Separation and purification of tea polysaccharides and analysis by gas chromatography-mass spectrometry

Abstract: Tea polysaccharides are mainly composed of about 20 kinds of common amino acids in the protein part, the sugar part is mainly composed of arabinose, xylose, fucose, glucose, galactose, etc. The mineral elements are mainly calcium, magnesium, iron and manganese And a small amount of trace elements, such as rare earth elements.

Objective To extract the water-soluble crude polysaccharide from willow tea for the first time and to separate, purify and analyze its composition. Methods Willow tea was extracted by hot water, ethanol precipitation, deproteinized by Sevag method, decolorized by H2O2, and dialyzed against countercurrent water to obtain polysaccharide refined products. After derivatizing refined polysaccharides, their chemical components were analyzed by gas chromatography-mass spectrometry. The results determined that the water-soluble polysaccharides in willow tea were composed of ribose, mannose, glucose and galactose, among which ribose, glucosose and galactose were the main components. Conclusion This method is simple, sensitive, and can accurately determine various monosaccharides contained in polysaccharides from willow tea.

[Keywords] tea willow polysaccharide; purification; gas chromatography-mass spectrometry; monosaccharide

Abstract: ObjectiveToisolateandpurifythewater-solublepolysaccharidefromLiuchaandanalyzethechemicalcompositionofliucha.MethodsTheliuchawasextractedinhotwater, thenthepolysaccharideinthefiltratewasfractionallyprecipitatedbyalcohol, afterthattheproteinsintheprecipitatedproductwereremovedbySevagreagent, hydrogenperoxidedecolor, thehydrolysateofpolysaccharidewasdialyzedbythereverseflowtogetthefineproduct.Finally, thechemicalcompositionsofthefineproductafterderivatizationwereexaminedbythegaschromatography-massspectrometricanalysis.ResultsThepolysaccharidesarecomposedofribose, mannose, glucoseandgalactose.Themajormonosaccharidecomponentsareribose, galactoseandglucose.ConclusionThemethodreportedinthispaperissimple, andcanbeusedforanalysisofmonosaccharideshydrolyzedfromLiuchapolysaccharide.

Keywords: Liuchapolysaccharide; Purify; Gaschromatography-massspectrometry; Monosaccharide

Willow tea is a commonly used folk medicine of Tibetans. It is based on the branches and leaves of the Rosaceae (Sibiraea) plant Sibeiraeaangustata (Rehd.) Hand-Mazz. And the Sibeiraealaecigata (L) Maxim. The fruit branches of the fruit) are used in medicine, mainly for digestion, stasis, and evacuation of wind and heat; according to the Tibetan medicine literature, willow tea mainly treats fever and epidemic diseases [1]. Now Tibetan folks often make tea to treat indigestion caused by many reasons such as bloating after eating, and it often works. In addition to volatile oil and trace elements, willow tea also found rich polysaccharides through experiments. Polysaccharides have been accepted by many people as health products to improve the body's immune function. However, many polysaccharide products only stay in the health products stage and have not been developed into drugs. Therefore, this article aims to study the extraction, purification, and component analysis of willow tea polysaccharides and discuss their nutritional and health care functions, in order to provide a reference for the development and comprehensive utilization of willow tea.

1 Instruments and materials

1.1 Instrument UV-2450 ultraviolet spectrophotometer (Shimadzu, Japan); NE2155 electronic balance (Germany Sartorius Instrument Company); Agilent 5793I-6890 gas chromatography-mass spectrometry (GC-MS); SE-54 elastic quartz capillary column (50m × 0.25mmid × 0.50μm); high purity helium (purity ≥99.999%); rotary evaporator (Shanghai Qingpu Luxi Instrument Factory).

1.2 Materials The leaves of the Xianbei genus are collected from the suburb of Gannan Tibetan Autonomous Prefecture, Gansu Province, at an altitude of more than 3200m. Professor Yang Yongjian, Institute of Pharmacy, School of Pharmacy, Lanzhou University, identified it as the leaves of the narrow-leaved Xianbei Sibiraeaangustata (Rehd.) Hand-Mazz. And the Sibiraealaecigata (L) Maxim. Of the Rosaceae (Sibiraea) plant.

Monosaccharide standard glucose, arabinose, ribose, mannose, xylose, fructose, galactose standard (Fluka), hydroxylamine hydrochloride, pyridine, trifluoroacetic acid, acetic anhydride, chloroform and other reagents are all domestically produced Analytically pure, distilled water is made in the laboratory.

2 methods

2.1 Extraction and purification of polysaccharides

2.1.1 Extraction of crude polysaccharide Take a certain amount of willow tea in order to remove fat-soluble impurities with petroleum ether and ether. Add 10 times the amount of water and extract 3 times at 90 ~ 100 ℃, 3h / time. Combine the water extracts and filter. Concentrate to a proper amount under reduced pressure in a 70 ° C water bath, add 5 times the amount of 95% ethanol for pure analysis, let stand overnight, centrifuge and filter, and place in a vacuum freeze-drying to obtain crude willow tea polysaccharide.

2.1.2 Purification of crude polysaccharide [2] Dissolve the crude polysaccharide in distilled water, remove insoluble matter, deproteinize according to the Sevag method (chloroform: n-butanol = 4: 1), repeat more than 10 times, and then move to a dialysis bag for use Ionized water was dialysed countercurrently for 24 hours until UV spectrum analysis detected no protein absorption. Adjust the pH to 8 with ammonia water, add H2O2 dropwise, incubate at 50 ° C for 2 h, concentrate under reduced pressure, add 5 times the amount of absolute ethanol, let stand overnight, collect the precipitate by centrifugation, rinse the precipitate with anhydrous ethanol, acetone and ether in sequence 2 times, freeze-dried to get pure proteoglycan.

2.1.3 UV spectrum analysis Take appropriate amount of willow tea polysaccharide, add water to dissolve, and prepare into a polysaccharide aqueous solution with a concentration of 1.0g / L. Scan by UV-Vis spectrophotometry in the range of 190-700nm. The spectrum shows that the tea willow polysaccharide has the characteristic UV absorption spectrum of polysaccharide only at 200nm. There is a sharp absorption peak, and there is no obvious UV absorption above 250nm, which is a flat background. The results show that the extracted and purified polysaccharide does not contain nucleic acids, proteins and other impurities.

2.2 Determination of willow tea polysaccharides by gas chromatography mass spectrometry

2.2.1 Preparation of test solution (glyconitrile acetyl ester derivative) Weigh 20 mg of purified polysaccharide sample from tea will be dissolved in 2 ml of 2mo1 / L TFA, seal the tube, hydrolyze at 100 ° C for 6 h, and remove TFA under reduced pressure. Add 20mg hydroxylamine hydrochloride and 1ml pyridine to the hydrolysate, seal the tube and react at 90 ° C for 30min. After cooling to room temperature, 1 ml of acetic anhydride was added, the tube was sealed and reacted at 90 ° C for 30 min. After cooling, the supernatant was transferred out, and concentrated to dryness under reduced pressure. After dropping 0.5 ml of chloroform to dissolve, gas chromatography-mass spectrometry analysis was performed.

2.2.2 Preparation of reference substance solution Precisely weigh monosaccharide standard glucose, arabinose, ribose, mannose, xylose, fructose, galactose reference substance 20mg, use the above method to prepare acetylated derivatives, after chloroform is dissolved Perform gas chromatography-mass spectrometry analysis.

2.3 GC-MS analysis conditions

2.3.1 Chromatographic conditions SE-54 capillary column, gasification chamber temperature 270 ° C, pre-column pressure 17.8kPa, split ratio 30: 1, constant flow mode carrier gas He flow 1.2ml / min, sample injection volume 1μl, temperature programmed : The initial temperature is maintained at 100 ° C for 6 minutes, raised to 220 ° C at 20 ° C / min for 14 minutes, and raised to 290 ° C at 5 ° C / min.

2.3.2 Mass spectrometry conditions electron bombardment source (EI), electron energy 70eV; ion source temperature: 230 ° C; quadrupole temperature: 150 ° C; transmission rod temperature: 280 ° C; scanning range: 14-400 amu.

3 results

3.1 GC-MS analysis of monosaccharide standard products and willow tea polysaccharides

3.1.1 The total ion current diagram of the mixed monosaccharide standard acetylated derivative is shown in Figure 1. According to the GC-MS total ion chromatogram of acetylated derivatives of monosaccharide standards, the peak order of GC-MS total ion chromatogram of 7 mixed monosaccharide standards was determined to be fructose, ribose, arabinose, xylose , Mannose, glucose and galactose.

3.1.2 The total ion current diagram of the acetylated derivatives of willow tea polysaccharides is shown in Figure 2. According to the total ion chromatogram of tea tea polysaccharides, the retention time of each peak and the standard mass spectrometry Nist spectral library (NIST) control analysis can determine that it contains ribose, mannose, glucose and galactose. Table 1.

4 Discussion

To analyze the composition of monosaccharides in polysaccharides, the primary task is to thoroughly hydrolyze the polysaccharide samples. Because if the hydrolysis effect is not ideal, it is difficult for the polysaccharide to be completely hydrolyzed to monosaccharides or only a small amount of hydrolysis, resulting in a small peak of the hydrolyzed derivatized product in the gas chromatographic analysis, which affects the determination of monosaccharides with less polysaccharide content. Through comprehensive investigation and analysis of various factors that affect the hydrolysis effect, it is concluded that for the hydrolysis of willow tea polysaccharides, 2ml2mo1 / L TFA should be used to hydrolyze for 6h. Under this hydrolysis condition, the polysaccharide can be hydrolyzed relatively completely. Sulfuric acid can also be used for the hydrolysis of polysaccharides, but the effect of using low-concentration sulfuric acid for hydrolysis is not ideal, and high-concentration sulfuric acid can easily cause carbonization of polysaccharides. And because the boiling point of sulfuric acid is relatively high, the remaining acid after hydrolysis is not easy to be volatile and removed, and it must be neutralized with barium carbonate, which is easy to cause the loss of sample components.

Table 1 Monosaccharide content in polysaccharides from willow tea

When the tea polysaccharide is extracted or dried in the air, the color will deepen. It can be decolorized with hydrogen peroxide or activated carbon. The amount of activated carbon used for decolorization is large, and the sample loss is large. Therefore, H2O2 is used for decolorization. After decolorizing with hydrogen peroxide, it must be dialyzed until no hydrogen peroxide is detected. H2O2 is detected by potassium permanganate method. In addition, covering the sample with a thin film during alcohol washing, ether washing, and suction filtration can reduce air contact and prevent darkening of the color. During decolorization, the temperature is kept at 50 ℃ for 2h. The short time is too much pigment to affect the detection result, and the longer the time, there is no better decolorization effect.

In addition to various chemical components such as volatile oil, trace elements, triterpenoids, etc., the tea group found that the polysaccharides contained in it are also one of its main active ingredients; the application of polysaccharides as medicines was in the 1940s and since the 20th century Since the 1960s, people have discovered that polysaccharides have more biological activities. It not only has immunoregulatory functions, but also can resist infection, lower blood sugar and anti-tumor. With the deepening of research on polysaccharide compounds, it has shown broad application prospects in the clinical treatment of anti-tumor and viruses. This paper is the first to study the chemical composition of willow tea polysaccharides, which has certain significance for the development and utilization of local resources. Further study of its chemical structure will help elucidate the relationship between its activity and its composition.

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