CN-121994962-A - Method for quantitatively detecting plant polysaccharide in biological sample

CN121994962ACN 121994962 ACN121994962 ACN 121994962ACN-121994962-A

Abstract

The invention provides a method for quantitatively detecting plant polysaccharide in a biological sample, wherein the biological sample is rat plasma or rat tissue, the content of characteristic monosaccharide in the biological sample is quantitatively detected, and then the content of the plant polysaccharide in the biological sample is calculated according to the ratio of the characteristic monosaccharide in the plant polysaccharide, and the characteristic monosaccharide comprises rhamnose and arabinose. The method avoids the interference and structural damage caused by exogenous markers, has the excellent performances of high specificity, high sensitivity (LLOQ signal to noise ratio > 10:1), high accuracy (accuracy is 85-115%), high precision (RSD is < 15%), and the like, and the method is proved to be successfully applied to quantitative analysis of target characteristic monosaccharide content in rat plasma and various tissues of intravenous plant polysaccharide through comparison verification with a FITC labeling method, and is successfully applied to in vivo pharmacokinetics and tissue distribution research of hedysarum polysaccharide, thereby providing a key methodology tool for elucidating in vivo processes and pharmacological mechanisms of hedysarum polysaccharide.

Inventors

ZHAO LIANGGONG
SUN SONG
LIU XIAOHUA
Fu Hongyin
ZHAO LIBO
WANG FU
CHEN XINYUE

Assignees

连云港福瑞生物科技有限公司
兰州大学第二医院

Dates

Publication Date: 20260508
Application Date: 20260225

Claims (10)

1. A method for quantitatively detecting plant polysaccharide in a biological sample, wherein the biological sample is rat plasma or rat tissue, is characterized by quantitatively detecting the content of characteristic monosaccharide in the biological sample, and then calculating the content of the plant polysaccharide in the biological sample according to the ratio of the characteristic monosaccharide in the plant polysaccharide, wherein the characteristic monosaccharide comprises rhamnose and arabinose.
2. The method of claim 1, wherein the rat tissue comprises one of heart, liver, spleen, lung, kidney, thymus tissue, and/or The plant polysaccharide comprises one of hedysari polysaccharide, astragalus polysaccharide and honeysuckle polysaccharide, and/or The calculating method for calculating the content of the plant polysaccharide in the biological sample according to the ratio of the characteristic monosaccharide in the plant polysaccharide comprises the steps of calculating the content of the plant polysaccharide in the biological sample=the measured value (mg) of the characteristic monosaccharide in the biological sample/the mass fraction of the characteristic monosaccharide in the plant polysaccharide.
3. A method for quantitatively detecting plant polysaccharide in biological samples according to claim 1 or 2, wherein the quantitative detection of the content of characteristic monosaccharides in biological samples comprises the following steps: (1) Adding deionized water into biological sample, removing protein in plant polysaccharide by hot water precipitation method, collecting supernatant, and freeze drying; (2) Carrying out acid hydrolysis on the dried powder by trifluoroacetic acid so as to crack polysaccharide into monosaccharide; (3) Carrying out oximation and acetylation two-step derivatization reaction on the product 1 by taking phytic acid ester as an internal standard, dissolving the product in chloroform again to obtain a product 2 containing the internal standard and chloroform, filtering, and collecting filtrate; (4) Separating and detecting the filtrate by adopting a gas chromatography-hydrogen flame ionization detector, and obtaining the content of the characteristic monosaccharide in the biological sample according to a pre-established standard curve of the corresponding characteristic monosaccharide by detecting a chromatographic response signal of the characteristic monosaccharide; The retention time of rhamnose was 8.51 min, the retention time of arabinose was 8.78 min and the retention time of the internal standard was 15.10 min.
4. The method according to claim 3, wherein in step (1), the volume ratio of deionized water to biological sample is 5:1-1:5, preferably the volume ratio of deionized water to biological sample is 2:1, and/or The tissue concentration was 0.2 g/mL.
5. The method according to claim 3, wherein in the step (1), the hot water precipitation method is carried out under the specific conditions of 60-100 ℃, the stirring time is 5-30min, the rotating speed is 3-10rpm/min, and preferably, the hot water precipitation method is carried out under the specific conditions of 95+ -1 ℃, 10+ -1 min and the rotating speed is 10 rpm.
6. The method according to claim 3, wherein in the step (2), the specific conditions of the acid hydrolysis are that 1-5 mol/L trifluoroacetic acid solution is adopted, the temperature is 100-150 ℃ and the hydrolysis time is 1-5h; Preferably, 2 mol/L trifluoroacetic acid is used, the temperature is 120 ℃, and the hydrolysis time is 3h.
7. The method according to claim 3, wherein in the step (3), the product 1 is subjected to two-step derivatization reactions of oximation and acetylation in sequence by using the phytyl acetyl ester as an internal standard: Adding an internal standard solution, hydroxylamine hydrochloride and pyridine into the product 1, swirling, heating for oximation, cooling, adding acetic anhydride, and continuing heating reaction to complete acetylation; preferably, the reaction conditions of the oximation and the acetylation are 90+/-5 ℃ and 45+/-5 min.
8. The method according to claim 3, wherein in the step (4), the gas chromatography condition is that an HP-5 capillary column or an OV-101 quartz capillary chromatography column is adopted, the sample inlet temperature is 200-250 ℃, the detector is FID, and the detector temperature is 250-300 ℃; The temperature programming is adopted: The initial temperature was 175 ℃ without hold, raised to 210 ℃ at a rate of 15 ℃/min and held at 5min, raised to 260 ℃ at a rate of 5 ℃/min and held at 5min, total run time 21.5 minutes; Preferably, an OV-101 quartz capillary chromatographic column is used, the sample inlet temperature is 210 ℃, and the detector temperature is 260 ℃.
9. The method according to claim 1 to 8 for studying phytopolysaccharide pharmacokinetics or tissue distribution in a biological sample, which is rat plasma or rat tissue, Preferably, the rat tissue comprises one of heart, liver, spleen, lung, kidney, thymus tissue of the rat, and/or The plant polysaccharide comprises one of hedysarum polysaccharide, astragalus polysaccharide and honeysuckle polysaccharide.
10. A method for studying the pharmacokinetic or tissue distribution of plant polysaccharides in a biological sample, which is plasma of a rat or tissue of a rat, comprising quantitatively analyzing plant polysaccharides in a biological sample using the method according to any one of claims 1 to 8; preferably, the rat tissue comprises one of heart, liver, spleen, lung, kidney, thymus tissue of the rat, and/or The plant polysaccharide comprises one of hedysarum polysaccharide, astragalus polysaccharide and honeysuckle polysaccharide.

Description

Method for quantitatively detecting plant polysaccharide in biological sample Technical Field The invention belongs to the technical field of pharmaceutical analytical chemistry, in particular to a method for quantitatively detecting plant polysaccharide in a biological sample, and particularly relates to a method for quantitatively detecting the content of the plant polysaccharide in a rat biological sample by using characteristic monosaccharide as an endogenous tracer through gas chromatography. Background The plant polysaccharide is a natural biological macromolecule extracted from higher plants, algae and fungi, is formed by connecting more than ten monosaccharides through glycosidic bonds, and is an important active substance in the fields of traditional Chinese medicines and functional foods. The hedysarum polybasic polysaccharide, the honeysuckle polysaccharide, the astragalus polysaccharide and the like have proved to have various pharmacological activities of immunoregulation, antioxidation, anti-tumor, anti-inflammatory, blood sugar reduction, gastrointestinal mucosa protection and the like, and have wide development prospect and application potential. However, although plant polysaccharides exhibit significant biological activity in vitro and in animal models, they still face many challenges in terms of pharmacokinetic profile and tissue distribution in vivo. This restricts the development of new dosage forms, efficacy evaluation and safe and reasonable clinical medication. At present, quantitative analysis of plant polysaccharide components in biological samples mainly comprises the following methods, but has certain problems that the labeling process of fluorescent labeling is easy to damage polysaccharide space conformation, so that pharmacokinetic behavior is distorted, the method is harsh in condition and low in reproducibility, the traditional phenol-sulfuric acid method is based on color reaction, has poor specificity and is easy to be interfered by endogenous substances, accurate quantification of target components in complex biological matrixes is difficult to realize, and the problems of easy loss of target materials, enzyme degradation and the like in operation of the conventional pretreatment technology of ethanol precipitation introduce errors, so that detection accuracy is influenced. At present, a method for realizing high-sensitivity and high-specificity quantitative analysis of plant polysaccharide in a biological sample on the premise of keeping the natural structure of the plant polysaccharide is not known, and the method becomes a key bottleneck for in-vivo metabolism and pharmacodynamic mechanism of the biological sample. Therefore, it is necessary to develop a quantitative analysis method of plant polysaccharide in a biological sample, which is free of labeling, anti-interference, simple and convenient to operate, accurate and reliable. Disclosure of Invention The invention aims to overcome the technical defects and shortcomings of the existing exogenous labeling method that the natural structure of plant polysaccharide is damaged and the specificity of the traditional chemical analysis method is poor, and provides a method for quantitatively detecting plant polysaccharide in biological samples (in the blood plasma or tissues of rats) with strong specificity, high sensitivity, accuracy and reliability. The invention takes characteristic monosaccharides (arabinose and rhamnose) which are naturally existed in plant polysaccharide and can not be synthesized in a rat body as endogenous tracers, thereby realizing accurate quantification of the plant polysaccharide in biological samples (in the plasma or tissues of the rat). The invention provides a method for quantitatively detecting plant polysaccharide in a biological sample, wherein the biological sample is rat plasma or rat tissue, the content of characteristic monosaccharide in the biological sample is quantitatively detected, and then the content of the plant polysaccharide in the biological sample is calculated according to the ratio of the characteristic monosaccharide in the plant polysaccharide, and the characteristic monosaccharide comprises rhamnose and arabinose. Preferably, the rat tissue comprises one of heart, liver, spleen, lung, kidney, thymus tissue of the rat, and/or The plant polysaccharide comprises one of hedysari polysaccharide, astragalus polysaccharide and honeysuckle polysaccharide, and/or The calculating method for calculating the content of the plant polysaccharide in the biological sample according to the ratio of the characteristic monosaccharide in the plant polysaccharide comprises the steps of calculating the content of the plant polysaccharide in the biological sample=the measured value (mg) of the characteristic monosaccharide in the biological sample/the mass fraction of the characteristic monosaccharide in the plant polysaccharide. Preferably, the quantitative determination of the content of