A method for preparing yellow tea polysaccharide with immune activity

Abstract

The present invention relates to a method for preparing yellow tea polysaccharide with immunological activity, comprising: decocting a yellow tea sample with hot water, precipitating with alcohol, and freeze-drying to obtain a primary yellow tea crude polysaccharide; subjecting the primary yellow tea crude polysaccharide to graded precipitation to obtain a secondary yellow tea crude polysaccharide, and then subjecting the primary yellow tea crude polysaccharide to anion exchange column chromatography, elution, dialysis, and freeze-drying to obtain the yellow tea polysaccharide. The obtained yellow tea polysaccharide is composed of a uniform polysaccharide, and its monosaccharide composition is pyranose, pyranose glucose, furanose, and pyranose galacturonic acid, and the molar ratio of pyranose, pyranose, furanose, and pyranose galacturonic acid is 0.7:0.7:1.2:0.5, and the glycosidic bond linking mode in the polysaccharide is clearly identified, providing safety guarantee for the medicinal quality control of the yellow tea polysaccharide.

Description

Technical Field

[0001] The invention belongs to the field of health food, and particularly relates to a method for preparing yellow tea polysaccharide with immune activity. Background Art

[0002] Tea is a widely consumed beverage worldwide, and its extracts are used as medicine and dietary supplements in many countries, including China, Japan, and the United States. Tea contains a variety of bioactive compounds, including tea polyphenols, theanine, and tea polysaccharides, which are beneficial to health. Tea polysaccharides are a group of protein-bound heteropolysaccharides extracted from the leaves, flowers, and seed skins of the tea plant. Studies have shown that tea polysaccharides can not only improve immunity, but also have a variety of biological activities, such as anti-oxidation, anti-tumor, anti-hyperglycemia, and anti-inflammatory.

[0003] Huangda tea, also known as Anhui Huangda tea, is produced in Huoshan, Jinzhai, Yuexi and other places in Anhui Province. In addition to its unique tea fragrance, its tea soup also has a variety of health functions. Its extract is called Huangda tea polysaccharide. At present, the specific pharmacological material basis of the biological activity of Huangda tea polysaccharide components is still unclear. In addition, due to the structural complexity, variety and component heterogeneity of the existing Huangda tea polysaccharide, it is difficult to ensure the quality of Huangda tea polysaccharide as medicine.

[0004] Therefore, the present invention provides a method for preparing yellow tea polysaccharide with immunological activity, and obtains polysaccharide with uniform composition and immunological activity. Summary of the invention

[0005] In view of the above problems, the present invention provides a method for preparing yellow tea polysaccharide with immunological activity, the preparation method comprising:

[0006] The yellow tea sample is decocted with hot water, precipitated with alcohol, and freeze-dried to obtain the crude yellow tea polysaccharide;

[0007] The primary yellow tea crude polysaccharide is subjected to graded precipitation to obtain the secondary yellow tea crude polysaccharide, and then subjected to anion exchange column chromatography, elution, dialysis and freeze-drying to obtain the yellow tea polysaccharide.

[0008] Furthermore, the yellow tea sample is dried yellow tea leaves.

[0009] Furthermore, when decocting with hot water, the volume ratio of the yellow tea sample to the hot water is 1:4-6.

[0010] Furthermore, the hot water decocting operation is performed 2-5 times.

[0011] Furthermore, the mass concentration of ethanol used in the alcohol precipitation is 80%, the volume ratio of the filtrate obtained by water decoction to 80% ethanol is 1:4-7, the alcohol precipitation temperature is 25-30°C, and the alcohol precipitation time is 12 hours.

[0012] Furthermore, the mass concentration of ethanol used in the graded precipitation is 20%, 40%, 60%, and 80%, and the alcohol precipitation time for each time is 10-24 hours.

[0013] Furthermore, the concentration of the secondary yellow tea crude polysaccharide solution in the anion exchange column chromatography is 5-10 mg / mL.

[0014] Furthermore, the eluent during the elution is a NaCl solution with a concentration of 0 mol / L.

[0015] Furthermore, the eluent during the elution is a NaCl solution with a concentration of 0.2 mol / L.

[0016] Furthermore, the flow rate of the eluent is 2.0 mL / min.

[0017] Furthermore, the relative molecular mass of the yellow tea polysaccharide is 6.1 kDa.

[0018] Furthermore, the monosaccharide composition of the yellow tea polysaccharide is pyranose mannosyl, pyranose glucose, furanose xylose and pyranose galacturonic acid, and the molar ratio of pyranose mannosyl, pyranose glucose, furanose xylose and pyranose galacturonic acid is 0.7:0.7:1.2:0.5.

[0019] Furthermore, the main glycosidic bond connection mode in the molecule of the yellow tea polysaccharide is β-D-Glcp(3→,→3)-α-D-Xylp(4→,→1)-β-D-Manp(3→,→3)-α-D-Xylp(4→and→2)-α-D-GalAp(3→.

[0020] Beneficial effects of the present invention:

[0021] The yellow tea polysaccharide obtained by the present invention is a uniform polysaccharide, and its monosaccharide composition is pyranose glucose, pyranose mannose, furanose xylose and pyranose galacturonic acid, and the molar ratio of pyranose glucose, pyranose mannose, furanose xylose and pyranose galacturonic acid is 0.7:0.7:1.2:0.5, and the glycosidic bond linking mode in the polysaccharide is clearly shown, which provides safety guarantee for the medicinal quality control of the yellow tea polysaccharide.

[0022] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The process flow chart of preparing yellow tea polysaccharide according to an embodiment of the present invention is shown;

[0025] Figure 2 The DEAE-52 anion exchange column chromatography elution diagram of the yellow tea polysaccharide according to the embodiment of the present invention is shown;

[0026] Figure 3 The DPPH antioxidant test diagram of the yellow tea polysaccharide according to the embodiment of the present invention is shown;

[0027] Figure 4 The ultraviolet spectrum of yellow tea polysaccharide according to the embodiment of the present invention is shown;

[0028] Figure 5 The HPGPC diagram of the yellow tea polysaccharide according to the embodiment of the present invention is shown;

[0029] Figure 6 The infrared spectrum of the yellow tea polysaccharide according to the embodiment of the present invention is shown;

[0030] Figure 7 The GC-MS diagram of monosaccharide composition analysis of yellow tea polysaccharide according to the embodiment of the present invention is shown;

[0031] Figure 8 The results show that the polysaccharide of yellow tea in the embodiment of the present invention 1 H-NMR spectrum;

[0032] Fig. 9 The results show that the polysaccharide of yellow tea in the embodiment of the present invention 13 C-NMR spectrum;

[0033] Fig.10 The results show that the polysaccharide of yellow tea in the embodiment of the present invention 1 H- 1 H COSY diagram;

[0034] Fig.11 The HSQC diagram of the yellow tea polysaccharide according to the embodiment of the present invention is shown;

[0035] Fig.12 The HMBC diagram of the yellow tea polysaccharide according to the embodiment of the present invention is shown;

[0036] Fig.13 The structural diagram of the yellow tea polysaccharide according to the embodiment of the present invention is shown;

[0037] Fig.14 The in vitro cell experiment diagram of yellow tea polysaccharide according to the example of the present invention is shown. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] like Figure 1 As shown, a method for preparing yellow tea polysaccharide with immunological activity comprises the following steps:

[0040] The yellow tea sample is decocted with hot water, precipitated with alcohol, and freeze-dried to obtain the crude yellow tea polysaccharide;

[0041] The primary yellow tea crude polysaccharide is subjected to graded precipitation to obtain the secondary yellow tea crude polysaccharide;

[0042] The secondary yellow tea crude polysaccharide is subjected to anion exchange column chromatography, elution, dialysis and freeze-drying to obtain yellow tea polysaccharide.

[0043] Example 1

[0044] Preparation of yellow tea polysaccharide:

[0045] (1) boiling dried leaves of Huang Da Cha with hot water, wherein the volume ratio of Huang Da Cha to hot water is 1:4, the temperature of the hot water is 100° C., the boiling time is 1 hour, and filtering to obtain a filtrate;

[0046] (2) the filtrate is precipitated with 80% ethanol, wherein the volume ratio of the filtrate to 80% ethanol is 1:4, the alcohol precipitation temperature is room temperature 25-30° C., the alcohol precipitation time is 12 hours, and the precipitate is collected by centrifugation at 5000 rpm / min for 15 minutes, and the precipitate is freeze-dried to obtain the crude polysaccharide of primary yellow tea;

[0047] (3) The crude polysaccharide of yellow tea was precipitated by graded alcohol with ethanol of mass concentration of 20%, 40%, 60% and 80%, respectively, the precipitation temperature was room temperature 25-30°C, the precipitation time was 12 hours, centrifuged at 5000 rpm / min for 15 minutes, the precipitate was collected, freeze-dried, and the best component was screened by DPPH antioxidant test, and the secondary crude polysaccharide of yellow tea obtained by graded alcohol precipitation with ethanol of mass concentration of 60% was the best;

[0048] (4) The crude polysaccharide of the second yellow tea leaf obtained by graded alcohol precipitation with 60% ethanol by mass concentration was made into a 10 mg / mL solution, and the solution was chromatographed on a DEAE-52 anion exchange column (3.5×30 cm) and eluted, and the polysaccharide-containing eluate was collected to obtain the yellow tea leaf polysaccharide eluate, wherein the eluent was 0, 0.1, 0.2, and 0.5 mol / L NaCl solution, and the flow rate of the eluent was 2.0 mL / min. The polysaccharide content in the eluate was detected by phenol-sulfuric acid colorimetry (see Figure 2 ), and two polysaccharide components were obtained, namely HDCP-1 eluted by 0 mol / L NaCl solution and HDCP-2 eluted by 0.2 mol / L NaCl solution. No polysaccharide components were obtained with other concentrations of NaCl solution.

[0049] Example 2

[0050] The polysaccharide content in different eluents in Example 1 was detected by phenol-sulfuric acid colorimetry. The measurement results are as follows: Figure 2 As shown, two components were obtained, namely, HDCP-1 eluted with 0 mol / L NaCl solution and HDCP-2 eluted with 0.2 mol / L NaCl solution;

[0051] The obtained yellow tea polysaccharide HDCP-2 has a uniform composition, consisting of glucose, mannose, xylose and galacturonic acid, and has a relative molecular mass of 6.1 kDa.

[0052] Example 3

[0053] DPPH free radical scavenging experiment: The freshly prepared reaction mixture DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) (0.2 mM methanol solution, 2.0 mL) and various concentrations of yellow tea polysaccharide (2.0 mL) solution (0.2-1.0 mg / mL) were reacted continuously in the dark at room temperature (25°C) for 30 min, and then the absorbance of the mixture was measured at 517 nm, and the scavenging rate was calculated according to the following formula.

[0054]

[0055] In the above formula, A blank is the absorbance of the blank solution, A sample is the absorbance value of the sample solution, A control is the absorbance value of the control solution.

[0056] The best components were screened based on free radical scavenging efficiency. Figure 3 The DPPH radical scavenging rates of HDCP-2 eluted with 0.2 mol / L NaCl solution and HDCP-1 eluted with 0 mol / L NaCl solution (i.e. pure water) are given. Figure 3It can be seen that HDCP-2 is a better component.

[0057] According to the ratio of the amount of the refined polysaccharide obtained to the amount of the dried leaves of Camellia ternata used, the extraction rate of Camellia ternata polysaccharide HDCP-2 was 0.19%.

[0058] Example 4

[0059] The physicochemical properties of yellow tea polysaccharide are analyzed as follows:

[0060] (1) Determination of carbohydrate content of yellow tea polysaccharide freeze-dried powder by anthrone-sulfuric acid method

[0061] Take 33 mg of anhydrous glucose reference substance that has been dried to constant weight, accurately weigh it, place it in a 100 mL volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain (each 1 mL contains 0.33 mg of anhydrous glucose). Accurately measure 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the reference substance solution, place them in 10 mL centrifuge tubes respectively, add water to 1.0 mL each, shake well, slowly drop 4 mL of 0.2% anthrone-sulfuric acid solution in an ice water bath, mix well, cool it, and heat it in a 100°C boiling water bath for 20 min. Take it out, immediately cool it in an ice water bath for 10 min, measure the absorbance at a wavelength of 582 nm, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa;

[0062] Weigh 10 mg of each yellow tea polysaccharide freeze-dried powder (HDCP-2), dissolve it in pure water to form a 0.5 mg / mL polysaccharide solution, take 0.4 mL of each solution, place it in a 10 mL test tube, and measure the absorbance of each sample solution.

[0063] The polysaccharide content was calculated and the carbohydrate content in HDCP-2 was 56.18%. The instrument used was SHIMADZU UV-2550 UV-visible spectrophotometer.

[0064] (2) Determination of uronic acid content of yellow tea polysaccharide freeze-dried powder by m-hydroxybiphenyl method

[0065] Weigh 0.955g of sodium tetraborate and dissolve it in 100mL of concentrated sulfuric acid. Weigh 0.015g of m-hydroxybiphenyl and dissolve it in 0.5% NaOH solution, dilute to 10mL, accurately weigh 5mg of anhydrous galacturonic acid, dissolve it in pure water and dilute to 10mL with a 10mL volumetric flask to obtain a 0.5mg / mL galacturonic acid standard solution.

[0066] Accurately pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0 mL of the above anhydrous galacturonic acid standard solution into 10 mL test tubes, add double distilled water to each tube to 1.0 mL, place the test tube in an ice bath and add 5 mL of cooled borate solution dropwise, shake and mix well, place it in a boiling water bath for 20 min, take it out and cool it to room temperature, add 100 μL of m-hydroxybiphenyl solution to each tube, mix well and place it for 30 min. Accurately weigh 10 mg of yellow tea polysaccharide freeze-dried powder (HDCP-2), dissolve it in pure water to form a 0.5 mg / mL polysaccharide solution, take 0.4 mL of each solution, place it in 10 mL test tubes, and measure the absorbance value at a wavelength of 525 nm.

[0067] The content of uronic acid in the obtained yellow tea polysaccharide HDCP-2 was determined to be 4.28%. The instrument used was SHIMADZU UV-2550 ultraviolet visible spectrophotometer.

[0068] (3) UV scanning was used to determine the freeze-dried powder of crude polysaccharide of yellow tea (corresponding to Figure 4 polysaccharides in), secondary yellow tea crude polysaccharide freeze-dried powder obtained by 60% ethanol graded alcohol precipitation (corresponding to Figure 4 60% alcohol precipitation component in the yellow tea polysaccharide freeze-dried powder (HDCP-1 (corresponding to Figure 4 Pure water in) and HDCP-2 (corresponding to Figure 4 The UV absorption spectrum of 0.2 mol / L NaCl) is as follows Figure 4 As shown, the UV scanning spectrum of the obtained yellow tea polysaccharide HDCP-2 has no absorption peaks of pigments, proteins and nucleic acids. The instrument used is a SHIMADZU UV-2550 UV-visible spectrophotometer with a scanning range of 200 to 800 nm.

[0069] Example 5

[0070] The homogeneity and relative molecular weight of HDCP-2 polysaccharide obtained from yellow tea were determined by HPGPC method

[0071] Test conditions: Agilent 1260 Infinity system; SRT SEC-150 (7.8×300 mm) chromatographic column; mobile phase: double distilled water; injection volume: 10 μL; flow rate: 1.0 mL / min; column temperature: 35°C; differential refractometer.

[0072] Test results see Figure 5 After testing, the molecular weight determination standard curve was LogMw = -0.50x + 9.13, R 2 =0.998 (Mw: molecular weight), the peak position is 8.723 minutes, and the obtained yellow tea polysaccharide HDCP-2 is a uniform polysaccharide with a relative molecular mass of 6.1 kDa.

[0073] Example 6

[0074] The characteristic groups of HDCP-2 polysaccharide from yellow tea were analyzed by infrared spectrometer

[0075] Weigh about 2.0 mg of dried yellow tea polysaccharide HDCP-2 sample, mix with 200 mg of KBr powder, press into tablets, and then scan and analyze on L1600400Spectrum TWO FT-IR Fourier transform infrared spectrometer with a scanning range of 4000 cm -1 ~400cm -1 .

[0076] Test results see Figure 6 After testing, the obtained yellow tea polysaccharide HDCP-2 has the characteristic peaks of polysaccharides, with a broad peak at 3400cm -1 , belonging to -OH in polysaccharide, 2936cm -1 represents the CH asymmetric stretching vibration. The peak is about 1614cm -1 represents -C=O stretching vibration, and a weak peak at 1415 cm -1 The position is due to the stretching vibration of C=O. 1018cm -1 、1100cm -1 and 1143cm -1 The absorption peak at 833cm represents the asymmetric stretching vibration of COC. -1 It indicates that there is an α-glycosidic bond.

[0077] Example 7

[0078] The chemical structure of the yellow tea polysaccharide HDCP-2 is identified as follows:

[0079] (1) Determination of the monosaccharide composition of the yellow tea polysaccharide HDCP-2 obtained in Example 1 by GC-MS

[0080] The dried polysaccharide sample (30 mg) was reacted with 3 mL 4 M TFA at 120 ° C for 4 h, centrifuged, added with 1 mL methanol, and dried under reduced pressure (3-5 times); the hydrolyzed sample was dissolved in 2 mL anhydrous DMSO, ultrasonically dissolved for 5 min, added with 90 mg NaOH, stirred at 35 ° C for 1 h, and slowly added 2 mL CH 3 I. Block the reaction at 35°C in the dark for 12 h, add 2 mL of distilled water to stop the reaction, extract the methylated sample with 3 mL of dichloromethane, wash with the same volume of distilled water for 3 times, remove water with anhydrous sodium sulfate, dry under reduced pressure, dilute with dichloromethane and perform GC-MS detection.

[0081] Test conditions: BR-17 chromatographic column (0.25μm×250μm×30m), carrier gas: high-purity helium (purity 99.999%) and high-purity nitrogen (purity 99.999%): Nanjing Shangyuan Industrial Gas Plant; column flow rate: 1.0mL / min; split injection, inlet temperature 250℃. The temperature program was set as follows: initial temperature 60℃, 20℃ / min to 100℃, hold for 2min, then 4℃ / min to 180℃. Mass spectrometry conditions: EI ion source, electron energy 70eV, ion source temperature 220℃, transmission line temperature 280℃, quadrupole temperature 40℃. Full scan mode, scan range m / z 40~600, mass spectrometry standard library: NIST 11.L.

[0082] GC-MS test results are as follows Figure 7 As shown, the yellow tea polysaccharide HDCP-2 obtained in Example 1 is composed of pyranose mannosyl (Manp), pyranose glucose (Glcp), furanose xylose (Xylp) and pyranose galacturonic acid (GalAp), and the molar ratio thereof is 0.7:0.7:1.2:0.5.

[0083] (3) Glycosidic bond types of HDCP-2 polysaccharide from yellow tea obtained by methylation analysis

[0084] Weigh 15 mg of fully dried polysaccharide, add 2 mL of anhydrous dimethyl sulfoxide (DMSO) to fully dissolve, then add 90 mg of NaOH powder, stir and react for 2 hours, then add 2 mL of anhydrous iodomethane, react at 35°C in a sealed and light-proof place for 12 hours, add an appropriate amount of distilled water to terminate the reaction, and obtain a completely methylated sample.

[0085] Take the completely methylated sample and place it in a 10mL ampoule, add 3mL of 4mol / L trifluoroacetic acid, seal the tube, hydrolyze at 120℃ for 6h, cool at room temperature, transfer the hydrolyzate to a rotary evaporator and evaporate to dryness, then add appropriate amount of methanol to evaporate to dryness to completely remove the residual trifluoroacetic acid, then add 1.5mL 0.05M NaOH solution and 45mg sodium borohydride, react for 12h at room temperature, neutralize with 100μL of acetic acid to neutrality, and place the reaction solution on a rotary evaporator to evaporate to dryness; add 1mL of acetic anhydride and 1mL of pyridine to the reaction solution in sequence, seal it and react at 90℃ for 2h; after the reaction, extract the acetylation product with dichloromethane, remove residual water from the extract with anhydrous sodium sulfate, and perform GC-MS determination.

[0086] Detection conditions: BR-17 chromatographic column (0.25 μm × 250 μm × 30 m), carrier gas: high-purity helium (purity 99.999%) and high-purity nitrogen (purity 99.999%): Nanjing Shangyuan Industrial Gas Plant; column flow rate: 1.0 mL / min; split injection, inlet temperature 250 ° C. The temperature program was set as follows: initial temperature 50 ° C, 25 ° C / min to 100 ° C, hold for 3 min, and then 4 ° C / min to 180 ° C.

[0087] Mass spectrometry conditions: EI ion source, electron energy 70 eV, ion source temperature 220 ° C, transmission line temperature 280 ° C, quadrupole temperature 40 ° C. Full scan mode, scan range m / z 40-600, mass spectrometry standard library: NIST 11.L.

[0088] After testing, the obtained methylation product of yellow tea polysaccharide HDCP-2 is shown in Table 1, and the specific methylation structure analysis is shown in Table 1.

[0089] Table 1 Methylation analysis of HDCP-2 polysaccharide from yellow tea

[0090]

[0091] (3) Chemical structural characteristics of the yellow tea polysaccharide HDCP-2 obtained by NMR

[0092] Take 30 mg of polysaccharide and completely dissolve it in 99.9% D 2 O, placed in a nuclear magnetic tube, and subjected to NMR measurement.

[0093] Detection conditions: VNMRS600 superconducting nuclear magnetic resonance spectrometer; One NMR probe (5mm); 1 H spectrum operating frequency 599.81MHz; 13 C spectrum working frequency 150.84MHz; measured at 25℃ 1 H spectrum, measured at 25°C 13 C, COSY, HSQC, and HMBC spectra.

[0094] See also Figure 8-Figure 12 The obtained yellow tea polysaccharide HDCP-2 is composed of →1)-β-D-Manp(3→,β-D-Glcp(3→,→3)-α-D-Xylp(4→ and →2)-α-D-GalAp(3→, wherein, Figure 8 The 298K hydrogen spectrum ( 1 H NMR spectrum), Fig. 9 is the carbon spectrum ( 13 C NMR spectrum), Fig.10 is the homonuclear chemical shift correlation spectrum ( 1 H-1 H COSY diagram), Fig.11 is the heteronuclear single quantum relationship spectrum (HSQC diagram), Fig.12 The HMBC diagram is a multi-bond carbon-hydrogen relationship diagram. The chemical shift analysis of the five sugar residues is shown in Table 2. The specific NMR spectrum structure analysis is shown in Table 2.

[0095] Table 2 NMR analysis of Polygonatum cyrtonema polysaccharide PKP 2-1

[0096]

[0097] Based on the above analysis, the following results can be obtained: the composition of the yellow tea polysaccharide HDCP-2 is uniform, and the relative molecular mass is 6.1 kDa. The monosaccharide composition analysis and methylation results show that the yellow tea polysaccharide HDCP-2 of the present invention contains pyranose mannosyl (Manp), pyranose glucose (Glcp), furanose xylose (Xylp) and pyranose galacturonic acid (GalAp), and the molar ratio is 0.7:0.7:1.2:0.5; the results of nuclear magnetic resonance analysis show that the configuration of the sugar residues is α and β type, and its primary structural unit contains five sugar residues: →1)-β-D-Manp(3→, β-D-Glcp(3→, →3)-α-D-Xylp(4→ and →2)-α-D-GalAp(3→, and the connection method of the five sugar residues is as follows Fig.13 shown.

[0098] The anti-rheumatoid arthritis effect of Polygonatum sibiricum polysaccharide with anti-inflammatory immune activity is analyzed as follows:

[0099] (1) Cell culture

[0100] DMEM cell culture medium containing 10% FBS and 1% double antibody, 37°C, 5% CO 2 , cultured in a saturated humidity constant temperature incubator. When MH7A cells grew to cover 70%-80% of the bottom of the culture flask, washed three times with PBS solution using a 3mL pipette, digested the cells with 0.25% trypsin digestion solution, terminated cell digestion with serum-containing DMEM complete medium, and cell passaged at a ratio of 1:2 to complete the relevant experiments.

[0101] (2) Cell proliferation activity assay

[0102] Take the MH7A cells that grow normally in the logarithmic phase, digest them, collect the cells by centrifugation, count them under a counting plate, and make 1×10 5 100 μL of single cell suspension was inoculated into each well of a 96-well plate and incubated at 37°C with 5% CO 2After overnight culture, the cells were allowed to adhere to the wall for 24 hours, and then stimulated with TNF-α (10 ng / mL) and treated with different concentrations (50, 100, 200 μg / mL) of Polygonatum sibiricum polysaccharide PKP 2-1 for 24 hours. 10 μL of CCK-8 solution was added to each well and the cells were placed in the incubator for another 1 hour. The absorbance (OD) value of each well at 450 nm was measured with an ELISA reader. The experiment was repeated 3 times, the average was taken, and the cell survival rate was calculated.

[0103] like Fig.14 As shown, the concentrations of the normal group, model group and three HDCP-2 groups were 50, 100, and 200 μg / mL, respectively, and the cells were treated with MH7A cells for 24 hours. The cell viability of the normal group was 100%. When TNF-α (10 ng / mL) stimulated MH7A cells for 1 hour, the corresponding cell viability was calculated to be 127%, indicating inflammation promotion. After HDCP-2 acted on MH7A cells at concentrations of 50, 100, and 200 μg / mL for 24 hours, the cell survival rates were 102%, 91%, and 87%, respectively. Therefore, with the increase of HDCP-2 concentration, the survival rate of MH7A cells decreased.

[0104] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing yellow tea polysaccharide with immunological activity, characterized in that: include: (1) boiling dried leaves of Huang Da Cha with hot water, wherein the volume ratio of Huang Da Cha to hot water is 1:4, the temperature of the hot water is 100° C., the boiling time is 1 hour, and filtering to obtain a filtrate; (2) The filtrate was precipitated with 80% ethanol, wherein the volume ratio of the filtrate to the 80% ethanol was 1:4, the precipitation temperature was room temperature 25-30°C, the precipitation time was 12 hours, and the precipitate was collected by centrifugation at 5000 rpm for 15 minutes, and the precipitate was freeze-dried to obtain the crude polysaccharide of yellow tea; (3) The crude polysaccharide of primary yellow tea was precipitated with ethanol of mass concentration of 20%, 40%, 60%, and 80%, respectively. The precipitation temperature was room temperature (25-30°C) and the precipitation time was 12 hours. The precipitate was collected and freeze-dried at 5000 rpm for 15 min. The best component was screened by DPPH antioxidant test. The crude polysaccharide of secondary yellow tea obtained by ethanol of mass concentration of 60% was the best. (4) The crude polysaccharide of camellia sinensis obtained by secondary alcohol precipitation with 60% ethanol by mass concentration was made into a 10 mg / mL solution, and the solution was chromatographed and eluted with a DEAE-52 anion exchange column. The polysaccharide-containing eluate was collected to obtain the polysaccharide eluate of camellia sinensis. The size of the DEAE-52 anion exchange column was 3.5×30 cm, and the eluent was 0, 0.1, 0.2, and 0.5 mol / L NaCl solution. The flow rate of the eluent was 2.0 mL / min. The polysaccharide eluate of camellia sinensis obtained by eluting with the 0.2 mol / L NaCl solution was dialyzed and freeze-dried to obtain polysaccharide of camellia sinensis.

2. The method for preparing the yellow tea polysaccharide with immunological activity according to claim 1, characterized in that: The monosaccharide composition of the yellow tea polysaccharide is mannose, glucopyranose, xylofuranose and galactopyranose, and the molar ratio of mannose, glucopyranose, xylofuranose and galactopyranose is 0.7:0.7:1.2:0.

5.

3. The method for preparing the yellow tea polysaccharide with immunological activity according to claim 1, characterized in that: The main glycosidic bond connection mode in the molecule of the yellow tea polysaccharide is β-D-Glcp(3→,→3)-α-D-Xylp(4→,→1)-β-D-Manp(3→,→3)-α-D-Xylp(4→ and→2)-α-D-GalAp(3→.

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