CN106124607B - The new method of saccharide complex content in a kind of measurement biological sample - Google Patents
The new method of saccharide complex content in a kind of measurement biological sample Download PDFInfo
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Abstract
Due to lacking sensitive detection method applied widely, sugared compound detection and tracing method in the biological sample has become the bottleneck problem in its pharmacokinetic.The present invention relates to include the content assaying method of glycoprotein, polysaccharide, glycolipid.After iodo derivative by preparing saccharide complex, the content that its iodine in the biological sample is measured with ICP-MS calculates the content of determination sample kind saccharide complex further according to the content of iodine in saccharide complex, is integrated innovation method.This method high sensitivity, result be accurate, easy to operate, "dead" pollution, applied widely.
Description
Technical field
That the present invention relates to saccharide complexes (polysaccharide, glycoprotein, glycolipid) is simple, sensitive, accurate in the biological sample, without radiation
Contact scar, detection applied widely and tracing method.Belong to Pharmaceutical Analysis technical field.
Background technology
Saccharide complex is the important large biological molecule of the another class in organism other than isolating protein and nucleic acid, is had a variety of
Various biological function, the alienation biological metabolism toxicity that it has almost without micromolecular compound, use is safer, and
The interaction of other drugs is fewer.Have due to its These characteristics and to many diseases for lacking medicine at present good
Foreground, it has also become extremely valued a kind of compound in life science and new drug development.But very due to its structure
Complexity isolates and purifies difficulty, lacks simple, sensitive detection method, the research of bioavilability and pharmacokinetics is very
Difficulty seriously constrains its research and development.
Bioavilability and pharmacokinetics are by principle of dynamics to the absorption of drug, distribution, metabolism and exclusion
(ADME) process is studied.It is the edge for integrating the subjects such as pharmacological exploitation, pharmaceutical chemistry, analytical chemistry, mathematics
Section.It, which carries, illustrates mechanism of drug action and scientific meaning；Design and preferred dosage regimen；Promote new drug development dosage form preferred
And quality control；In the research and development of drug, play an important role in and position.
The bioavilability of saccharide complex and the research relative difficulty of pharmacokinetics, main cause, which is the absence of, preferably to exist
Detection method in biological sample.The detection method in saccharide complex biological sample mainly has at present：The same position of chromatography, radioactivity
Plain labelling method, fluorimetry and bioassary method.The characteristics of being metabolized due to carbohydrate structure and its in vivo, sample and metabolism
Product is one group of structure and the approximate compound of molecular weight.Chromatography, bioanalysis, fluorescent marker method are to medicine generation of saccharide compound
Dynamics research, or because detection method sensitivity is not high, interferes by force or because biological sample processing is difficult, is influenced on measurement result
The reasons such as serious make its application be severely limited.Isotope-labelling method is applied more, and the high sensitivity of this method is applicable in
Range is wide, and application is more.But this method is harsh and of high cost to experiment condition requirement, easily causes radioactive pollution.Find and
Simply and easily tracer and assay method are established, is the pass in saccharide complex pharmacokinetics, including in bioavailability study
One of key scientific problems urgently to be resolved hurrily in key technology and saccharide complex research.
Because it contains amido or tyrosine with iodine substitution reaction can occur for glycoprotein, compound for the sugar without containing amido
Object after fluorescein derivative that can be by preparing it, then carries out iodide reaction, generate it is stable, carbohydrate structure is influenced it is smaller
Iodo compound.It is multiple to sugar with the radioactive isotope of iodine in the pharmacokinetic that isotope-labelling method carries out sugar
It is method that is classical and being applicable in close object and be marked.
Inductivity coupled plasma mass spectrometry (Inductively coupled plasma mass spectrometry,
ICP-MS), it is new analysis and testing technology that developed recently gets up.It can not only replace traditional inorganic analysis technology such as electricity
Feel coupled plasma spectroscopy technology, sampling Graphite Furnace Atomic Absorption etc., carries out the accurate survey of qualitative and quantitative analysis and isotopic ratio
Amount etc., the also detectable above method are difficult to the element detected, such as halogen；Simultaneously it can also with other technologies such as HPLC,
HPCE, GC are combined the analysis of the form into row element, distribution character etc., have the characteristics that high sensitivity, result are stablized.By iodo
Polysaccharide is combined with ICP-MS detection techniques, integrated innovation, is expected to establish a kind of new simple, convenient, and reliable, applied widely
Saccharide complex tracer and assay method.
The present invention found no before completing close iodo saccharide complex be combined with ICP-MS measure it is sugared compound in biological sample
The report of object content, the present invention are with a wide range of applications.
Invention content
An object of the present invention is the difference for preparing iodo saccharide complex according to saccharide complex chemical property, is carried out not
Same substitution reaction
One, it for containing tyrosine saccharide complex, directly by chemical reaction, is connect with iodine with covalent bond, generates iodo
Saccharide complex.The reaction can be catalyzed by toluene-sodium-sulfonchloramide, directly carry out the substitution reaction of iodine, generate iodo polysaccharide
Two, the ingredient of substitution reaction can directly be occurred with iodine by not contained to polysaccharide, glycolipid class, prepare itself and fluorescein first
Derivative, then by iodine and luciferin reaction, prepare iodo- fluorescein-polysaccharide compound.
The second object of the present invention is to inductivity coupled plasma mass spectrometry (Inductively coupled plasma
Mass spectrometry, ICP-MS) measure iodo saccharide complex content.
ICP-MS is the new analysis and testing technology that developed recently gets up.It can not only replace traditional inorganic analysis skill
Art such as inductively coupled plasma spectrometry technology, sampling Graphite Furnace Atomic Absorption etc., progress qualitative and quantitative analysis and isotopic ratio
Accurate measurement etc., the also detectable above method are difficult to the element detected, such as halogen；It can also be with other technologies such as simultaneously
HPLC, HPCE, GC are combined the analysis of the form into row element, distribution character etc., have the characteristics that high sensitivity, result are stablized.
The present invention is the content of iodine according to measurement, calculates the content of saccharide complex in each sample.By iodo polysaccharide and ICP-
MS detection techniques are combined, and establish in new biological sample the method for measuring polyoses content, are integrated innovation, and this method is letter
It is single, conveniently, the tracer of saccharide complex reliable, applied widely and assay method, have tangible outstanding feature and significant
Technological progress.
Concrete operations are：
1, a kind of new method measuring polyoses content in biological sample, the method are：
(1) contain free amino in glycoprotein, under the catalysis of catalyst, the hydrogen in free amino can be replaced by iodine, raw
At iodo polysaccharide, concrete operation method is：Glycoprotein sample 0.2g is taken, is dissolved with water 5ml, using toluene-sodium-sulfonchloramide as catalyst, is added dense
Degree is the potassium iodide iodine 1ml of 0.5mol/L, carries out iodide reaction, and reaction product is purified through gel filtration chromatography, collects glycoprotein
Compound part, freeze-drying obtain iodo glycoprotein；
(2) for polysaccharide, glycolipid without containing free amine group, the derivative of they and fluorescein can be prepared first, then with
Iodine carries out substitution reaction, and concrete operation method is：Sample 1g, dissolving dimethyl sulfoxide (DMSO) is taken to add pyridine 10 to drip in 10ml, be added
Fluorescein 0.1g, adds dibutyl tin laurate 20mg, and 95 DEG C of Hybrid Heatings 2 hours are added absolute ethyl alcohol 50mL, put
It sets, incline supernatant, is cleaned repeatedly with ethyl alcohol, washes away extra dyestuff, and centrifugation obtains fluorescein-polysaccharide compound, which presses
Iodide reaction is carried out according to described in step (1), iodo polysaccharide or iodo glycolipid is made；
(3) by the iodo product of the saccharide complex prepared by step (1) or (2), animal or cell experiment are carried out, is collected
The sample that need to be measured is converted into the content of iodine in ICP-MS determination samples according to the labelled amount of iodine in sample after digestion
The content of saccharide complex in each sample.
The particular content of assay method of the present invention is described in detail by taking white fungus glycoprotein as an example below.Due to without commercially available
Sample is available, we need to make white fungus glycoprotein by oneself, the sample as research.
Saccharide complex includes glycoprotein, polysaccharide and glycolipid.Glycoprotein contains a certain amount of tyrosine, can directly be sent out with iodine
Raw substitution reaction；Polysaccharide and glycolipid are since without containing can be with the group that iodine directly reacts, need to be made can replace with iodine
After the derivative of reaction, iodo derivative is generated with Iod R.The iodo saccharide complex of generation, after measuring content of iodine, into action
Object or cell experiment prepare the biological sample of needs；After resolution, the content of wherein iodine is measured with ICP-MS, according in sample
Content of iodine calculates the content of saccharide complex in sample.
The preparation of one, iodos glycoprotein-
1. our previous studies that prepare of white fungus glycoprotein show using method appropriate, can white fungus be prepared by white fungus
Glycoprotein solves the problems, such as no suitable commercial product reagent.Therefore glycoprotein with tremella polysaccharides compound be represent preparation methods as:
One kilogram of Tremella fuciformis spores polysaccharide is taken, 5 times of amount pure water are added, is heated up to 70 DEG C, stirs 2 hours, centrifuge (3000rpm, 10min.), collect
Supernatant is evaporated under reduced pressure to 700ml, and stirring is lower to add absolute ethyl alcohol 4000ml, is centrifuged after standing overnight, and collects sediment fraction, cold
It is lyophilized dry.Crude white fungus glycoprotein collects the part of molecular weight 10,000 through pressing gel filtration chromatography in Sephadex G-100, concentration,
Freeze-drying, obtains white fungus glycoprotein.
2. total reducing sugar, uronic acid, protein are respectively with phenol-sulphur in the sample after the analysis of white fungus glycoprotein sample is dry
Acid system, hydroxyl diphenol method, Lowery methods measure, and mannose, glucuronic acid and bovine serum albumin(BSA) are standard items, the results showed that
Wherein the content of total reducing sugar is 80.2%, glucuronic acid content 6.5%, protein content 7.8%；Molecular weight distribution is with HPGPC
Method measures, using serial dextran as molecular weight reference substance, OHPAK-SB804HQ chromatographic columns, and RID-10A detectors, mobile phase
It for 0.7% metabisulfite solution, is as a result calculated by GPC softwares, the peak molecular weight of tremella polysaccharides compound is 10.23KDa；
Composition glycan analysis determines sample using PMP derivatization methods according to the retention time and peak area of each standard monosaccharide and analyte derivative object
Sugar composed type and ratio in product, the results showed that, sugared part is by xylose, mannose, grape alditol in the polysaccharide compound
Acid and glucose group at；Protein part is by 9 kinds of amino acid groups such as arginine, tyrosine, alanine, tryptophan, phenylalanine
At.Analysis result shows that white fungus glycoprotein obtained meets the requirements.
3. the iodo sample 500mg of white fungus glycoprotein sample is dissolved with 5ml pure water, NaI solution (0.5mol/L) is added
3ml is mixed well, and toluene-sodium-sulfonchloramide solution (0.1g/ml) 3ml is added, and mixes well rear concussion reaction 1min, is added and is laid particular stress on sulfurous acid
Potassium iodide 5ml (0.1g/ml) is added after mixing, mixes well by sodium 3ml (0.1g/ml), after centrifugation (5000r/min, 10min)
Supernatant is taken, is freeze-dried, obtains iodo white fungus glycoprotein.
4. isolating and purifying for the purifying iodo white fungus glycoprotein of iodo white fungus glycoprotein is enterprising in middle pressure gel permeation chrommatograph
Row.Iodo white fungus glycoprotein 200mg is loaded to Sephadex G-50 gel chromatographic columns (40cm x5cm) after being dissolved with pure water,
With water elution, eluent (10ml/ pipes) is collected, beginning and end acquisition time is determined according to the interior void volume of the chromatographic column.
The sugared content in each pipe is measured with phend-sulphuric acid, using sugared content as ordinate, pipe number is mapped for abscissa, according to elution profile
(see Fig. 1), collects 10-25 pipes, and concentration, freeze-drying obtain purifying iodo white fungus glycoprotein.
5. the analysis of iodo white fungus glycoprotein
Spectrum analysis has been carried out by iodo white fungus glycoprotein made from iodine substitution reaction.Before ultraviolet spectra shows iodo
Its absorption maximum does not change in 234nm afterwards.Infrared spectrum shows to be substantially change before and after iodo, after iodo most
It is absorbed as greatly:3363,3267,1631,1527,1396,1303,1087,902,810,624,536cm-1, and before iodo, white fungus
The absorption maximum of glycoprotein is:3309,2931,1462,1381,1319,1261,1084,1022,729,624cm-1, produces
Apparent halogenated alkane peak (536cm-1), show that iodine is keyed to by chemistry in sugar.Iodo white fungus glycoprotein passes through ICP-MS
It is 0.55% to measure its iodo rate, is further demonstrated that, iodide reaction success.
6. iodo glycoprotein stability study
Whether the saccharide complex after iodine labeling is stablized when carrying out animal, cell experiment, is studied with following experiments：
Iodo white fungus glycoprotein 10mg is taken, is dissolved in 10ml cell culture fluids (), is placed in 37C0 constant temperature oscillations, exists respectively
It draws 2ml within 1,2,3 hour, gel filtration chromatography (30cm X 1cm) is carried out by Sephadex G-50, water elution to receive automatically
Storage collects (1ml/ pipes), and content of iodine therein is measured with ICP-MS pipes, the results showed that, iodo white fungus glycoprotein and through cultivating
The chromatography curve of 1,2,3 hour iodo white fungus glycoprotein is essentially identical, is nearby not detected in the flow point (Ve) of small-molecular-weight
Free iodine shows that the iodine in iodo white fungus glycoprotein does not crack, and iodo product is stablized.
The processing of two, samples is by the biological sample comminution after iodo saccharide complex sample or freeze-drying, in headspace sampling bottle
It is accurately weighed in (10ML) in right amount, 3ml TMAH and 0.1ml H is sequentially added after the wetting of 4ml ultra-pure waters is added2O2, mixing screws
Head cover, ultrasonication 30min at 80 DEG C, is settled to 25ml.With 0.45um membrane filtrations, filtrate carries out ICP-MS measurement.
The measurement of three, samples is measured in inductively coupled plasma mass spectrometry (ICP-MS) (Agilent JL-713-
IP001-YQ030 is carried out on)
The condition such as following table of ICP-MS settings：
The operating condition reference of 1 ICP-MS instruments of table
Four, measurement results
1 method detection limit and the range of linearity
1%TMAH solution prepares the NaI standard solution of 1mg/ml as solvent, be diluted to respectively 5ng/ml, 10ng/ml,
50ng/ml, 100ng/ml, 300ng/ml iodine standard working solution.The normal equation for measuring working curve (see Fig. 2) is y=
0.0075 ×+0.0232, R=0.9996, the range of linearity are wider.
2 method precisions
0.05g iodo white fungus glycoprotein accurately is weighed, sample handling processes are the same, replication 10 times, carry out precision
Experiment, experimental result see the table below (table 2), it is found that relative standard deviation (RSD) value that sample measures is 3.89% from result.
2 iodo white fungus glycoprotein precision measurement result (n=10) of table
3 method reproducibility
0.05g iodo white fungus glycoprotein accurately is weighed, totally 6 parts, every part of processing procedure is same, carries out method reproducibility experiment,
Experimental result is shown in Table 3, it is found that the relative standard deviation that sample measures is 4.5% from result.
3 iodo white fungus glycoprotein reproducibility measurement result of table
4 method stability
50mg iodo white fungus glycoprotein samples measured once after resolution every two hours, as a result such as table 4.Show at 8 hours
Interior sample is stablized.
4 iodo white fungus glycoprotein reproducibility measurement result of table
5. recovery of standard addition
This method carries out rate of recovery experiment by sample of iodo white fungus glycoprotein, accurately weighs iodo white fungus glycoprotein, with
Pure water dissolves, and totally 6 parts of 50mg/3.5ml solution is made, and every part is added 1mg/ml iodate sodium standard solution 0.5ml, and processing procedure is same
7, the dilute 1000 times of experimental results of filtrate are shown in Table 5, are calculated according to experimental result, recovery of standard addition between 92%-101%,
RSD values are 5.16%.
5 iodo white fungus glycoprotein recovery of standard addition measurement result of table
Five, white fungus glycoprotein content in the biological sample and distribution
The preparation method of 5.1 biological samples
SD rats are tested after adapting to environment 3 days.Fasting 12h, free water before blood sampling.This experiment is with iodo white fungus
Glycoprotein is sample, is divided into experimental group and blank control group, every group of 3 rats, and experimental group, which is given, gives iodo white fungus glycoprotein, empty
White group gives physiological saline, and dosage is 0.2g/, and only for 12m L/, gastric infusion, it is eyeball to take blood mode to administration capacity
Take blood and abdominal aortic blood；It is rat left eye that 1h, which takes blood point, and it is rat right eye that 2h, which takes blood point, and it is rat abdomen active that 3h, which takes blood point,
Arteries and veins, every rat take blood totally 3 times, by blood 0.5ml in addition to last 10 times of dilution, after the same of other processing procedures draws medicine 3h
The rat heart, liver, spleen, lung, kidney distinguish vacuum freeze drying, weigh, and grinding is uniform, weighs sample 100mg, need not except last filtrate
Dilution is outer, other processing procedures are the same.
5.2 experimental result
5.2.1 white fungus glycoprotein Content measurement result in different time points rat blood
Under normal circumstances, the 7.4% of the blood volume percentage of liveweight of rat, rat average weight 190g, therefore whole blood in rat body
Amount calculates white fungus glycoprotein Content in a rat whole blood with 12.6ml.It is visible (table 6) by result, with the time after administration
Increase, is absorbed into blood volume and is also increasing, after 3 hours, nearly 5% white fungus glycoprotein is deposited in blood.
Tremella polysaccharides content in 6 different time points rat whole blood of table
5.2.2 after iodo white fungus glycoprotein is administered 3 hours, content results are shown in Table 7 in rat main organs；
Amount containing iodo white fungus glycoprotein in 7 viscera in rats of table
By measurement result as it can be seen that this method high sensitivity, as a result stablizes.
Iodo polysaccharide is combined by the present invention with ICP-MS detection techniques, is established in new biological sample and is measured polyoses content
Method, be integrated innovation, have tangible outstanding feature and significant technological progress.
Description of the drawings
The present invention is further described with embodiment below in conjunction with the accompanying drawings.
Fig. 1 is that iodo white fungus glycoprotein isolates and purifies elution profile；
Fig. 2 is method detection limit and range of linearity curve graph.
With reference to embodiment, invention is further described in detail.But following embodiments is only the letter of the present invention
Easy example, does not represent or limits the scope of the present invention, and protection scope of the present invention is subject to claims.
Specific implementation mode
For the present invention is better described, it is easy to understand technical scheme of the present invention, of the invention is typical but non-limiting
Embodiment is as follows.
The measurement of 1 white fungus glycoprotein of embodiment
Its method is identical with foregoing summary as result, and details are not described herein.
The measurement of 2 iodo polysaccharide of embodiment, glycolipid
The compound of many sugar does not contain tyrosine in natural products, substitution reaction cannot directly occur with iodine.Needing will
They connect with the group that substitution generation can occur with iodine first, then carry out the substitution reaction of iodine.
One, fluorescein (FITC) marks polysaccharide
Fluorescein can generate fluorescein-polysaccharide compound under alkaline condition with the generation substitution reaction in polysaccharide.This is multiple
It closes object in physiological conditions to stablize, is the common method of fluorescent marker polysaccharide.
1, polysaccharide sample
We select Polysaccharide in Pleurotus eryngii (PEPI) as sample.PEPI is that laboratory is homemade
One polysaccharide does not contain albumen.
2, fluorescein marks PEPI
Sample (1g) is dissolved in 10ml dimethyl sulfoxide (DMSO)s, adds 10 drop pyridines, and FITC0.1g is added, adds tin dilaurate
Dibutyl tin 20mg, 95 DEG C of Hybrid Heating 2h.Absolute ethyl alcohol 50ML. is added to place, incline supernatant, cleans repeatedly with ethyl alcohol, washes
Extra dyestuff is removed, centrifuges, obtains FITC-PEPI compounds.
3, the purifying of iodo fluorescein-PEPI and sample
The iodine substitution reaction method of fluorescein-PEPI is with the iodo method of glycoprotein, and details are not described herein.
Purification process of the purification process of iodo polysaccharide with iodo white fungus glycoprotein.Assay method with white fungus glycoprotein,
This is repeated no more.
The method of the processing of sample and the measurement of sample with tremella polysaccharides albumen, details are not described herein.
Two, Polysaccharide in Pleurotus eryngii content in the biological sample and distribution
1, the preparation method of biological sample
Experiment packet and dosage are the same as white fungus glycoprotein.
2, experimental result
Polysaccharide in Pleurotus eryngii assay result in 2.1 different time points rat bloods
Under normal circumstances, the 7.4% of the blood volume percentage of liveweight of rat, rat average weight 190g, therefore whole blood in rat body
Amount calculates white fungus glycoprotein Content in a rat whole blood with 12.6ml.It is visible (table 8) by result, when 1h, Polysaccharide in Pleurotus eryngii
Concentration in blood peaks, and with the increase of time after administration, is absorbed into oligemia.
Polysaccharide in Pleurotus eryngii content in 8 different time points rat whole blood of table
After 2.2 Polysaccharide in Pleurotus eryngii are administered 3 hours, content results are shown in Table 9 in rat main organs.
Amount containing iodo Polysaccharide in Pleurotus eryngii in 9 viscera in rats of table
Claims (1)
1. a kind of new method measuring polyoses content in biological sample, the new method are：
(1) for polysaccharide, glycolipid without containing free amine group, the derivative of they and fluorescein can be prepared first, then with iodine into
Row substitution reaction, concrete operation method are：Sample 1g, dissolving dimethyl sulfoxide (DMSO) is taken to add pyridine 10 to drip in 10ml, fluorescence is added
Plain 0.1g, adds dibutyl tin laurate 20mg, and 95 DEG C of Hybrid Heatings 2 hours are added absolute ethyl alcohol 50mL, place, incline
Supernatant is removed, is cleaned repeatedly with ethyl alcohol, extra dyestuff is washed away, centrifuges, obtains fluorescein-polysaccharide compound；
(2) fluorescein-polysaccharide compound sample 0.2g prepared by step (1) is taken, is dissolved with water 5ml, is catalysis with toluene-sodium-sulfonchloramide
The potassium iodide iodine 1ml of a concentration of 0.5mol/L is added in agent, carries out iodide reaction, and reaction product is purified through gel filtration chromatography, collects
Glycoprotein compound part, freeze-drying obtain iodo glycoprotein；
(3) by the iodo glycoprotein prepared by step (2), animal or cell experiment are carried out, collects the sample that need to be measured, through digestion
Afterwards, with the content of iodine in ICP-MS determination samples, according to the labelled amount of iodine in sample, it is converted into containing for saccharide complex in each sample
Amount.
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Non-Patent Citations (6)
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| ICP-MS对六味地黄多糖部位微量元素含量及其溶出特性的研究;胡军华等;《中国中药杂志》;20150228;649-653 * |
| Iodination of proteins, proteomes and antibodies with potassium triodide for LA-ICP-MS based proteomic analyses;Larissa Waentig.et al.;《Journal of Analytical Atomic Spectrometry》;20111231;1610–1618 * |
| Labelling of proteins by use of iodination and detection by ICP-MS;Norbert Jakubowski.et al.;《Journal of Analytical Atomic Spectrometry》;20080828;1487–1496 * |
| Magnetic quantitative analysis for multiplex glycoprotein with polymer-based elemental tags;Hanyong Peng.et al.;《Journal of Analytical Atomic Spectrometry》;20141231;1112－1119 * |
| Selective analysis of human serum albumin based on SEC-ICP-MS after labelling with iophenoxic acid;Julie Maria Dersch.et al.;《Analytical and Bioanalytical Chemistry》;20150204;2829–2836 * |
| 体积排阻色谱与电感耦合等离子体质谱联用技术在富硒金针菇硒多糖形态分析中的应用研究;铁梅等;《分析科学学报》;20071031;497-501 * |
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