CN114280182A

CN114280182A - HPLC-FLD detection method for homogeneous polysaccharides of radix pseudostellariae

Info

Publication number: CN114280182A
Application number: CN202111589715.1A
Authority: CN
Inventors: 胡娟; 庞文生; 杨斌; 栗园; 陈锦龙; 阚永军
Original assignee: FUJIAN COLLEGE OF TRADITIONAL CHINESE MEDICINE
Current assignee: FUJIAN COLLEGE OF TRADITIONAL CHINESE MEDICINE
Priority date: 2021-12-23
Filing date: 2021-12-23
Publication date: 2022-04-05

Abstract

The invention discloses an HPLC-FLD detection method of homogeneous radix pseudostellariae polysaccharide, which is characterized in that 2-aminobenzidine is used for carrying out fluorescence labeling on the homogeneous radix pseudostellariae polysaccharide, glucan is used as a standard substance, and the homogeneous radix pseudostellariae polysaccharide is quantitatively analyzed by HPLC-FLD and used for measuring the homogeneous radix pseudostellariae polysaccharide in a biological sample cell culture solution. The method is simple, sensitive and accurate, and has wide application prospect.

Description

HPLC-FLD detection method for homogeneous polysaccharides of radix pseudostellariae

Technical Field

The invention belongs to the technical field of polysaccharide fluorescence detection, and particularly relates to an HPLC-FLD detection method for homogeneous polysaccharides of radix pseudostellariae.

Background

Polysaccharide is one of three substances constituting life science and widely exists in traditional Chinese medicines. The polysaccharide has a plurality of biological activities of immunoregulation, anti-tumor, blood sugar reduction, blood fat reduction, oxidation resistance, aging resistance, virus resistance and the like, and pharmacological research is widely proved.

The difficulty of detection caused by the lack of chromophore (weak absorption under UV condition) of polysaccharide makes the research of saccharide molecules in the field of biochemistry be impossible, and the molecular form absorption, transportation and transmission mechanisms of polysaccharide in vivo are not clear.

The detection of the polysaccharide is generally carried out by a mass spectrum or a fluorescence detector after the fluorescence labeling of a reducing end, and all sugar chains with the semi-carboxyl aldehyde hydroxyl can realize the fluorescence derivatization through a reductive amination reaction. The commonly used fluorescent derivatization reagent 2-aminobenzidine (2-AB) has strong applicability and wide application, and the labeling kit is commercialized.

Disclosure of Invention

The invention aims to provide an HPLC-FLD detection method of heterophylly falsestarwort root homopolysaccharide, which is characterized in that 2-aminobenzidine is used for carrying out fluorescence labeling on the heterophylly falsestarwort root homopolysaccharide, and a high performance liquid chromatography-fluorescence detector (HPLC-FLD) detection method is established, is applied to the determination of biological sample cell culture solution, and is used for researching the absorption and transportation characteristics of the heterophylly falsestarwort root homopolysaccharide in Caco-2 cells.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for detecting homogeneous polysaccharide of radix Pseudostellariae by HPLC-FLD comprises performing fluorescence labeling on homogeneous polysaccharide of radix Pseudostellariae with 2-aminobenzidine, and quantitatively analyzing the homogeneous polysaccharide of radix Pseudostellariae by HPLC-FLD with dextran as standard.

The detection method is applied to a biological sample cell culture solution.

The invention has the beneficial effects that: 2-aminobenzidine is used for carrying out fluorescence labeling on heterophylly falsestarwort homogeneous polysaccharide, a high performance liquid chromatography-fluorescence detector (HPLC-FLD) detection method is established, and the method is applied to the determination of biological sample cell culture solution and the study of the absorption and transport characteristics of the biological sample cell culture solution in Caco-2 cells. The method is simple and accurate, and has wide application prospect.

Drawings

FIG. 1 is a thin layer chromatography identification of polysaccharide 2-ab. The left graph T-1 is radix Pseudostellariae homogeneous polysaccharide, T-2 is dextran control, and T-3 and T-4 are sodium borohydride and blank solvent; the middle panel shows the fluorescent spots of dextran T-500, T-70, T-40 and T-10 controls after 2-AB labeling; the right rectangle shows the fluorescent spots of the homopolysaccharide after 2-AB labeling, and the uppermost spot was the 2-AB fluorescent spot in each TCL plate.

FIG. 2 is the chemical structure of 2-AB derivatized polysaccharide. 2-AB dextran standard (A), and 2-AB radix Pseudostellariae homogeneous polysaccharide (B).

FIG. 3 is a linear plot of peak emergence time versus logM.

FIG. 4 is an HPLC chromatogram of 2-AB fluorescently labeled dextran standard and polysaccharide samples.

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.

Heterophylly falsestarwort root homogeneous polysaccharide (PHP) 2-AB fluorescent label

1.2-AB-PHPs preparation

350. mu.L of DMSO was dissolved in 350mg of TBAF, and 120. mu.L of Ac was added. Mixing, adding 5mg 0.5M polysaccharide, vortex for 30min, and dissolving at 65 deg.C for 1 hr until clear; adding 20mg of 2-AB, and mixing; 10mg of sodium cyanoborohydride are added and mixed and incubated for 4h at 65 ℃. The thin layer chromatogram is shown in FIG. 1, and the chemical structure is shown in FIG. 2A.

2.2-AB-Glucan preparation

DMSO was dispensed in 350. mu.L, and 150. mu.L of Ac was added and mixed. Dissolving 25mg of 2-AB by using the mixed solution, adding 30mg of sodium cyanoborohydride, uniformly mixing, adding 5mg of glucan, and incubating for 4 hours at 65 ℃; and (3) treating the marked polysaccharide: after the reaction is finished, adding 3.5mL of absolute ethyl alcohol for quenching, and stopping the reaction; 3000r/min, centrifugate for 10min, remove the supernatant, add 1mL distilled water, redissolve the polysaccharide. Repeating the above steps three times until 2-AB is not detected in the supernatant; the reaction product is lyophilized and then assayed. The thin layer chromatogram is shown in FIG. 1, and the chemical structure is shown in FIG. 2B.

Molecular weight determination of di, 2-AB labeled fluorescent polysaccharide

1. Chromatographic conditions

Shimadzu LC, UltA rahydrogel 1000 column, Shimadzu RF-10AXL detector; the mobile phase is 0.1mol/LNaNO₃An aqueous solution; the column temperature was 35 ℃; the flow rate is 0.5 ml/min; the amount of the sample was 20. mu.L.

Determination of molecular weight

Preparing 2-AB-PHPs into 240 mu g/mL solution; 2-AB-T-10 is prepared into a solution of 12 mu g/mL; 2-AB-T-40 was formulated as a 120. mu.g/mL solution, 2-AB-T-70, 2-AB-T-500 was formulated as a 240. mu.g/mL solution and subjected to exclusion chromatography.

The peak output time and molecular weight of the fluorescent marked uniform sample and the standard product, and the linear relationship graph of the peak output time and logM are respectively shown in Table 1 and FIG. 3. According to the formula logM ═ a + bRt, the average molecular weight of the homogeneous polysaccharide of pseudostellaria root labeled by two batches of 2-AB is 46355, which is determined by fluorescence gel chromatography.

Analysis of 2-AB-polysaccharide by HPLC-FLD

A240. mu.g/mL solution of polysaccharide-2-ab was prepared. A control solution was also prepared having a concentration of T40-2-AB of 240. mu.g/mL. T40 was chosen as a standard because of its molecular weight similarity to PHP and its higher sensitivity. Establishes HPLC-FLD quantitative determination of polysaccharide-2-AB. The mobile phase (sodium nitrate), T40 solution, did not fluoresce under uv light induction. Fluorescence characteristic peaks are respectively found in chromatogram maps of PHP-2-AB and PHP-2-AB, and retention time of the fluorescence characteristic peaks is 13.5min and 94.0 min. The quantitative analysis of PHP 0-2-AB was carried out with T40-2-AB as a control, and the detection limit of PHP-2-AB was 8.84. mu.g/mL, and the linear range was 29.45-683.28. mu.g/mL, as shown in FIG. 4.

Fourth, study of homogeneous polysaccharide of radix pseudostellariae in transport and absorption of Caco-2 cell model

Inoculating cell, counting the number of cells in the single cell suspension to 2 × 10⁵one/mL of the cell fluid was inoculated on the AP side of a Transwell polycarbonate membrane 24-well plate, 0.5mL of the medium was added to the AP side of the polycarbonate membrane per well, and 1mL of the medium was added to the BL side of the polycarbonate membrane per well until day 21.

The sample is fixed to the volume of 100, 200 and 400 mu by HBSS solutiong/mL. The TEER value was adjusted to 500. omega. cm²The left and right cell well culture solution is discarded, the preheated HBSS solution is added into the cell well for cleaning, the process is repeated for 2 times, and the balance is carried out for 30 min. HBSS solution was discarded, and sample solutions were added at concentrations of 0, 100, 200, 400. mu.g/mL, 3 wells per concentration, respectively. To the supply side, i.e., the AP side, 0.5mL of the sample solution was added. The amount of drug transported to the basal plane (BL) by absorption from the chorion plane (AP) was measured by adding 1mL of blank HBSS solution to the receptor side, i.e., BL side. The amount of drug transferred to the nap surface (AP) by absorption from the basal surface (BL) was measured by adding 1mL of the sample solution to the supply side, BL side, and 0.5mL of the blank HBSS solution to the receiving side, AP side. 100 mu L of the solution to be measured is taken out from the receiving test at 30min, 60 min and 120min respectively, and 100 mu L of the preheated HBSS solution is added into the receiving test. The solution to be tested was centrifuged at 1000rpm for 5min and the samples were stored at-80 ℃ for HPLC-FLD detection.

Under the same liquid phase condition, the concentration ratio of the fluorescent polysaccharide is consistent with the ratio of the liquid phase detection peak area, when one side added with the fluorescent polysaccharide is detected, the peak area at the moment represents the concentration added with the fluorescent polysaccharide, when one side not added with the fluorescent polysaccharide is detected, the fluorescence intensity is zero, the number of the polysaccharide penetrating through cells in a certain time period can be obtained by detecting the peak intensity of the BL surface after 30min, 60 min and 120min, and the transport capacity and the rule of cell transport can be obtained. The results are shown in Table 2.

As can be seen from Table 2, the bidirectional transport of PHP, P_APPThe value AP → BL is greater than BL → AP, indicating that the mode of transport for PHP is passive transport. Papp value greater than 1 × 10⁶It shows that the polysaccharide has strong permeability and absorption capacity to cells.

The detection method established by the invention can be used for measuring the homogeneous polysaccharide of the radix pseudostellariae in the cell culture solution.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. An HPLC-FLD detection method of radix pseudostellariae homogeneous polysaccharide is characterized in that: the homogeneous polysaccharide of the radix pseudostellariae is subjected to fluorescence labeling by using 2-aminobenzidine, and is quantitatively analyzed by using HPLC-FLD (high performance liquid chromatography-flash chromatography) by using glucan as a standard substance.

2. The method of claim 1, wherein: the chemical structural formula of the 2-aminobenzidine fluorescence labeled radix pseudostellariae homogeneous polysaccharide is as follows:

。

3. use of the assay of claim 1 in a biological sample cell culture.