CN102539220B - Analysis method for metabolin of A-type proanthocyanidins - Google Patents
Analysis method for metabolin of A-type proanthocyanidins Download PDFInfo
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- CN102539220B CN102539220B CN201210019913.9A CN201210019913A CN102539220B CN 102539220 B CN102539220 B CN 102539220B CN 201210019913 A CN201210019913 A CN 201210019913A CN 102539220 B CN102539220 B CN 102539220B
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Abstract
The invention discloses an analysis method for metabolin of A-type proanthocyanidins, which comprises the steps of collection of a biological sample, preparation of metabolin solution, detection of the metabolin and the like, wherein the preparation of the mtabolin solution comprises preparation of hydrolysate, preparation of crude extraction of the metabolin and purification of the metabolin crude extraction. The analysis method for the metabolin of the A-type proanthocyanidins is good in separating purification effect, efficient, fast, high in result accuracy and good in reproducibility, and not only facilitates understanding of a metabolic mechanism and the metabolin, but also plays an important role in further utilization of the A-type proanthocyanidins.
Description
Technical field
The present invention relates to a kind of analytical approach of A-type procyanidin metabolic product.
Background technology
Procyanidin (procyanidins, PC) be the general name of the extensive large class polyphenol compound existing in plantage, the procyanidin of occurring in nature is to be combined into by the catechin of varying number and epicatechin, because of monomer whose connected mode difference, can be divided into A-type (C2-O-C7 or C2-O-C5 connect) and two kinds, B-type (C4-C8 or C4-C6 are connected).A-type procyanidin has the antioxidation activity of stronger removing free radical, antibacterial, reducing blood lipid, inhibition atherosclerotic etc., has great development and utilization and is worth.A-type procyanidin enters blood through animal intestinal absorption in vivo, exists with monomer and the form of methylating thereof, and after arrival colon, is degraded by enteric microorganism, has formed multiple phenolic acid and derivant thereof and has discharged by urine.
At present, in pharmacokinetics research, often and external research method interior by animal body, discloses medicine dynamic rule in vivo, obtains the analytic metabolism parameter of medicine.The biological sample that biopharmaceutical analysis adopts comprises blood, urine, saliva, hair, organs and tissues, milk, seminal fluid, bile, gastric juice, lymph liquid, ight soil etc., wherein modal is serum and urine, can well embody the relation between drug concentration and therapeutic action.Target substance in biological sample must carry out separation and purification just can detect analysis, for medicine or its metabolin endogenous material in body be combined generate conjugate, before separation and purification, also needing is hydrolyzed makes it discharge.Determination method comprises high performance liquid chromatography (HPLC), Liquid Chromatography-Mass Spectrometry (HPLC-MS), vapor-phase chromatography (GC), ultraviolet spectrophotometry, fluorescence polarization immunoassay etc.
Up to the present, also there is not yet about the metabolic product of A-type procyanidin and the report of Methanogenesis method thereof both at home and abroad.The metabolic product of single flavanol compound material can directly utilize organic solvent to carry out extract and separate to enzymolysis product, then organic phase is detected, and this is current the most frequently used sample treatment.If but metabolic product component is comparatively complicated, when object content is less, will cause recovery of extraction low, object is interfered.A-type procyanidin complicated components, compared with single tested material, only utilizing organic solvent to extract can not be fully by metabolic product separation and purification, and is difficult for detecting quantitatively.
Summary of the invention
The object of this invention is to provide a kind of analytical approach of A-type procyanidin metabolic product.
The object of the invention is to be achieved through the following technical solutions:
A kind of analytical approach of A-type procyanidin metabolic product, described analytical approach comprises collection, the preparation of A-type procyanidin metabolic product solution and the detection of A-type procyanidin metabolic product of biological sample, and the preparation of described A-type procyanidin metabolic product solution comprises the following steps:
(1) prepare hydrolysate: the pH value to 5.5 that regulates biological sample with the acetum of 0.2-1.0mmol/L, add GRD beta-glucuronidase, 37 DEG C of hydrolysis are after 1-3 hour, by the rear cessation reaction of hydrochloric acid solution adjusting pH value to 2 of 2-6mol/L, at 4 DEG C centrifugal 10 minutes, get supernatant, obtain hydrolysate;
(2) prepare metabolic product crude extract: hydrolysate is extracted with ethyl acetate, merges ester phase, Vacuum Concentration redissolves in methanol aqueous solution after removing ethyl acetate, obtains metabolic product crude extract;
(3) purifying of metabolic product crude extract: metabolic product crude extract is through C18 solid-phase extraction column adsorption and purification, clean with clear water, then use methanol-eluted fractions constant volume, obtain A-type procyanidin metabolic product solution.
Described biological sample is animal blood serum and/or urine.
Centrifugal described in step (1) is the high speed centrifugation of 10000-15000 rev/min.
The flow velocity of the methanol-eluted fractions described in step (3) is that 1-3ml/ divides.
Beneficial effect of the present invention:
1, because A-type procyanidin metabolic product component is comparatively complicated, the present invention adopts the method for Solid-Phase Extraction to carry out separation and purification to crude extract, pack mutually C18 filler into miniature pillar as fixing, when contain medicine biological sample upper prop time, owing to being subject to " absorption ", " distribution " or other effects, medicine is retained in fixingly to be gone up mutually, cleans with pure water, taking methyl alcohol as eluant, eluent wash-out, thereby reach the object of separation and purification.The method has effectively been avoided the emulsion of water and organic phase in liquid-liquid extraction, has reduced impurity and has introduced and environmental pollution.
2, the present invention can be used in the A-type procyanidin metabolic product of analyzing in multiple biological sample, and metabolic product after treatment is carried out to HPLC and HPLC-MS analysis, and result accuracy is high, favorable reproducibility.
3, analytical approach of the present invention is efficiently quick, not only contributes to understand metabolic mechanism and the metabolic product of A-type procyanidin, also for further utilizing A-type procyanidin to play an important role.
Brief description of the drawings
Fig. 1: the HPLC chromatogram of A-type procyanidin.
Fig. 2: the HPLC chromatogram before urine metabolism product Solid-Phase Extraction.
Fig. 3: the HPLC chromatogram after urine metabolism product Solid-Phase Extraction.
Fig. 4: the HPLC chromatogram after blood serum metabolic product Solid-Phase Extraction.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described:
Embodiment 1
An analytical approach for A-type procyanidin metabolic product, comprises the following steps:
1, the collection of biological sample
(1) grouping: get 15 of SD rats, male, body weight 200 ± 20g, is purchased from Tongji Medical College, Huazhong Science and Technology Univ.'s Experimental Animal Center.Experiment divides 3 groups, normal group, low dosage (150mg/kg) and high dose (300mg/kg) A-type procyanidin group, every group of 5 rats.
(2) administration: SD rat is raised after the week at SPF level Animal House, started experiment.Cryodesiccated A-type procyanidin powder 150mg, 300mg are dissolved in 10ml physiological saline and are made into suspension, give low dosage and high dose group rat oral gavage, normal group directly substitutes with physiological saline.Before gavage, rat fasting feedwater 12-18h, starts gavage at 4 in afternoon, after gavage, rat is placed in to metabolic cage, collects the urine of discharging in its 18h ,-80 DEG C of freezing preservations before analysis.
(3) blood sampling: cryodesiccated A-type procyanidin powder 300mg is dissolved in 10ml physiological saline and is made into suspension, and normal group is with physiologic saline for substitute.Rat fasting feedwater 12-18h before gavage, in 4 gavages in afternoon, after gavage 1h, to the local anaesthesia of rats by intraperitoneal injection chloral hydrate, neck dislocation is put to death, and extracts blood from rats'liver portal vein, at 4 DEG C, high speed centrifugation 10min gets supernatant ,-80 DEG C of freezing preservations before analysis.
2, the preparation of metabolic product:
(1) prepare hydrolysate: the pH value to 5.5 that regulates biological sample with the acetum of 0.2-1.0mmol/L, add GRD beta-glucuronidase, 37 DEG C of hydrolysis are after 1-3 hour, by the rear cessation reaction of hydrochloric acid solution adjusting pH value to 2 of 2-6mol/L, at 4 DEG C centrifugal 10 minutes, get supernatant, obtain hydrolysate;
(2) prepare metabolic product crude extract: hydrolysate is extracted with ethyl acetate, merges ester phase, Vacuum Concentration redissolves in methanol aqueous solution after removing ethyl acetate, obtains metabolic product crude extract;
(3) purifying of metabolic product crude extract: metabolic product crude extract is through C18 solid-phase extraction column adsorption and purification, clean with a small amount of clear water, then use methanol-eluted fractions constant volume, obtain A-type procyanidin metabolic product solution.
Described GRD beta-glucuronidase is Type H-5 type, in every 100 microlitre serum, adds 200-500U, in every milliliter of urine, adds 0.5-1.5KU to be hydrolyzed.
3, the detection of metabolic product
(1) HPLC of metabolic product detects
Chromatographic column: ZORBAX Eclipse XDB-C
18(150mm × 4.6mm, 5 μ are m); Column temperature: 28 DEG C; Detect wavelength: 280nm; Flow velocity: 1mL/min; Mobile phase: A is 0.4% aqueous acetic acid, and B is acetonitrile.Gradient: 0-40min, 5-35%B; 40-45min, 35-50%B; 45-50min, 50-80%B; 50-55min, 80-5%B, column equilibration 10min, sample size 20 μ l.
(2) HPLC-MS of metabolic product detects
Liquid phase chromatogram condition is the same, and mass spectrum is selected negative ion mode, and electron spray air pressure is 20psi, and dry gas flow velocity is 7.0L/min, and baking temperature is 325 DEG C.Ion scan scope: 50-1200m/z, pays close attention to the small-molecule substance of molecular weight 300 left and right, carries out two-stage crushing simultaneously.
(3) HPLC of A-type procyanidin medicine detects
Rat oral gavage medicine A-type procyanidin is carried out to HPLC analysis, and chromatographic condition is with the detection of metabolin, sample size 10 μ l.
The analysis result of A-type procyanidin metabolic product:
The metabolic product of A-type procyanidin in urine and blood, detect and analyze through HPLC and HPLC-MS: after the metabolism of A-type procyanidin mainly with 3-hydroxyl phenylacetic acid, 3-hydroxy phenylpropionic acid, 4-hydroxy phenylpropionic acid, p-coumaric acid, m-coumaric acid, caffeic acid, vanillic acid, the form of the little molecule phenolic acid such as shikimic acid and derivant thereof is discharged by urine; And the material that only finds that there is a small amount of procyanidin monomers and the form that methylates thereof in serum exists.
1, the HPLC of urine metabolism product analyzes
Due to the metabolic product more complicated of A-type procyanidin, as shown in Figure 2, do not pass through the urine metabolism product of solid-phase extraction column purifying, can not effectively separate by C18 chromatographic column, conditions of streaking is very serious, cannot carry out interpretation of mass spectra to product wherein.After C18 post adsorption and purification, from Fig. 3, obviously can see, occur that more than 10 plant material between 10-35min, wherein major part all has maximum absorption band in 277nm left and right, and it belongs to flavanol compound material deducibility.The detectability of greatly having optimized metabolic product after purifying is described, and accuracy is higher.
2, the qualification of metabolic product in urine
Utilize HPLC-MS to analyze the metabolic product of A-type procyanidin in urine, by with gavage former state (as shown in Figure 1) and with the comparing of phenolic acid standard items, find that A-type procyanidin has produced multiple phenolic acid and derivant thereof through metabolism, the procyanidin that a part is not absorbed and used excretes by urine with its parent or methylated form.Catechin, epicatechin, A2 and the tripolymer { epicatechin-(4 β → 8,2 β → O → 7)-epicatechin-(4 β → 8)-epicatechin } and the derivant etc. thereof that wherein exist are identified especially, in table 1.The phenolic acid of having identified at present comprises: 3-hydroxyl phenylacetic acid, 3-hydroxy phenylpropionic acid, 4-hydroxy phenylpropionic acid, p-coumaric acid, m-coumaric acid, caffeic acid, vanillic acid and shikimic acid.After repeatedly repeating, result reappearance is high.Proceed quantitative test, can obtain the discharge rate of each metabolin.
3, the metabolic product of procyanidin in blood
From Fig. 4, be not difficult to find out, in rat blood serum, contain a certain amount of epicatechin monomers (table 2), can be by calculating its concentration, and as evaluating A-type procyanidin a kind of means of absorptivity in vivo.
The Methanogenesis of A-type procyanidin in table 1 rat urine
The Methanogenesis of A-type procyanidin in table 2 rat blood serum
Claims (4)
1. the analytical approach of an A-type procyanidin metabolic product, it is characterized in that described analytical approach comprises collection, the preparation of A-type procyanidin metabolic product solution and the detection of A-type procyanidin metabolic product of biological sample, the preparation of described A-type procyanidin metabolic product solution comprises the following steps:
(1) prepare hydrolysate: the pH value to 5.5 that regulates biological sample with the acetum of 0.2-1.0mmol/L, add GRD beta-glucuronidase, 37 DEG C of hydrolysis are after 1-3 hour, by the rear cessation reaction of hydrochloric acid solution adjusting pH value to 2 of 2-6mol/L, at 4 DEG C centrifugal 10 minutes, get supernatant, obtain hydrolysate;
(2) prepare metabolic product crude extract: hydrolysate is extracted with ethyl acetate, merges ester phase, Vacuum Concentration redissolves in methanol aqueous solution after removing ethyl acetate, obtains metabolic product crude extract;
(3) purifying of metabolic product crude extract: metabolic product crude extract is through C18 solid-phase extraction column adsorption and purification, clean with clear water, then use methanol-eluted fractions constant volume, obtain A-type procyanidin metabolic product solution.
2. the analytical approach of A-type procyanidin metabolic product as claimed in claim 1, is characterized in that described biological sample is animal blood serum or urine.
3. the analytical approach of A-type procyanidin metabolic product as claimed in claim 1, is characterized in that the centrifugal high speed centrifugation for 10000-15000 rev/min described in step (1).
4. the analytical approach of A-type procyanidin metabolic product as claimed in claim 1, the flow velocity that it is characterized in that the methanol-eluted fractions described in step (3) is that 1-3ml/ divides.
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