligosaccharide, Flos Mume polysaccharide, Radix Isatidis glycopeptide and
Polysaccharide-peptide and the similarity of mannatide standard spectrogram are respectively：0.1016th, 0.5604, -0.0398 and 0.0457.Compare
Under, there is preferably specificity using correlation coefficient process.
The similarity of the test sample of table 4 and reference substance spectrogram
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention
Enclose being defined of being defined by claims.
Claims (3)
- A kind of 1. method of nuclear magnetic resonance identification mannatide, it is characterised in that by the hydrogen nuclear magnetic resonance spectrogram of testing sample Compared with the peak type and spectral peak shift value of the hydrogen nuclear magnetic resonance spectrogram of mannosan poly saccharide peptide standard product, be used as specificity in selected spectrogram The spectral regions of identification, and calculate the similarity of selected spectral regions；Mannatide sample is similar to mannosan poly saccharide peptide standard product Spend ρ>0.95；The spectral regions as specificity identification, it is δ 4.89ppm~δ 5.90ppm anomer hydrogen area；Test sample is treated in acquisition The step of hydrogen nuclear magnetic resonance spectrograms of product and the hydrogen nuclear magnetic resonance spectrogram of mannosan poly saccharide peptide standard product, includes：(1) preparation of sample：Mannosan poly saccharide peptide standard product and each 30mg of testing sample accurately are weighed, is dissolved in 0.5mL D2It is ultrasonic to completely molten in O solution Solution, 10 μ L 0.2% TSP heavy aqueous solutions are added, ultrasonic mixing is uniform, is transferred in 5mm nuclear magnetic tube；(2) NMR is determined：1H NMR resonant frequency is 400MHz, and sampling parameter is as follows：Pulse train is zgcppr, sampling number 64k, is scanned Number is 64, and it is 4 that sky, which sweeps number, spectrum width 8012.8Hz, pulse angle 45, sampling time 3.7s, reception gain 10, Test temperature is 303K, and the relaxation delay time is 1.0s, and window function linewidth factor is 1Hz.
- 2. according to the method for claim 1, it is characterised in that δ 5.29, δ 5.14, δ 5.04, δ 4.90 respectively correspond to 1,2-, 1,3-, 1- connect the anomer hydrogen signal of mannose residue with 1,6-；δ 5.12 connects mannose residue with the corresponding 1,2,6- of δ 5.08 Anomer hydrogen signal.
- 3. according to the method for claim 1, it is characterised in that the similarity ρ is calculated using correlation coefficient process.
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