| As a traditional Chinese medicine,Dendrobium officinale is rich in polysaccharide components with Glucomannan in the majority.Dendrobium officinale polysaccharides are biologically active substances with prospects for drug development and product development.In order to find a better and more accurate method for the measurement of water-soluble polysaccharides,the traditional phenol-sulfuric acid method was used for the determination of water-soluble polysaccharides in Dendrobium officinale.We found that the method has the disadvantages of environmental pollution and danger to the operator due to the use of sulfuric acid and the large deviation in the measurement.Using sulfuric acid as reagent,it is dangerous and the results have much bigger errors.Therefore,Refractive Index Detector was chosen to quantify the water-soluble polysaccharide in Dendrobium officinale.By comparison and validation of two methods,RID method for the quantification of water-soluble polysaccharides in Dendrobium officinale is not only applicable to the quantification of water-soluble components in Dendrobium,but also applicable to the quantification of water-soluble polysaccharides in natural drugs.I.Measurement with phenol-sulfuric acid methodIn the determination of polysaccharide by phenol sulfuric acid method,the quantitative curve was close to the theoretical line when the concentration was lower than 90μg/m L by absorbance at OD490（Optical Density,490 nm）.The standard curve is well fit with the correlation coefficient greater than 0.9990,while the quantitative site of sugar decreased at high concentration and the quantitative linear curve was biased.The common standard derivatives of monosaccharides had obvious absorption differences and the slopes of the quantitative curves differed widely.By measuring the yield of furfural,it was found that the yield of the intermediate product 5-hydroxymethylfurfural（5-HMF）started to decrease at high concentration in glucose of 90μg/m L or more,leading to a decrease in the OD490 value.The effective molecular weight effect and functional group effect of polysaccharides and monosaccharides on the quantitative results was analyzed by comparing that of dextran and glucose.Molecular weight effects between polysaccharides and monosaccharides were clearly observed from its kinetic monitoring results,and the intrinsic reasons for the decrease of furfural and the decrease of yield were analyzed by comparing the stabilization time of OD490 value.II.Establishment of the RID methodRID detector was chosen to investigate the detection of water-soluble polysaccharides based on the review of relevant literature and previous studies.By testing the effects of temperature,molecular weight and functional groups on the quantification of RID detector,RID method was designed and verified the feasibility test of quantifying natural water-soluble polysaccharides.The results showed that the RID signal acquisition system had similar quantification curves for glucose and dextran of different molecular weights（1.0-50.0 k Da）in0.312-10.0 mg/m L at temperature of 40℃with the correlation coefficients all greater than 0.9990.Since the RID detector is only sensitive to changes in the mass concentration of the sample,it is not affected by the molecular weight of natural water-soluble polysaccharides.RID detector is only sensitive to changes in the mass concentration of the sample and is not affected by the molecular weight of natural water-soluble polysaccharides,various substituents can be accurately quantified in this quantitative range.In the methodological validation,this study and compared the experimental results of both methods,the phenol-sulfuric acid method showed a higher recovery of 107.66 with an RSD value of6.72%,while the total recovery of RID was 100.08%with an RSD value of2.09%.RID showed higher precision and stability,while the interference level of water-soluble impurities should be controlled below 6%（w/w）.III.Measurement of water-soluble polysaccharide in Dendrobium officinaleThe water-soluble polysaccharides in Dendrobium officinale were extracted,separated and purified by grading,and the concentration of the eluted fraction by column chromatography on Cellulose-DEAE-52（1.0 m L/min）and Sephadex G-100 chromatographic column（0.6 m L/min）,and the same fraction was collected to obtain the water-soluble polysaccharides in Dendrobium officinale.The purified polysaccharide fraction（Dendrobium officinale purified polysaccharide,DOPP）was analyzed by high performance gel permeation chromatography（HPGPC）with the number average molecular weight of 116.0k Da,the molecule weight average molecular weight of 214.1 k Da and the distribution coefficient of 1.85.The water-soluble polysaccharides were quantified by phenol sulfuric acid method and RID method,respectively.The results showed that the absorption values of DOPP were lower than the glucose standard curve in the phenol sulfuric acid method,while the RID method showed stable and consistent absorption of glucose and DOPP.After partial methylation treatment of DOPP,the result analysis concluded that its monosaccharide residues were composed of glucose and mannose.DOPP showed significant absorption at 1642.41 cm-1 and 1726.60 cm-1 in IR spectra,indicating the presence of glyoxylate groups and acetylated groups in the DOPP fraction.The m-hydroxylated biphenyl method showed that the DOPP fraction contained 2.46%glyoxylate groups,and the nuclear magnetic resonance（NMR）method measured 40.2%acetyl groups in the DOPP fraction,which indicated that the presence of glyoxylate and acetyl groups was the main reason for the lower quantification of DOPP in phenol sulfuric acid method.In this study,the RID method is established and serves as more reliable measure than phenol-sulfuric acid method.RID analytical method avoids the use of solvents such as sulfuric acid or phenol which are polluting to the environment,and more importantly,it provides an efficient,reliable and green method for the quantitation of water-soluble polysaccharides... |
