Extraction and Application of Liubao Tea Polysaccharide

Abstract

The present invention belongs to the technical field of polysaccharide extraction. More specifically, it relates to the extraction and application of Liubao tea polysaccharide with in vitro antioxidant activity, blood glucose-lowering and blood lipid-lowering activities. The present invention obtained the Liubao tea polysaccharide component TPS-5 by extraction with water extraction and alcohol precipitation methods, purification with DEAE-52 cellulose column and Sephadex G-200 gel column, and identified and analyzed its structure. In vitro biological activities confirmed that TPS-5 has antioxidant, bile salt-binding, α-amylase and α-glucosidase inhibitory activities. HepG2 cell experiments proved that TPS-5 has good effects on lowering blood glucose, blood lipid and cholesterol. These results indicate that TPS-5 can be used as a natural antioxidant resource to regulate blood lipid and blood glucose, and may also be used as a natural ingredient for treating diabetes. The present invention lays a theoretical foundation for the application of functional active Liubao tea polysaccharide in health foods or functional foods.

Description

Technical Field

[0001] The present invention belongs to the technical field of polysaccharide extraction, and more specifically, relates to the extraction and application of Liubao tea polysaccharide with in vitro antioxidant activity, blood sugar lowering and blood lipid lowering activities. Background Art

[0002] Liubao tea is a dark tea produced in Wuzhou, Guangxi, China, with a history of more than 1,500 years and is one of the top 24 famous teas in China. Liubao tea is made from fresh leaves of local large-leaf tea trees and processed through processes such as pile fermentation, initial steaming, piling, re-steaming, pressing into baskets, drying, and natural aging. Pile fermentation and natural aging are the key processes for forming the unique flavor of Liubao tea. In a humid and hot environment, components such as polyphenols, carbohydrates, proteins, and amino acids in the raw materials undergo a series of complex chemical and biological transformations, ultimately forming Liubao tea with a dark red and thick soup color, mellow fragrance, rich and mellow taste, and a unique betel nut aroma. According to the records of Li Shizhen's Compendium of Materia Medica in the Ming Dynasty, Liubao tea has the effects of relieving heat, refreshing the spleen, warming the stomach, clearing the palate, aiding digestion, treating heatstroke and colds, etc. Recent research has also confirmed that Liubao tea has physiological active functions such as antioxidant, hypoglycemic, blood lipid lowering, anticoagulant, and improving intestinal flora.

[0003] Dark tea contains rich tea polysaccharides, which are one of the important active substances reflecting the efficacy of dark tea. Compared with unfermented tea, fermented tea polysaccharides have higher biological activities. Dark tea polysaccharides have good antioxidant activity and α-glucosidase inhibitory activity. However, compared with green tea polysaccharides, dark tea polysaccharides are formed through microbial transformation. Due to the special piling and aging processes, most dark tea polysaccharides are acidic heteropolysaccharides, combined with other components such as polyphenols and proteins, with a more complex structure and relatively difficult to analyze.

[0004] The research on Liubao tea polysaccharide started relatively late. In previous studies, the basic structural characteristics and in vivo and in vitro biological activities of Liubao tea polysaccharide have been studied. However, in terms of the structural analysis of Liubao tea polysaccharide, current research is still limited to aspects such as molecular weight range, monosaccharide composition and ratio, and morphological characterization. Clear structural information such as the types of glycosidic bonds in the main chain and side chains, the connection sequence and position of monosaccharides, etc. has not been studied yet. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the above problems existing in the prior art, and first provide a Liubao tea polysaccharide extracted from Liubao tea.

[0006] The second object of the present invention is to provide a preparation method of the above Liubao tea polysaccharide.

[0007] The third object of the present invention is to provide the application of the above Liubao tea polysaccharide

[0008] The object of the present invention is achieved by the following technical solutions:

[0009] Liubao tea polysaccharide TPS-5, which is composed of rhamnose, arabinose, galactose, glucose, mannose, and galacturonic acid, and its molar ratio is 0.077∶0.120∶0.191∶0.143∶0.023∶0.398; the main chain of TPS-5 is composed of →2,4)-α-L-Rhap-(1→4)-α-D-GalAp-(1→, and the repeating unit structure of the main chain is Formula (I) and Formula (II). The branched chain is composed of →5)-α-L-Araf-(1→5,3)-α-L-Araf-(1→3)-β-D-Galp-(1→3,6)-β-D-Galp-(1→, and the repeating unit structure of the branched chain is Formula (III) and Formula (IV);

[0010]

[0011] The Liubao tea polysaccharide obtained in the present invention is an acidic polysaccharide with a pectin structure and a molecular weight of 48.289 kDa.

[0012] The present invention also provides a preparation method of the Liubao tea polysaccharide, comprising the following steps:

[0013] S1. Extract the crude Liubao tea polysaccharide by water extraction-alcohol precipitation method;

[0014] S2. Separate the crude Liubao tea polysaccharide with DEAE-52 cellulose, elute it successively with distilled water and NaCl aqueous solution with gradient concentration dilution, the flow rate is 15 mL / h, and collect the system components eluted with 0.4 mol / L NaCl aqueous solution, that is, obtain Liubao tea polysaccharide TPS5;

[0015] S3. Purify the Liubao tea polysaccharide TPS5 with Sephadex G-200 gel, elute it with pure water, the elution flow rate is 15 mL / h, collect it at 20 min / tube, and obtain Liubao tea polysaccharide TPS-5.

[0016] Preferably, the preparation method of the Liubao tea polysaccharide is characterized in that the operation of step S1 is:

[0017] S11. Add 95% ethanol to the Liubao tea powder at a solid-liquid ratio of 1:10 w / v and soak for 18 hours, stir to remove pigments;

[0018] S12. Then mix the tea leaves and pure water at a ratio of 1:10 w / v, extract at 90 °C for 2 h, centrifuge, and collect the supernatant;

[0019] S13. Concentrate the supernatant by rotary evaporation, then add three times the amount of absolute ethanol for alcohol precipitation, and refrigerate overnight;

[0020] S14. Wash the precipitated polysaccharide with absolute ethanol, dissolve it in pure water, centrifuge, and collect the polysaccharide supernatant for freeze-drying; remove the protein in the sample and decolorize it to obtain crude polysaccharide from Liubao tea.

[0021] Preferably, in step S2, the sample loading concentration is 20.0 mg / mL, the sample loading volume is 10 mL, the flow rate is 0.25 mL / min, and the eluent is distilled water, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L aqueous NaCl solutions.

[0022] Preferably, in step S3, the sample loading concentration is 10.0 mg / mL, the sample loading volume is 5 mL, the flow rate is 0.25 mL / min, and the eluent is pure water.

[0023] The present invention also provides the application of the Liubao tea polysaccharide in the preparation of a functional product, and the functional product has at least one of the following functions:

[0024] (1) Scavenging DPPH free radicals and ABTS + free radicals;

[0025] (2) Inhibiting α-amylase and α-glucosidase;

[0026] (3) Binding to sodium taurocholate and glycocholate;

[0027] (4) Reducing the glucose concentration in vivo;

[0028] (5) Reducing the TG content and TC content in vivo.

[0029] Preferably, the functional product is a functional food or a tea product.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention extracted by water extraction and alcohol precipitation method, purified by DEAE-52 cellulose column and Sephadex G-200 gel column to obtain the Liubao tea polysaccharide component TPS-5, and its structure was identified and analyzed. In vitro biological activity confirmed that TPS-5 has antioxidant, bile salt binding, α-amylase and α-glucosidase inhibitory activities. HepG2 cell experiments proved that TPS-5 has good effects on reducing blood sugar, lipid and cholesterol. These results indicate that TPS-5 can be used as a natural antioxidant resource to regulate blood lipid and blood sugar, and may also be used as a natural ingredient for treating diabetes. The present invention lays a theoretical foundation for the application of functional active Liubao tea polysaccharide in health food or functional food. Description of the Drawings

[0032] Figure 1It is the elution curve of DEAE-52 cellulose column chromatography;

[0033] Figure 2 It is the elution curve of Sephadex G-200 gel column chromatography;

[0034] Figure 3 It is the ultraviolet absorption spectrum of TPS-5;

[0035] Figure 4 It is the FT-IR spectrum of TPS-5;

[0036] Figure 5 It is the THPGPC-RID diagram of TPS-5;

[0037] Figure 6 It is the HPAEC-ED chromatogram composed of standard monosaccharide (A) and TPS-5 monosaccharide (B);

[0038] Figure 7 It is the nuclear magnetic resonance spectrum of TPS-5;

[0039] Figure 8 It is the structure of the repeating unit of TPS-5;

[0040] Figure 9 It is the scanning electron micrograph of TPS-5;

[0041] Figure 10 It is the antioxidant activity diagram of TPS-5;

[0042] Figure 11 It is the in vitro hypoglycemic activity diagram of TPS-5;

[0043] Figure 12 It is the effect of different concentrations of TPS-5 on HepG2 cells;

[0044] Figure 13 It is the effect of TPS-5 on oil red O staining of HepG2 cells; among them, Figures A-E are the blank control group, the glycolipid metabolism disorder model group, the positive acarbose group, the positive lovastatin group, and the TPS-5 treatment group respectively;

[0045] Figure 14 It is the effect of TPS-5 on GLU in HepG2 cells;

[0046] Figure 15 It is the effect of TPS-5 on TG in HepG2 cells;

[0047] Figure 16 It is the effect of TPS-5 on TCHO in HepG2 cells. Specific implementation methods

[0048] The following further describes the specific embodiments of the present invention. It should be noted here that the description of these embodiments is for helping to understand the present invention, but does not limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0049] Unless otherwise specified, the test methods used in the following examples and experimental examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels; the equipment used, unless otherwise specified, are all conventional experimental equipment.

[0050] Example 1 Extraction, Purification and Content Determination of Liubao Tea Polysaccharide

[0051] The Liubao tea was crushed and passed through a 80-mesh sieve, and 95% ethanol was added at a solid-liquid ratio of 1:10 (w / v) and soaked for 18 hours, with stirring from time to time to remove pigments. The crude polysaccharide of Liubao tea was prepared by hot water extraction-ethanol precipitation method. The tea leaves and pure water were mixed at a ratio of 1:10 (w / v), extracted at 90 °C for 2 h, centrifuged, and the supernatant was collected. The above operation was repeated 3 times. The supernatant was rotary evaporated and concentrated, then three times the amount of absolute ethanol was added for alcohol precipitation, and it was placed in the refrigerator overnight. The precipitated polysaccharide was washed with absolute ethanol, dissolved in pure water again, centrifuged, and the polysaccharide supernatant was collected and freeze-dried. The Sevag method was used to remove proteins from the sample, and polyamide resin was used for dynamic adsorption and decolorization to obtain the crude polysaccharide.

[0052] The crude polysaccharide sample was redissolved with distilled water. Then, the polysaccharide solution was loaded onto a DEAE-52 cellulose column (2.6 cm x 30 cm), and eluted with 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 1 M NaCl solutions respectively. The elution flow rate was 15 ml / h, and one tube was collected every 20 min. The total sugar content and uronic acid content in each tube were measured. An elution curve was plotted with the tube number as the abscissa and the absorbance as the ordinate, and the results are as Figure 1 shown. Six polysaccharide fractions were separated by the DEAE-52 cellulose column. After collecting the elution peaks, concentrating, dialyzing, freeze-drying and weighing, the yields of the six polysaccharides were calculated based on the mass of the crude polysaccharide loaded as follows: TPS1 (7.76%), TPS2 (5.37%), TPS3 (8.22%), TPS4 (14.77%), TPS5 (20.07%), TPS6 (7.67%). TPS5 with the highest yield was selected for further purification using a Seperdex-200 column.

[0053] Weigh 150 mg of TPS5, dissolve it in 10 mL of distilled water, centrifuge at 12,000 rpm for 10 min, load the solution onto a Sephadex G-200 gel column (1.7 cm x 70 cm), elute with pure water at an elution flow rate of 15 mL / h, collect one tube every 20 min, and track and determine the total sugar and uronic acid contents in the eluate. Draw an elution curve with the tube number as the abscissa and the absorbance as the ordinate, collect the main part at the symmetric peak and freeze-dry it to obtain a pure polysaccharide named TPS-5 for further analysis. The results are as Figure 2 shown. The symmetric single-peak shape indicates that TPS-5 is homogeneous. Chemical composition analysis shows that the contents of neutral sugar, uronic acid, bound phenol, and protein in TPS-5 are 53.32 ± 3.13%, 35.31 ± 2.23%, 0.83 ± 0.47%, and 0.48 ± 0.23% respectively. Further analysis was carried out on the pure polysaccharide of TPS-5.

[0054] Example 2 Structural Identification of Liubao Tea Polysaccharide

[0055] 1. Ultraviolet Spectroscopy Analysis

[0056] Dissolve the sample in distilled water and record the ultraviolet absorption spectrum of the solution in the wavelength range of 200 - 800 nm.

[0057] The ultraviolet spectrum of TPS-5 shows extremely weak absorption peaks at 260 and 280 nm, indicating that TPS-5 does not contain proteins and nucleic acids. The results are as Figure 3 shown.

[0058] 2. Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

[0059] Use the FT-IR method to measure the infrared spectrum of TPS-5. Take 2.0 mg of the sample and add 150 mg of potassium bromide to make pellets. Record the FT-IR spectrum of TPS-5 with an FT-IR spectrometer in the wavelength range of 4000 cm -1 —400 cm -1 wavelength range.

[0060] As Figure 4 shown, the FT-IR spectrum of TPS-5 shows a strong broadband at 3361 cm -1 , which is attributed to the stretching vibration of the O-H bond. The stretching and bending vibrations of C-H result in a weak band at 2934 cm -1 . These two intervals are characteristic absorption peaks of polysaccharides. The infrared detection results are consistent with the uronic acid detection results in the sample. The strong peak near 1098 cm -1 is the characteristic absorption of the pyranose ring C-O-C and C-O-H. 898 cm -1 and 813 cm -1The peaks indicate that TPS-5 has β-type and α-type glycosidic bonds between sugar units respectively. The results of infrared spectrum scanning show that TPS-5 is a complex acidic polysaccharide.

[0061] 3. Molecular weight determination and monosaccharide composition analysis

[0062] The molecular weight of TPS-5 was determined by high performance gel permeation chromatography (HPGPC) using a Shimadzu LC-10A chromatograph, an Ultrahydrogel TM linear BRT105 - 104 - 102 column (20 μl, 8 mm x 300 mm) and a differential refractive index detector. 20 μL of the sample was injected into the chromatographic column, eluted with a 0.05 M NaCl solution at 40 °C at a flow rate of 0.6 mL / min. The molecular weight of TPS-5 was calculated using the calibration curve of the t-series dextran standard molecular weight (Mw). According to the retention time and Figure 5 the calibration curve, based on the calibration curve of dextran standard and retention time, the Mw of TPS-5 was 48.289 kDa.

[0063] The monosaccharide composition of TPS-5 was determined using an ion chromatograph. 10 mg of the sample was taken, 10 ml of 3 M trifluoroacetic acid (TFA) was added, and hydrolysis was carried out at 121 °C for 3 h. The acid hydrolysis solution was dried with nitrogen to completely remove TFA. 5 ml of deionized water was added, vortexed and mixed evenly, 100 μL was taken and added to 900 μL of deionized water, and centrifuged at 12000 rpm for 5 min. 5 μL of the supernatant was taken for IC analysis. The chromatographic method was: Dionex TM ICS-5000+ high pressure ion chromatography system (HPLC). Based on high performance anion exchange chromatography, a CarboPac TM PA20 chromatographic column (3 x 150 mm) was used, the column temperature was 30 °C, and a pulsed amperometric detector was used. At the same time, three mobile phases of A: H2O, B: 250 mM NaOH, C: 50 mM NaOH and 500 mM NaOAC were eluted with a gradient of 0.3 mL / min. The monosaccharide composition was analyzed by HPAEC-ED. As Figure 6 shown in A and B, TPS-5 is composed of Rha, Ara, Gal, Glu, Man, and GalA with a molar ratio of 0.077∶0.120∶0.191∶0.143∶0.023∶0.398. The results show that TPS-5 is a typical acidic heteropolysaccharide containing galacturonic acid, with Ara and Glu accounting for the largest proportions.

[0064] 4. Methylation analysis

[0065] 2–3 mg of TPS-5 powder was dried under vacuum at 80 °C for 5 h and then dried overnight with P2O5. Then, the dried sample was dissolved in 0.5 mL of anhydrous dimethyl sulfoxide and sonicated at 45 °C for 4 h followed by a water bath at 85 °C with continuous stirring for 1 h to ensure complete dissolution of TPS-5. 20 mg of NaOH (dried under vacuum at 80 °C for 5 h and dried overnight with P2O5) was added to the solution and stirred continuously at room temperature for 3 h. Then, the methylation reaction was initiated by adding 0.3 mL of CH3I and stirred continuously at room temperature for 2.5 h. The mixture was extracted with 2 mL of CH2Cl2 and separated three times with deionized water (3–5 mL). The separated organic phase was filtered through an anhydrous Na2SO4 column, eluted twice with CH2Cl2, and the methylated TPS-5 was dried and recovered by evaporation under a N2 flow at room temperature. Under a nitrogen atmosphere, it was further hydrolyzed in 0.5 mL of 4 M trifluoroacetic acid with continuous stirring at 100 °C for 6 h. After that, the hydrolysis product was cooled to room temperature and dried with a continuous N2 flow. Then, 0.3 mL of deionized water, one drop of 1% (v / v) NH4OH, and 1–5 mg of NaBD4 were added successively and stirred overnight to reduce the hydrolyzed sample. Then, before N2 evaporation, the reduced product was acetylated with 0.5 mL of acetic anhydride with constant stirring at 100 °C for 2 h.

[0066] The obtained partially methylated alditol acetates (PMAAs) were analyzed using a GC-MS-GCMS-QP 2010 chromatographic column (30 m × 0.25 mm × 0.25 μm). The column temperature was set at 120 °C during injection and increased to 250 °C at a rate of 3 °C / min and held for 5 min. Helium was used as the carrier gas with a flow rate of 1 mL / min. The injection port temperature and the detector temperature were both 250 °C.

[0067] Before methylation analysis, GalA or its methyl-esterified form in TPS-5 was reduced to the corresponding Gal residue. The resulting PMAAs were subjected to GC-MS detection. Based on the retention time and mass spectrometry in the CCRC spectral database, the GC-MS detected information of PMAA, and there were 10 peaks corresponding to the characteristics of methylated sugars. The molar ratio of glycoside residues was estimated according to the peak area. The linkage pattern of TPS-5 was summarized (Table 1), in which GalAp-1-4 accounted for a relatively large proportion (64.60%). Araf-1-5 (5.35%), Rhap-1-2-4 (5.17%), and Galp-1-3-4 (14.36%) had relatively high proportions and were speculated to be part of the branches of TPS-5. The ratio of terminal units (Araf-1, Galp-1) and branch points (Araf-1-3-5, Rhap-2-4, GalAp-1-3-4, Galp-1-3-6) was 68%. The results showed that TPS-5 had both linear units and branched units, and had a relatively large number of branches. The degree of branching (DB) of TPS-5 was 34.97%, and it was speculated that TPS-5 might be a pectin polysaccharide with Gal and GalA as the main chain and multiple branches.

[0068] Table 1 Linkage analysis of TPS-5 determined by methylation and GC–MS

[0069]

[0070] 5. Nuclear magnetic resonance spectroscopy

[0071] Pure TPS-5 (50 mg) was dissolved in D2O (0.5 mL, 60 °C), hydrated overnight at 4 °C, and freeze-dried at -50 °C. The completely dissolved sample was placed in 0.5 mL of D2O and tested on a Bruker Avance III 600 MHz NMR spectrometer to obtain 1H, 13C, COSY (1H-1H correlation), HSQC (1H-13C heteronuclear single quantum coherence), and HMBC (1H-13C heteronuclear multiple bond correlation) spectra, and the chemical shift (δ) was expressed in ppm.

[0072] Nuclear magnetic resonance (NMR) revealed the precise structural information of TPS-5. The anomeric carbons of the 13C NMR spectrum of TPS-5 showed that the residues were assigned according to the NMR spectrum and the data in Table 2. The signals at δ 108.19 ppm, δ 93.63 ppm, δ 100.39 ppm, δ 97.49 ppm, δ 105.39 ppm, Δ 110.63 ppm, Δ 108.91 ppm, and δ 99.81 ppm corresponded to C-1 of A, B, C, E, G, L, M, and O, the signal at δ 176.51 ppm was attributed to C-6 of A, and the signal at δ 170.80 ppm was attributed to C-6 of B and E.

[0073] In the DEPT135 spectrum, the inverted signals at δ63.87 ppm, δ70.76 ppm, δ62.41 ppm, δ62.5 ppm, δ67.81 ppm, and δ68.28 ppm can be attributed to C-5 / 6 of G, H, K, L, M, and N. The downfield shifts of the peaks at δ70.76 ppm, δ67.81 ppm, and δ68.28 ppm indicate the presence of substitution at C5 / 6. In the 1H NMR spectrum of TPS-5 ( Figure 7 C), the signals at δ5.71 ppm, δ5.03 ppm, δ4.84 ppm, δ4.55 ppm, δ5.16 ppm, δ5.00 ppm, and δ5.21 ppm correspond to H-1 of A, C, D, G, L, M, and O. The signal at δ1.19 ppm may be the deoxy sugar -CH3 of Rha, and the δt signal may be the acetyl -CH3.

[0074] COSY( Figure 7 D) and HSQC( Figure 7 E) spectra were used to analyze the assignments of other signals in 1H NMR and 13C NMR. Considering many glycosidic linkages, residue C, i.e., →4-α-D-GalpA-1→, is taken as an example in this section. The cross-peaks δ5.03 / 3.68 ppm, δ3.68 / 3.94 ppm, δ3.94 / 4.33 ppm, and δ4.33 / 4.69 ppm were determined by COSY. The signal at δ5.03 ppm corresponds to H-1 of residue C, and δ3.68 ppm, δ3.94 ppm, δ4.33 ppm, and δ4.69 ppm are attributed to H-2, H-3, H-4, and H-5 of C. Based on the 1H NMR assignment results of the COSY spectrum, the carbon signals in the HSQC spectrum of C can be easily assigned. The cross-peaks δ5.03 / 100.39 ppm, δ3.68 / 69.41 ppm, δ3.94 / 70.06 ppm, δ4.33 / 79.16 ppm, and δ4.69 / 72.66 ppm correspond to H-1 / C-1, H-2 / C-2, H-3 / C-3, H-4 / C-4, and H-5 / C-5 of C. The signal at δ54.23 / 3.73 was assigned to the methyl group of residue C on O-6. Similarly, the other proton and carbon signals of the remaining residues were analyzed in the same way.

[0075] HMBC( Figure 7 F) and NOESY( Figure 7G) The spectrum was used to confirm the linkage sites and sequences between residues. The H-1 of residue C showed a strong cross-peak with its C4 (C H-1 / C C-4), the C-1 in residue C had a strong coupling with its H4 (C C-1 / C H-4), and there was also an inter-residue NOESY contact between H-1 and H4 (CH-1 / C H-4) in C, indicating the existence of C→. In addition, the H-1 of residue C showed a strong cross-peak with the C4 of residue D (C H-1 / D C-4), the C-1 of residue B had a strong coupling with the H4 of residue D (C C-1 / D H-4), and the H-11 of C also had an inter-residue contact with the H4 of residue D (C H-1 / DH-4), indicating the existence of C→D linkage. In addition, the C-1 of residue D had a strong coupling with its H4 (D C-1 / D H-4), indicating the existence of D→D linkage. In addition, the C-1 of residue D was also coupled with the H4 of residue C (D C-1 / C H-4), indicating the existence of D→C linkage. The H-1 of residue C showed a cross-peak with the C2 of residue O (C H-1 / O C-2), and the H-11 of C also had an inter-residue contact with the H2 of residue 0 (C H-1 / O H-2), indicating the existence of C→O linkage. The H-1 of residue O showed a cross-peak with the C4 of residue C (O H-1 / C C-4), and the C-1 in residue O was also coupled with the H4 in residue B (O C-1 / C H-4), and the H-1 in O also had an inter-residue contact with the H4 in residue C (O H-1 / CH-4), indicating the existence of O→. In summary, these results indicate that the backbone of TPS-5 consists of a C→C→D→D→C→O→C linkage. In addition, a cross-peak between the C-1 of residue A and the H-3 of residue D was confirmed, indicating that residue A is linked to residue D at the O-3 position.

[0076] In addition, there was an inter-residue contact between the H-1 of L and the H5 of residue N (L H-1 / NH-5). The H-1 of residue N was also coupled with the C5 of residue M (N H-1 / M C-5). There was an inter-residue contact between the H-1 of L and the H3 of residue M (L H-1 / M H-3). The C-1 of residue M was also coupled with the H3 of residue G (M C-1 / G H-3). The H-1 of G had an inter-residue contact with the H3 of residue H (G H-1 / H H-3). The C-1 of residue H was also coupled with the H4 of residue O (H C-1 / O H-4). Similarly, the side chain of TPS-5 consists of an L→N→M→G→H→ linkage. In addition, since their signals in NMRS were extremely weak, the residues of Man and Glc that could be detected by methylation analysis were not observed in this structure.

[0077] Based on the analysis of monosaccharide composition, methylation results, and NMR data, it can be inferred that the main chain is linked by →2,4)-α-L-Rhap-(1→4)-α-D-GalAp-(1→, and the branches are linked to the O-4 position of the main chain by →5)-α-L-Araf-(1→5,3)-α-L-Araf-(1→3)-β-D-Galp-(1→3,6)-β-D-Galp-(1→ through →2,4)-β-L-Rhap-(1→. Therefore, based on the above information, the possible structure of TPS-5 is proposed, as shown in Figure 8 shown. It has typical pectin structural characteristics.

[0078] Table 2 1H and 13C NMR chemical shifts of sugar residues in TPS-5

[0079]

[0080]

[0081] 6. Scanning electron microscopy analysis

[0082] The solid sample was coated with a thin layer of platinum and observed using a scanning electron microscope system at an acceleration voltage of 2.0 kV with image magnifications of 500×, 2000×, and 5000×.

[0083] At 500 times magnification, TPS-5 showed a granular and massive cluster structure with a large number of cavities inside ( Figure 9 A). At 2000 times magnification, a rough surface could be seen ( Figure 9 B). At 5000 times magnification, the surface of a granular aggregate closely stacked together could be seen ( Figure 9 C). These phenomena may be caused by the strong interaction between polysaccharide molecules.

[0084] Example 3 Determination of in vitro antioxidant activity of Liubao tea polysaccharide

[0085] 3.1 DPPH free radical scavenging assay

[0086] The reaction system included 1.5 ml of DPPH ethanol solution (0.1 mmol / l) and 1.5 ml of polysaccharides at different concentrations. After incubating in the dark at 25 °C for 20 min, the absorbance was measured at 517 nm. Ascorbic acid was used as the positive control, and ultrapure water was used as the blank control. The DPPH free radical scavenging rate was calculated as follows:

[0087] Scavenging rate (%) = [1 - (AS - Ac) / A0] × 100%;

[0088] where AS is the absorbance of the sample, Ac is the absorbance measured with 95% ethanol instead of DPPH, and A0 is the blank control.

[0089] 3.2 ABTS+ Radical Scavenging Assay

[0090] The reaction system consisted of 2.9 mL of ABTS+ dilution solution, 0.1 mL of TPS-5 samples at different concentrations, incubated in the dark at 30 °C for 10 minutes, and the absorbance at 734 nm was measured. The scavenging rate of ABTS+ radicals was calculated as follows:

[0091] Scavenging rate (%) == [1 - (As - Ar) / A0] × 100%;

[0092] where As is the absorbance of the sample, Ar is the absorbance of the control group, and A0 is the absorbance of the blank group.

[0093] 3.3 Ferric Reducing Antioxidant Power (FRAP) Assay

[0094] 150 μL of TPS-5 solutions at different concentrations were mixed with 4.5 mL of FRAP working solution. After reacting at 37 °C for 10 min, the absorbance was measured at 593 nm, substituted into the standard curve of iron reduction ability, and the iron reduction power was calculated. The antioxidant ability was expressed by the value of FeSO4 and calculated as follows:

[0095] Iron reduction ability (mmol / L FeSO4) = Iron reduction ability of the sample - Iron reduction ability of the blank.

[0096] Experimental results: The scavenging abilities of TPS-5 against DPPH radicals and ABTS+ radicals showed concentration dependence in the concentration range of 0.10 - 1.0 mg / mL. The IC50 values of TPS-5 and Vc against DPPH were 1.169 and 0.006 mg / mL respectively, and the IC50 values against ABTS+ radicals were 1.559 and 0.014 mg / mL ( Figure 10 A and B). In the concentration range of 0.05 - 0.2 mg / mL, the iron reduction ability of TPS-5 showed a positive concentration-gradient correlation. At 1.0 mg / mL, the FRAP value reached the maximum of 187.21 μmol / L, which was less than that of Vc ( Figure 10 C). The results indicated that TPS-5 had certain antioxidant effects on DPPH, ABTS+ and iron ion reduction, but was weaker than VC, which might be due to the differences in their molecular structures and antioxidant activity mechanisms.

[0097] Example 4 In vitro Hypoglycemic and Hypolipidemic Activities of Liubao Tea Polysaccharide

[0098] 4.1 Inhibitory Activities of TPS-5 against α-Amylase and α-Glucosidase

[0099] 4.1.1 α-Amylase Inhibitory Activity

[0100] To 2.0 mg / mL TPS-5 solution, add 0.25 mL of 0.1 M phosphate buffer (pH 5.6) containing α-amylase (1 U / mL), and incubate at 37 °C for 10 minutes. Then, add 0.5 mL of 1% starch solution to the mixture and react at 37 °C for 10 minutes. Add 1 mL of DNS reagent to stop the reaction. Then, incubate the mixture in a boiling water bath for 5 min, and add 5 mL of distilled water to dilute the mixture. Measure the absorbance at 540 nm. The α-amylase inhibition rate is calculated according to the following formula:

[0101] Inhibition rate (%) = [(A1 - A2) - (A3 - A4)] / (A1 - A2) × 100;

[0102] where A1, A2, A3, and A4 are the absorbances of the solution without inhibitor and enzyme, the solution with inhibitor and enzyme, the solution with inhibitor, and the solution without enzyme, respectively.

[0103] 4.1.2 α-Glucosidase inhibition activity

[0104] In a 96-well plate, dissolve 50 μL of the extract and 100 μL of the reaction mixture of α-glucosidase (0.7 U / mL) in 100 mM phosphate buffer at pH 6.9, and pre-incubate at 37 °C for 10 min. Then, use 50 μL of p-nitrophenyl-α-D-glucoside as the substrate, incubate at 37 °C for 20 min, and add 1 M Na2CO3 solution to terminate the reaction. Record the absorbance of the mixture at 405 nm, using acarbose as the positive control and water as the blank control. The results are expressed as the α-glucosidase inhibition rate, and the calculation formula is as follows:

[0105] Inhibition rate (%) = [(ΔA O405 - A e405 ) / ΔAO405] × 100;

[0106] In the formula, ΔA O405 is the absorbance without sample; A e405 is the absorbance of the sample.

[0107] Experimental results: α-Amylase is a key enzyme for starch digestion, and α-glucosidase can release α-glucose to promote gastrointestinal absorption and is also a key enzyme for controlling blood glucose levels.

[0108] TPS-5 has a certain dose-effect relationship with the inhibition rates of α-amylase and α-glucosidase. When the concentration of TPS-5 is 5 mg / mL, the inhibition rates of α-amylase and α-glucosidase are as high as 46.68 and 38.94% ( Figure 11 ). The IC 50The values were 4.923 and 0.035 mg / mL respectively, and the IC 50 values were 5.078 and 0.48 mg / mL respectively.

[0109] 4.2 In vitro bile salt binding capacity

[0110] Prepare sodium taurocholate and glycochenodeoxycholate solutions with concentrations of 0, 0.05, 0.10, 0.15, 0.20, and 0.30 mmol / L respectively using deionized water. Then, take 2 mL of each solution and put it into a test tube, add 4 mL of 60% H2SO4, heat the mixed solution in a water bath at 70 °C for 20 min, measure the absorbance at 387 nm after cooling, plot the standard curve of bile salt content, and obtain the regression equation. The standard curves of sodium glycochenodeoxycholate and sodium taurocholate were established.

[0111] Take 3 mL of 2.0 mg / mL TPS-5 solution and put it into a 100 mL Erlenmeyer flask, then add 3 mL of 10 mg / mL pepsin solution, and successively add 1 mL of 0.01 mol / L HCl. The above system was continuously shaken at 37 °C (simulating the gastric digestion environment) for 1 hour. After adjusting the pH to 6.3 with 0.1 mol / L NaOH solution, add 4 mL of 10 mg / mL trypsin solution. Shake the above system at 37 °C for 1 h (simulating the intestinal environment). Thus, the in vitro simulated gastrointestinal digestion sample (SGDS) of TPS-5 was prepared.

[0112] Add 2 mL of 0.4 mmol / L sodium taurocholate and sodium glycocholate to 2.0 mL of SGDS respectively, and react with shaking at 37 °C for 1 hour. Centrifuge for 10 minutes (6000 r / min) and collect the supernatant. Add 4 mL of 60% H2SO4 to 2 mL of the supernatant, heat in a water bath at 70 °C for 20 min, and measure the absorbance at 387 nm after cooling. The residual bile acid content was calculated by the standard curve method. 1 mg / mL cholestyramine replaced the sample solution as a positive control. The bile salt binding rate was calculated as follows:

[0113] Bile salt binding rate (%) = (C0 - C1) / C0 × 100; where C0 is the amount of bile salt, μmol; C1 is the residual bile salt, μmol

[0114] Experimental results: Inhibiting the reabsorption and conversion of bile salts into cholesterol plays an important role in reducing blood lipid. After binding with tea polysaccharide, bile salts can be excreted from the body. Two bile salts (sodium taurocholate and sodium glycocholate) were used to investigate the potential blood lipid-lowering ability of TPS-5. The results showed that TPS-5 had a certain binding ability to bile salts and had application potential in reducing blood lipid. The binding rate of sodium taurocholate (31.46%) was higher than that of sodium glycocholate (10.50%). When the cholestyramine concentration was 100% the same, the relative binding rates of TPS-5 with sodium taurocholate and sodium glycocholate were 43.10 and 12.07% respectively (Table 3).

[0115] Table 3 Binding ability of bile salts and TPS-5

[0116]

[0117] Example 5 Effects of Liubao tea polysaccharide on glycolipid metabolism in HepG2 cells

[0118] Determination of polysaccharide concentration: Human hepatoma HepG2 cells were cultured and treated with 7 doses of TPS-5 (0, 0.2, 0.4, 0.8, 1.2, 1.6, 2.0 mg / mL) for 24 h, and then the cell viability was detected by CCK8 to select a suitable concentration for subsequent experimental treatment (with the standard of not inhibiting cell proliferation).

[0119] Establishment of abnormal glycolipid metabolism cell model: A FFA solution was prepared using a mixture of oleic acid:palmitic acid = 2:1. Under the condition of DMEM culture, 20 mmol / L glucose, 15 mmol / L fructose, 1 mmol / L FFA were additionally used for combined induction treatment for 24 h. In addition, cells were routinely cultured with DMEM medium, and 25 mmol / L glucose and 1% BSA were used as the control group.

[0120] Cell treatment: Corresponding treatments were given according to the grouping. Cells in the cell control group were given 25 mmol / L glucose; cells in the model group were given the corresponding glucose, fructose, and FFA for 24 h; cells in the acarbose treatment group were given 20 μmol / mL acarbose for 24 h after modeling; cells in the lovastatin group were given 0.2 μg / mL lovastatin for 24 h after modeling; cells in the TPS-5 treatment group were given 1 dose of TPS-5 for 24 h after modeling.

[0121] Cell detection: (1) Cell glucose uptake assay: After 24 h of cell treatment, the medium was replaced with DMEM medium containing 12.5 mmol / L. After culturing for 24 h, 5 μL of the supernatant was taken, and the glucose content was measured using a glucose kit to calculate the glucose consumption of the sample in 24 h; (2) Determination of triglyceride (TG) and total cholesterol (TC) contents: After 24 h of cell treatment, cells in each group were collected, and the contents of intracellular triglyceride (TG) and total cholesterol (TC) were detected using a biochemical kit; (3) After 24 h of cell treatment, cell smears were prepared for Oil Red O staining to detect lipid droplet formation.

[0122] Experimental results: The selected concentration of TPS-5 was used for subsequent cell experiments ( Figure 12 ). After Oil Red O staining of the cells in the model group, HepG2 cells were irregular ellipsoidal in shape, with rough edges and a certain gap between cells, and red lipid droplet particles were clear ( Figure 13 B). After adding TPS-5 to act on HepG2 cells, the red lipid droplets decreased to varying degrees ( Figure 13 E). As Figure 14 can be seen, after TPS-5 acted on HepG2 cells for 24 h, the intracellular glucose content decreased, indicating that TPS-5 can reduce the intracellular glucose concentration, thereby achieving the purpose of reducing blood sugar. As Figure 15 can be seen, compared with the cells in the model group, after TPS-5 acted on HepG2 cells for 24 h, the intracellular TG content decreased. As Figure 16 can be seen, after TPS-5 acted on HepG2 cells for 24 h, the intracellular TC content decreased, indicating that TPS-5 has good lipid-lowering and cholesterol-lowering effects on hyperlipidemic HepG2 cells.

[0123] In summary, the polysaccharide component TPS-5 of Liubao tea was obtained by extraction with water extraction and alcohol precipitation methods, purification by DEAE-52 cellulose column and Sephadex G-200 gel column, and its structure was identified and analyzed. The average molecular weight of TPS-5 was 48.289 kDa. Chemical structure analysis showed that TPS-5 was an acidic polysaccharide with pectin structural characteristics, composed of Rha, Ara, Gal, Glu, Man, and Gal A, with a molar ratio of 0.077∶0.120∶0.191∶0.143∶0.023∶0.398. The results of methylation and nuclear magnetic resonance spectroscopy analysis showed that the main chain of TPS-5 was composed of →2,4)-α-L-Rhap-(1→4)-α-D-GalAp-(1→, and the branches were composed of →5)-α-L-Araf-(1→5,3)-α-L-Araf-(1→3)-β-D-Galp-(1→3,6)-β-D-Galp-(1→. The branches were connected to the main chain through the O-4 bond of →2,4)-β-L-Rhap-(1→. In vitro biological activities confirmed that TPS-5 had antioxidant, bile salt binding, α-amylase and α-glucosidase inhibitory activities. HepG2 cell experiments proved that TPS-5 had good effects on reducing blood sugar, blood lipid and cholesterol. These results indicated that TPS-5 could be used as a natural antioxidant resource to regulate blood lipid and blood sugar, and might also be used as a natural ingredient for treating diabetes. This invention laid a theoretical foundation for the application of functional active Liubao tea polysaccharides in health foods or functional foods.

[0124] The above detailed description of the embodiments of the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principles and spirit of the present invention, various changes, modifications, substitutions and variations made to these embodiments still fall within the protection scope of the present invention.

Claims

1. Liubao tea polysaccharide TPS-5, characterized in that, TPS-5 is composed of rhamnose, arabinose, galactose, glucose, mannose, and galacturonic acid, and their molar ratio is 0.077∶0.120∶0.191∶0.143∶0.023∶0.398; the main chain of TPS-5 is composed of →2,4)-α-L-Rhap-(1→4)-α-D-GalAp-(1→, and the repeating unit structure of the main chain is formula (I) and formula (II). The branched chain is composed of →5)-α-L-Araf-(1→5,3)-α-L-Araf-(1→3)-β-D-Galp-(1→3,6)-β-D-Galp-(1→, and the repeating unit structure of the branched chain is formula (III) and formula (IV); the Mw of the said TPS-5 is 48.289 kDa; 2. The preparation method of the Liubao tea polysaccharide according to claim 1, wherein It includes the following steps: S1. Extract the crude polysaccharide of Liubao tea by water extraction - alcohol precipitation method; S2. Separate the crude polysaccharide of Liubao tea with DEAE-52 cellulose, elute it successively with distilled water and NaCl aqueous solution with gradient concentration dilution, the flow rate is 15 mL / h, collect the system components eluted by 0.4 mol / L NaCl aqueous solution, and then the Liubao tea polysaccharide TPS5 is obtained; S3. Purify the Liubao tea polysaccharide TPS5 with Sephadex G-200 gel, elute it with pure water, the elution flow rate is 15 mL / h, collect it every 20 min / pipe, and the Liubao tea polysaccharide TPS-5 is obtained.

3. The preparation method of the Liubao tea polysaccharide according to claim 2, wherein The operation of step S1 is as follows: S11. Add 95% ethanol to the Liubao tea powder at a solid-liquid ratio of 1:10 w / v and soak for 18 hours, stir to remove pigments; S12. Then mix the tea leaves and pure water at a ratio of 1:10 w / v, extract at 90 °C for 2 h, centrifuge, and collect the supernatant; S13. Rotate and evaporate the supernatant to concentrate it, then add three times of absolute ethanol for alcohol precipitation, and refrigerate overnight; S14. Wash the precipitated polysaccharide with absolute ethanol, dissolve it in pure water again, centrifuge and collect the polysaccharide supernatant for freeze-drying; remove the protein in the sample and decolorize it to obtain the crude polysaccharide of Liubao tea.

4. The preparation method of the Liubao tea polysaccharide according to claim 2, wherein In step S2, the sample loading concentration is 20.0 mg / mL, the sample loading volume is 10 mL, the flow rate is 0.25 mL / min, and the eluents are distilled water, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L NaCl aqueous solutions.

5. The preparation method of the Liubao tea polysaccharide according to claim 2, characterized in that, In step S3, the sample loading concentration is 10.0 mg / mL, the sample loading volume is 5 mL, the flow rate is 0.25 mL / min, and the eluent is pure water.

6. Use of the Liubao tea polysaccharide according to claim 1 in the preparation of a functional product, characterized in that, The said functional product has at least one of the following functions: (1) Scavenging of DPPH free radicals and ABTS + free radicals; (2) Inhibit α-amylase and α-glucosidase; (3) Bind sodium taurocholate and sodium glycocholate; (4) Reduce the glucose concentration in the body; (5) Reduce the content of TG and TC in the body.

7. The application according to claim 6, characterized in that, The said functional product is a functional food or a tea product.

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