CN110498865B - Selaginella maxima polysaccharide, preparation method thereof and application thereof in preparation of anticomplement medicines - Google Patents
Selaginella maxima polysaccharide, preparation method thereof and application thereof in preparation of anticomplement medicines Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicines, and relates to four homogeneous polysaccharides in sabina macrocarpa, a preparation method thereof and application thereof in preparation of anticomplement medicines. The invention separates four homogeneous polysaccharides YB-PS2, YB-PS3, YB-PS4 and YB-PS5 from the traditional Chinese medicine sabina chinensis for clearing heat and removing toxicity, and experiments prove that the homogeneous polysaccharides have obvious inhibition effect on complement activation and can be further used as active ingredients to prepare novel anticomplement medicines.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicines, relates to polysaccharide, a preparation method thereof and application thereof in preparation of products, and particularly relates to four natural homogeneous polysaccharides in sabina macrocarpa, a preparation method thereof and application thereof in preparation of anticomplement medicines.
Background
The complement system is an important component of the human immune system, and its normal activation plays an important role in exterminating foreign microorganisms, removing damaged or dead cells and tissues in the body, and maintaining the balance of the body. However, excessive activation of this system can cause excessive reaction of the human immune system, resulting in damage to normal tissues of the human body, such as rheumatoid arthritis, senile dementia, Systemic Lupus Erythematosus (SLE), and rejection after organ transplantation. Over-activation of complement also plays an important role in multiple organ failure syndromes, such as acute diseases like ischemic reperfusion, acute myocardial infarction, Acute Respiratory Distress Syndrome (ARDS) and the like.
At present, the immunosuppressive agents such as glucocorticoid, cyclophosphamide, methylamine pterine and the like which are widely used clinically have a certain treatment effect on certain diseases related to excessive complement activation, but because the drugs are not specific complement inhibitors, the long-term application can reduce the defense function of the organism, lead to the reduction of the anti-infection capability, facilitate secondary infection and spread potential focus, and generate various complications and side effects. Therefore, a novel complement inhibitor with high efficiency, low toxicity and specificity is urgently needed in clinic.
Active ingredients with anticomplementary effects widely exist in nature, Chinese medicine resources are rich, a plurality of Chinese medicines have obvious regulating effects on an immune system, and the method is a valuable resource for searching anticomplementary prodrug. During the period of resisting SARS, the Chinese medicine preparation has obvious SARS preventing and treating effect. Sabina chinensis, also known as Xiuba, Xuba, Jiaxiu and Xiu, is recorded in Jingzhu materia Medica, is used for treating pneumonia, rheumatic arthritis, rheumatoid arthritis, cholecystitis, nephritis, anthracnose, carbuncle furuncle pyogenic infections, yellow water disease and the like, is often used together with other Tibetan medicines, such as five-flavor manna decoction, ten-flavor myrobalan pill, fourteen-flavor antelope horn pill, twenty-eight-flavor areca pill and the like, and especially has good curative effect on arthritis frequently occurring in Qinghai-Tibet plateau as the medicinal bath with the characteristics of Tibetan medicines in the five-flavor manna decoction. The Selaginella macrocarpa (Sabina tibetica) is a common medicine for plants of Selaginella in Cupressaceae of Tibet plateau, and fruits and leaves can be used as medicine for clearing kidney heat, eliminating dampness, and removing toxic substance. The research finds that the crude polysaccharide of the sabina macrocarpa has obvious anticomplementary activity, CH50And AP5076 +/-9 mu g/ml and 87 +/-4 mu g/ml respectively, but the report on the separation and preparation of the homogeneous polysaccharide with the anticomplementary activity in the sabina macrocarpa is not found.
Based on the basis of the prior art, the inventor of the application tries to strengthen the research on the anti-complement active ingredients of the related traditional Chinese medicines and provides a material basis for the treatment of complement-related diseases, in particular to the sabina macrocarpa polysaccharide, the preparation method thereof and the application thereof in the preparation of anti-complement medicines.
Disclosure of Invention
The invention aims to provide active ingredients with anticomplementary effect in natural medicines based on the basis of the prior art, and particularly relates to sabina vulgaris polysaccharide, a preparation method thereof and application thereof in preparing anticomplementary medicines, in particular to four sabina vulgaris homopolysaccharides (YB-PS2, YB-PS3, YB-PS4 and YB-PS5), a preparation method thereof and application thereof in preparing anticomplementary medicines.
The invention separates the water extract of the traditional Chinese medicine sabina chinensis which can clear away heat and toxic material to obtain four homogeneous polysaccharides, and the four polysaccharides have obvious complement inhibition activity and can be developed as anticomplement medicines through in vitro experiments.
In the invention, the traditional Chinese medicine Sabina chinensis is the branches, leaves and fruits of Sabina chinensis Sabina tibetiica of Sabina plants
The structural characteristics of the sabina chinensis polysaccharide YB-PS2, YB-PS3, YB-PS4 and YB-PS5 are described as follows:
(1) YB-PS2 is a polysaccharide composed of three monosaccharides, and has a molecular weight of about 81.60 KDa; the total sugar content was 94.47%; the protein content was 3.25%; the uronic acid content is 1.89%; contains no sulfuric acid group. The monosaccharide composition showed that it was composed of arabinose, galactose and glucose in a molar ratio of 0.58:0.30: 0.12. The methylation reaction showed that it contained: 1,3, 5-linked arabinose, 1, 3-linked arabinose, terminal-linked arabinose, 1,2, 4-linked galactose, 1, 4-linked galactose, terminal-linked galactose and 1, 3-linked glucose in a molar ratio of 0.208:0.126:0.233:0.09:0.112:0.109: 0.132.
(2) YB-PS3 is a polysaccharide composed of five monosaccharides and has a molecular weight of about 40.47 KDa; total sugar content 95.21%; the protein content was 3.63%; the uronic acid content is 16.62%; contains no sulfuric acid group. The monosaccharide composition showed that it consisted of arabinose, galactose, rhamnose, galacturonic acid and 3-methyl galactose in a molar ratio of 0.16:0.27:0.24:0.17: 0.14. The methylation result shows that the structure contains: 1, 3-linked arabinose, terminal-linked arabinose, 1,2, 4-linked galactose, terminal-linked galactose, 1,2, 4-linked rhamnose, 1, 2-linked rhamnose, terminal-linked rhamnose, 1, 2-linked 3-methyl galactose, 1, 4-linked galacturonic acid in a molar ratio of 0.076:0.082:0.205:0.091:0.077:0.085:0.069:0.158: 0.147.
(3) YB-PS4 is a polysaccharide composed of five monosaccharides and has a molecular weight of about 218.47 KDa; total sugar content 96.69%; the protein content is 2.04%; the uronic acid content is 25.34%; contains no sulfuric acid group. The monosaccharide composition showed that it consisted of arabinose, galactose, rhamnose, galacturonic acid and 3-methyl galactose in a molar ratio of 0.21:0.27:0.19:0.20: 0.13. The methylation result shows that the structure contains: 1, 3-linked arabinose, terminal-linked arabinose, 1,2, 4-linked galactose, terminal-linked galactose, 1,2, 4-linked rhamnose, 1, 2-linked rhamnose, terminal-linked rhamnose, 1, 2-linked 3-methyl galactose and 1, 4-linked galacturonic acid in a molar ratio of 0.071:0.069:0.140:0.154:0.071:0.061:0.066:0.142: 0.198.
(4) YB-PS5 is a polysaccharide composed of two monosaccharides and has a molecular weight of about 153.33 KDa; the total sugar content was 95.23%; the protein content was 2.13%; the uronic acid content is 3.68%; contains no sulfuric acid group. Monosaccharide composition showed that it was composed of glucosamine, rhamnose, in a molar ratio of 0.44: 0.56. Methylation results showed that it contained end-set linked glucosamine, 1, 4-linked rhamnose and 1,2, 4-linked rhamnose in a molar ratio of 0.41:0.21: 0.38.
The conifer polysaccharide (YB-PS2, YB-PS3, YB-PS4 and YB-PS5) is prepared by the following method:
extracting Thuja Dolabrata with 95% ethanol, filtering, oven drying the residue, extracting with water solution, filtering the extractive solution, concentrating, adding 4 times of 95% ethanol, standing, centrifuging to remove supernatant, dissolving the precipitate with water, removing protein with trichloroacetic acid, centrifuging, adjusting the supernatant to neutral, concentrating, dialyzing, and freeze drying to obtain crude polysaccharide. Dissolving the crude polysaccharide with distilled water, and performing primary separation by DEAE-cellulose chromatography. Eluting with distilled water and NaCl solution of 0.1, 0.4, 0.8, 1.6 and 2.0mol/L, collecting each fraction, concentrating, dialyzing and lyophilizing to obtain 6 secondary components: WATER-EP-YB, 0.1M-EP-YB, 0.4M-EP-YB, 0.8M-EP-YB, 1.6M-EP-YB, and 2.0M-EP-YB.
Dissolving each secondary component in appropriate amount of mobile phase, centrifuging, and collecting supernatant with SephacrylTMSeparating by S300 (molecular weight cut-off of 2KDa-400KDa) chromatography, and collecting each flow. Detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with separate tube, mixing the fractions, concentrating, and freeze drying to obtain uniform polysaccharide
In vitro tests prove that YB-PS2, YB-PS3, YB-PS4 and YB-PS5 homogeneous polysaccharides obtained from sabina macrocarpa polysaccharide have obvious inhibition on cell hemolysis caused by activation of complement classical and alternative pathways, namely obvious anticomplementary effect.
CH for YB-PS2, YB-PS3, YB-PS4 and YB-PS550Values (concentration of test sample required for 50% inhibition of hemolysis by the classical pathway) were 461. + -. 10. mu.g/mL, 129. + -. 34. mu.g/mL, 94. + -. 9. mu.g/mL and 261. + -. 17. mu.g/mL, respectively; AP of YB-PS3 and YB-PS450The values (concentration of the test sample required for 50% inhibition of hemolysis by the alternative pathway) are 494. + -.80, 242. + -.20. mu.g/mL, respectively, and YB-PS2, YB-PS5 have no alternative pathway of the anticomplementary system.
The invention provides four homogeneous polysaccharides in sabina macrocarpa, a preparation method thereof and application thereof in preparing anticomplement medicines; the invention separates four homogeneous polysaccharides YB-PS2, YB-PS3, YB-PS4 and YB-PS5 from the traditional Chinese medicine sabina chinensis for clearing heat and removing toxicity, and experiments prove that the homogeneous polysaccharides have obvious inhibition effect on complement activation and can be further used as active ingredients to prepare novel anticomplement medicines.
Drawings
FIG. 1 shows HPGPC chromatograms of YB-PS2(A) and YB-PS3(B),
it shows a TSK-GEL GMPWXL GEL column (300X 7.6 mm); eluent: 0.01M NaCl; flow rate: 0.8 ml/min.
FIG. 2 HPGPC chromatograms of YB-PS4(A) and YB-PS5(B),
it shows a TSK-GEL GMPWXL GEL column (300X 7.6 mm); eluent: 0.01M NaCl; flow rate: 0.8 ml/min.
Detailed Description
EXAMPLE 1 preparation of Sabina grandiflora polysaccharides YB-PS2, YB-PS3, YB-PS4 and YB-PS5
Pulverizing 3Kg of Thuja Dolabrata, extracting with 95% ethanol, filtering, extracting the residue with water solution for 3 times, concentrating, centrifuging, adding 4 times volume of 95% ethanol into the supernatant, standing, centrifuging to remove supernatant, dissolving the precipitate with water, recovering ethanol under reduced pressure, and removing ethanol; removing free protein from the compound solution with trichloroacetic acid, centrifuging, adjusting the supernatant to neutral, dialyzing, concentrating, and freeze drying to obtain crude polysaccharide. Dissolving 100g of crude polysaccharide in distilled water, centrifuging, eluting the supernatant by DEAE-cellulose chromatography, eluting by distilled water and NaCl solutions of 0.1, 0.4, 0.8, 1.6 and 2.0mol/L, wherein the elution volume is more than 2 times of the column volume (about 3L), the flow rate is 10mL/min, collecting each fraction, detecting the absorbance value at 490nm (after the sulfuric acid-phenol method for color development) by a separating tube, merging the fractions, concentrating, dialyzing and freeze-drying according to the result of the sugar color development reaction and combining ultraviolet detection to obtain 6 secondary components: WATER-EP-YB, 0.1M-EP-YB, 0.4M-EP-YB, 0.8M-EP-YB, 1.6M-EP-YB and 2.0M-EP-YB;
dissolving 0.1M-EP-YB (6.0g) in distilled water, centrifuging, and separating supernatant with SephacrylTMSeparating by S300 chromatography (molecular weight cut-off of 2KDa-400KDa), eluting with 0.1mol/L NaCl solution at the flow rate of 0.8mL/min, collecting each fraction, detecting the absorbance value at 490nm (after color development by a sulfuric acid-phenol method) by a partition tube, combining the same fractions according to the detection result, concentrating, dialyzing and freeze-drying to obtain homogeneous polysaccharides YB-PS2(30mg) and YB-PS5(60 mg);
dissolving 0.4M-EP-YB (6.8g) in distilled water, centrifuging, and fractionating the supernatant with SephacrylTMS300, performing chromatographic separation, eluting with 0.1mol/L NaCl solution at the flow rate of 0.8mL/min, collecting each fraction, detecting the absorbance value at 490nm (after color development by a sulfuric acid-phenol method) by using a separating tube, combining the same fractions according to the detection result, and concentrating, dialyzing and freeze-drying to obtain homogeneous polysaccharide YB-PS3(100 mg);
dissolving 1.6M-EP-YB (1.9g) in distilled water, centrifuging, and fractionating the supernatant with SephacrylTMS300, performing chromatographic separation, eluting with 0.1mol/L NaCl solution at the flow rate of 0.8mL/min, collecting each fraction, detecting the absorbance value at 490nm (after color development by a sulfuric acid-phenol method) by using a separation tube, and combining the same fractions according to the detection resultConcentrating, dialyzing, and freeze-drying to obtain homogeneous polysaccharide YB-PS4(45 mg);
detecting that YB-PS2, YB-PS3, YB-PS4 and YB-PS5 are all uniform components by High Performance Gel Permeation Chromatography (HPGPC).
Example 2 structural characterization of Sabina macrocarpa polysaccharides (YB-PS2, YB-PS3, YB-PS4 and YB-PS5)
(1) Determination of molecular weight
The molecular weight is detected by adopting an 18-angle laser light scattering gel chromatography system, the basic principle is that homogeneous polysaccharide forms symmetrical chromatographic peaks through gel permeation chromatography, light scattering is formed after 18-angle laser irradiation, and light scattering signals are directly related to the molecular weight. The data were calculated with Astar (version 5.3.1) software to give the molecular weight directly;
the experimental method comprises the following steps: accurately weighing 5.0mg of homogeneous polysaccharide to prepare a solution of 10mg/ml, and passing through a 0.45-micron microporous filter membrane before sample injection, wherein the chromatographic conditions are as follows: the flow rate is 0.5mg/ml, the sample amount is 20 μ l, 0.1% NaCl solution is used as mobile phase, the column temperature is 25 deg.C, the laser wavelength is 685nm, and the refractive index parameter (dn/dc) is 0.138cm3/g；
(2) Determination of total sugar, uronic acid, protein and sulfate group content
The content of YB-PS2 total sugar is 94.47% by the sulfuric acid-phenol method; the total sugar content of YB-PS3 is 95.21%; the total sugar content of YB-PS4 is 96.69%; the total sugar content of YB-PS5 is 95.23%;
the content of uronic acid detected by an m-hydroxy biphenyl method is 1.89% of that of YB-PS 2; the uronic acid content of YB-PS3 is 16.62%; the uronic acid content of YB-PS4 is 25.34%; the uronic acid content of YB-PS5 is 3.68%;
protein content determination by Coomassie Brilliant blue method: the protein content of YB-PS2 is 3.25%; the protein content of YB-PS3 is 3.36%; the protein content of YB-PS4 is 2.04%; the protein content of YB-PS5 is 2.13%;
BaCl2the YB-PS2, the YB-PS3, the YB-PS4 and the YB-PS5 do not contain sulfate groups through the determination of a turbidimetric method;
(3) sugar composition analysis
Respectively carrying out full hydrolysis on YB-PS2, YB-PS3, YB-PS4 and YB-PS5 by 2mol/L TFA at 110 ℃ to obtain products, carrying out PMP derivatization, and carrying out liquid phase analysis;
YB-PS2 is polysaccharide composed of three monosaccharides, the mole ratio of the monosaccharide group is arabinose: galactose group: glucose is 0.58:0.30: 0.12;
YB-PS3 is polysaccharide composed of five monosaccharides, the mole ratio of the monosaccharide group is arabinose: galactose: rhamnose: galacturonic acid: 3-methyl galactose composition ═ 0.16:0.27:0.24:0.17: 0.14;
YB-PS4 is polysaccharide composed of five monosaccharides, the mole ratio of which is arabinose: galactose: rhamnose: galacturonic acid: 3-methyl galactose ═ 0.21:0.27:0.19:0.20: 0.13;
YB-PS5 is a polysaccharide composed of two monosaccharides, the molar ratio of the monosaccharides is glucosamine: rhamnose 0.44: 0.56;
(4) methylation analysis
Methylation of polysaccharides, depolymerization of the methylated product with 90% formic acid, perhydrolysis with 2mol/L TFA, NaBH, was carried out separately according to the literature methods (seeds PW, Selvendran RR. Avoiding oxidative degradation of polysaccharide sodium hydroxide/dimethyl-iodide carbohydrate reaction in dimethyl sulfoxide. carbohydrate Res.1993,245:1-10)4Reducing and acetylating acetic anhydride to prepare a partially methylated alditol acetate derivative, and then carrying out GC-MS analysis and judging by combining a standard map;
the methylation reaction of YB-PS2 shows that it contains: 1,3, 5-linked arabinose, 1, 3-linked arabinose, terminal-linked arabinose, 1,2, 4-linked galactose, 1, 4-linked galactose, terminal-linked galactose and 1, 3-linked glucose in a molar ratio of 0.208:0.126:0.233:0.09:0.112:0.109: 0.132;
the methylation result of YB-PS3 shows that the structure contains: 1, 3-linked arabinose, terminal-linked arabinose, 1,2, 4-linked galactose, terminal-linked galactose, 1,2, 4-linked rhamnose, 1, 2-linked rhamnose, terminal-linked rhamnose, 1, 2-linked 3-methyl galactose, 1, 4-linked galacturonic acid in a molar ratio of 0.076:0.082:0.205:0.091:0.077:0.085:0.069:0.158: 0.147;
the methylation result of YB-PS4 shows that the structure contains: 1, 3-linked arabinose, terminal-linked arabinose, 1,2, 4-linked galactose, terminal-linked galactose, 1,2, 4-linked rhamnose, 1, 2-linked rhamnose, terminal-linked rhamnose, 1, 2-linked 3-methyl galactose and 1, 4-linked galacturonic acid in a molar ratio of 0.071:0.069:0.140:0.154:0.071:0.061:0.066:0.142: 0.198;
the methylation result of YB-PS5 shows that it contains end-set linked glucosamine, 1, 4-linked rhamnose and 1,2, 4-linked rhamnose in a molar ratio of 0.41:0.21: 0.38.
Example 3 classical pathway complement inhibition assay
Collecting serum of 3-month-old guinea pig, and mixing with VBS2+Buffer (barbital buffer, pH 7.4, containing 0.5mM Mg2+And 0.15mM Ca2+) Dilution 1:100 was used as the complement source for this classical pathway. The rabbit anti-sheep red blood cell antibody is treated with VBS2+Diluting the buffer solution to 1:1000 to be used as hemolysin; sheep Red Blood Cells (SRBC) stored in Alsever solution were prepared into 2% SRBC, and 3mg of polysaccharide was precisely weighed and VBS was added2+The buffer was dissolved and diluted to 8 concentrations. After 200 mul of polysaccharide solution with different concentrations and 200 mul of complement which is diluted to 1:100 are preincubated for 10min at 37 ℃, 100 mul of hemolysin (1:1000) and 100 mul of 2% SRBC are sequentially added, and the mixture is put into a low-temperature high-speed centrifuge after being put into a water bath at 37 ℃ for 30min and then centrifuged for 10min at 5000rpm and 4 ℃. mu.L of supernatant was taken from each tube in a 96-well plate and absorbance was measured at 405 nm. The experiment was performed with a polysaccharide control group (200. mu.L of polysaccharide at the corresponding concentration plus 400. mu.L of VBS)2+Buffer), complement control group (at 200. mu.L VBS)2+Buffer instead of polysaccharide) and a whole blood group (100. mu.L of 2% SRBC in 500. mu.L of triple distilled water), the absorbance value of each concentration of polysaccharide group was subtracted from that of the corresponding polysaccharide control group to calculate the hemolysis inhibition. The logarithm of the polysaccharide concentration was plotted on the X-axis and the inhibition rate of hemolysis was plotted on the Y-axis, and the Concentration (CH) of the sample required for 50% inhibition of hemolysis was calculated from the obtained fitted curve50Values) with heparin as a positive control, the results are shown in table 1.
Example 4 alternative pathway complement inhibition assay
Serum of healthy male volunteers in adults is taken,VBS-Mg-EGTA buffer (barbital buffer, pH 7.4, containing 5mM Mg)2+And 8mM EGTA) diluted 1:8 as a source of complement for the alternative pathway. Rabbit red blood cells stored in 3.8% sodium citrate solution are prepared into 0.5% rabbit red blood cells by VBS-Mg-EGTA buffer solution, polysaccharide is precisely weighed to be about 3Mg, VBS-Mg-EGTA buffer solution is added, the solution is diluted to 8 concentrations in a double way, 150 mu L of polysaccharide solution with different concentrations and 150 mu L of complement with the ratio of 1:8 are preincubated for 10min at 37 ℃, 200 mu L of 0.5% rabbit red blood cells are added, the mixture is put into a low-temperature high-speed centrifuge after being subjected to water bath at 37 ℃ for 30min, and the centrifugation is carried out for 10min at 5000rpm and 4 ℃. Taking 200 mu L of supernatant per tube, placing in a 96-well plate, measuring absorbance at 405nm, setting a polysaccharide control group (150 mu L of polysaccharide solution with corresponding concentration and 350 mu L of VBS-Mg-EGTA buffer solution), a complement control group (150 mu L of LVBS-Mg-EGTA buffer solution replaces the polysaccharide solution) and a whole hemolyzed group (200 mu L of 0.5% rabbit red blood cells are dissolved in 300 mu L of triple distilled water) in the experiment, calculating the hemolysis inhibition rate after subtracting the absorbance value of the corresponding polysaccharide control group from the absorbance value of each polysaccharide group, using the logarithm of the polysaccharide concentration as an X axis and the hemolysis inhibition rate as a Y axis for plotting, and calculating the concentration of a sample (AP) required for inhibiting hemolysis by 50% (by using the obtained fitting curve)50Values) with heparin as a positive control, the results are shown in table 1.
TABLE 1 inhibition of complement activation by four thuja maxima polysaccharides
CH50And AP50The values are expressed as: mean ± SD (n ═ 3); NA: no active.
Claims (2)
1. The application of the polysaccharide of the sabina macrocarpa in preparing a complement inhibition medicament; the sabina macrocarpa polysaccharide is YB-PS2, YB-PS3, YB-PS4 or YB-PS5, and has the following structural characteristics respectively:
(1) YB-PS2 is polysaccharide composed of three monosaccharides, and the molecular weight is 81.60 KDa; the total sugar content was 94.47%; the protein content was 3.25%; the uronic acid content is 1.89%; free of sulfate groups, the monosaccharide composition shows that it consists of arabinose, galactose and glucose in a molar ratio of 0.58:0.30: 0.12;
(2) YB-PS3 is a polysaccharide composed of five monosaccharides, and the molecular weight is 40.47 KDa; total sugar content 95.21%; the protein content was 3.63%; the uronic acid content is 16.62%; free of sulfate groups, the sugar composition showed that it consisted of arabinose, galactose, rhamnose, galacturonic acid and 3-methyl galactose in a molar ratio of 0.16:0.27:0.24:0.17: 0.14;
(3) YB-PS4 is a polysaccharide composed of five monosaccharides, and the molecular weight is 218.47 KDa; total sugar content 96.69%; the protein content is 2.04%; the uronic acid content is 25.34%; free of sulfate groups, monosaccharide composition showed that it was composed of arabinose, galactose, rhamnose, galacturonic acid and 3-methyl galactose in a molar ratio of 0.21:0.27:0.19:0.20: 0.13;
(4) YB-PS5 is a polysaccharide composed of two monosaccharides and has a molecular weight of 153.33 KDa; the total sugar content was 95.23%; the protein content was 2.13%; the uronic acid content is 3.68%; free of sulfate groups, the monosaccharide composition showed that it was composed of glucosamine, rhamnose, in a molar ratio of 0.44: 0.56.
2. The use as claimed in claim 1, wherein the thuja maxima polysaccharide is prepared by the following steps:
extracting sabina vulgaris with ethanol, filtering, extracting the residue with hot water, filtering, concentrating, centrifuging, adding appropriate amount of ethanol into the supernatant to obtain ethanol with final concentration of 70-80%, standing, centrifuging to remove the supernatant, dissolving the precipitate with water, and removing free protein to obtain crude polysaccharide; dissolving crude polysaccharide in water, separating with DEAE-cellulose chromatography, sequentially eluting with distilled water, 0.1, 0.4, 0.8, 1.6 and 2.0mol/L NaCl solution, collecting each fraction, and mixing the same polysaccharide components according to sugar color reaction and liquid phase detection results;
dissolving 3 polysaccharide fractions obtained by eluting 0.1, 0.4 and 1.6 mol/L NaCl solution respectively with distilled water, further purifying by gel chromatography with molecular weight cutoff in the range of 2KDa-400KDa, eluting with 0.1mol/L NaCl solution, merging the same fractions according to the results of sugar color reaction and ultraviolet detection, and detecting the anticomplementary activity to finally obtain the coniferum macrocarpum polysaccharide which is the homogeneous polysaccharide with anticomplementary activity.
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| CN102653568A (en) * | 2012-05-28 | 2012-09-05 | 上海中医药大学 | Herba taraxaci homogeneous polysaccharide and preparation method and application thereof |
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