CN111484564B - Spreading hedyotis herb polysaccharide, preparation method thereof and application thereof in preparing anticomplement medicines - Google Patents
Spreading hedyotis herb polysaccharide, preparation method thereof and application thereof in preparing anticomplement medicines Download PDFInfo
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Abstract
The invention belongs to the field of traditional Chinese medicines, relates to oldenlandia diffusa polysaccharide, and particularly relates to five natural homogeneous polysaccharides in oldenlandia diffusa, a preparation method thereof and application thereof in preparation of anticomplement medicines. The five homogeneous polysaccharides HDP-3, HDP-4, HDP-5, HDP-6 and HDP-7 are separated from the heat-clearing and detoxifying traditional Chinese medicine oldenlandia diffusa, and experiments prove that the oldenlandia diffusa homogeneous polysaccharide has a remarkable inhibition effect on complement activation and can be further used as an active ingredient to prepare novel anticomplement medicines.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicines, relates to oldenlandia diffusa polysaccharide, and particularly relates to five natural homogeneous polysaccharides in oldenlandia diffusa, 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, in some cases, abnormal activation of the complement system causes an excessive reaction of the human immune system, resulting in damage to normal tissues of the human body itself, such as rheumatoid and rheumatoid arthritis, Systemic Lupus Erythematosus (SLE), Acute Respiratory Distress Syndrome (ARDS), acute pneumonia caused by influenza virus, and severe atypical pneumonia (SARS). It follows that inhibition of abnormal activation of the complement system is one of the important pathways for the treatment of the above-mentioned diseases.
At present, although the traditional immunosuppressant drugs such as glucocorticoid, cyclophosphamide, methotrexate and the like which are clinically allowed to be used have a certain relieving effect on diseases related to excessive complement activation, the drugs are not specific complement inhibitors, and long-term application can reduce the defense function of the body, so that the anti-infection capability is reduced, secondary infection is easy to occur, potential focus is diffused, and various complications and side effects are generated. Therefore, the search for new complement inhibitors with high efficiency and low toxicity is an urgent need for clinical treatment of such diseases. The medicinal plants widely contain components with anticomplementary activity, and most of the natural active components have low toxicity to the organism and can be directly digested and absorbed by the organism. Chinese medicine resources are rich, a plurality of Chinese medicines have obvious regulating effect on an immune system, and the Chinese medicine is a valuable resource for searching anticomplementary prodrug. During the period of resisting SARS, Chinese medicine and Chinese medicine have obvious effect of preventing and treating SARS, so that it is important to find new type of efficient anticomplementary matter from medicinal plant.
Oldenlandia diffusa (Hedyotis diffusa Wild.) is a whole herb with roots of an annual herbaceous oldenlandia diffusa of the genus Rubiaceae (Rubiaceae), is bitter, sweet and cold in nature and has the effects of clearing heat, promoting diuresis, detoxifying and the like. The oldenlandia diffusa has pharmacological effects of resisting tumor, inflammation and oxidation, regulating immunity, negative myocardium and the like. Herba Hedyotidis Diffusae contains tens of compounds such as iridoids, acid-containing compounds, anthraquinone compounds, volatile components, phenylpropanoids, coumarin and polysaccharides, and various trace elements. The polysaccharide component is effective immunomodulator, can obviously kill tumor cells, and has anti-aging effect, and the research shows that the herba Hedyotidis Diffusae crude polysaccharide has significant anticomplementary activity, CH50And AP5063 mug/ml and 87 mug/ml respectively, but the report about the separation and preparation of the homogeneous polysaccharide with anticomplementary activity in the oldenlandia diffusa is not found.
Based on the current situation and the foundation of the prior art, the inventors of the present application intend to provide spreading hedyotis herb polysaccharide, a preparation method thereof and a use thereof in preparing anticomplement medicines.
Disclosure of Invention
The invention aims to provide an active component with an anticomplementary effect in a natural medicament based on the current situation and the foundation of the prior art, and particularly relates to hedyotis diffusa polysaccharide, a preparation method thereof and application thereof in preparing a medicament for inhibiting a complement.
The invention separates the water extract of the traditional heat-clearing and detoxifying traditional Chinese medicine oldenlandia diffusa to obtain five homogeneous polysaccharides, namely HDP-3, HDP-4, HDP-5, HDP-6 and HDP-7, and the structure characteristics of the oldenlandia diffusa polysaccharide are as follows:
(1) the structure characteristics of spreading hedyotis herb polysaccharide HDP-3 are as follows: polysaccharides consisting of six monosaccharides, with a molecular weight of about 208 kDa; total sugar content about 90%; the protein content is lower than 1%; the uronic acid content is about 28%. The monosaccharide molar ratio is arabinose: rhamnose: mannose: glucose: galactose: galacturonic acid 1.1:1.8:1.1:0.9:1.4: 2.5; the connection mode mainly comprises terminal galacturonic acid, 1, 4-linked galactose and terminal rhamnose;
(2) the structure characteristics of spreading hedyotis herb polysaccharide HDP-4 are as follows: polysaccharides consisting of five monosaccharides, with a molecular weight of about 194 kDa; total sugar content about 93%; the protein content is lower than 1%; the uronic acid content is about 11%. Monosaccharide molar ratio mannose: rhamnose: glucuronic acid: glucose: galactose ═ 2.1:1.4:1.1:2.7: 2.8; the connection mode mainly comprises 1, 4-connecting galactose, 1,4, 6-connecting mannose, 1,3, 4-connecting galactose, 1, 3-connecting glucose, terminal galactose and terminal mannose;
(3) the structure characteristics of spreading hedyotis herb polysaccharide HDP-5 are as follows: polysaccharides consisting of six monosaccharides, with a molecular weight of about 742 kDa; total sugar content about 91%; protein content about 1%; uronic acid content of about 6%, monosaccharide molar ratio mannose: rhamnose: glucuronic acid: glucose: galactose: arabinose 6.8:3.5:2.7:15.2:9.9: 3.4; the connection mode mainly comprises terminal mannose, 1, 4-connecting glucose and 1,4, 6-connecting galactose;
(4) the structure characteristics of spreading hedyotis herb polysaccharide HDP-6 are as follows: polysaccharides consisting of six monosaccharides, with a molecular weight of about 238 kDa; total sugar content about 93%; the protein content is less than 1%. The molar ratio of the monosaccharides is mannose: rhamnose: glucose: galactose: arabinose is 3.6:2.4:9.0:5.3: 5.7; the connection mode mainly comprises 1, 4-connecting glucose, terminal mannose and 1, 3-connecting galactose;
(5) the structure characteristics of spreading hedyotis herb polysaccharide HDP-7 are as follows: polysaccharides consisting of five monosaccharides, with a molecular weight of approximately 166 kDa; total sugar content about 91%; the protein content is lower than 1%, and the monosaccharide molar ratio is mannose: rhamnose: glucose: galactose: arabinose 2.8:2.5:14.1:4.4: 3.0; the connection mode is mainly terminal glucose and 1, 4-connection glucose.
The invention provides a preparation method of oldenlandia diffusa polysaccharides HDP-3, HDP-4, HDP-5, HDP-6 and HDP-7, which comprises the following steps:
extracting herba Hedyotidis Diffusae with 95% ethanol, filtering, oven drying the residue, extracting with hot water, filtering the extractive solution, concentrating, adding 4 times volume 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, freeze drying to obtain crude polysaccharide, dissolving the crude polysaccharide with distilled water, performing primary separation with DEAE-cellulose column chromatography, eluting with distilled water, 0.1, 0.2, 0.4, 0.8 and 1.6mol/L NaCl solution, collecting fractions, concentrating, dialyzing (molecular weight cut-off of 14000Da) and freeze drying to obtain 6 secondary components: Water-HDP, 0.1M-HDP, 0.2M-HDP, 0.4M-HDP, 0.8M-HDP and 1.6M-HDP;
dissolving each secondary component in appropriate amount of mobile phase, centrifuging, separating the supernatant with gel chromatography Sephacryl S-200 and S-300, and collecting each fraction; detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with a separating tube, combining flow components, concentrating and freeze-drying to obtain homogeneous polysaccharide: HDP-3, HDP-4, HDP-5, HDP-6 and HDP-7.
In the invention, the traditional Chinese medicine oldenlandia diffusa is whole grass with roots of oldenlandia diffusa which belongs to the ear genus of rubiaceae.
The invention further provides the homogeneous polysaccharide prepared: the application of HDP-3, HDP-4, HDP-5, HDP-6 and HDP-7 in preparing a body inhibition drug.
The invention carries out in vitro tests, and the results prove that the oldenlandia diffusa polysaccharide HDP-3, HDP-4, HDP-5, HDP-6 and HDP-7 have obvious inhibition on cell hemolysis caused by the activation of the classical pathway and the alternative pathway of complement, namely have obvious anticomplementary action;
oldenlandia diffusa polysaccharides HDP-3, HDP-4, HDP-5, HDP-6 and CH of HDP-750Values (concentration of test sample required for 50% inhibition of hemolysis by the classical pathway) of about 1022. mu.g/mL, 84. mu.g/mL, 115. mu.g/mL, 53. mu.g/mL, and 154. mu.g/mL, respectively; AP of HDP-4, HDP-5, HDP-6 and HDP-750Values (concentration of test sample required for 50% inhibition of hemolysis by the alternative pathway) were approximately 176. mu.g/mL, 307. mu.g/mL, 74. mu.g/mL and 207. mu.g/mL, respectively, and HDP-3 had no alternative complement inhibitory activity.
The invention separates five homogeneous polysaccharides HDP-3, HDP-4, HDP-5, HDP-6 and HDP-7 from the heat-clearing and detoxifying traditional Chinese medicine oldenlandia diffusa, and experiments prove that the oldenlandia diffusa homogeneous polysaccharide has obvious inhibition effect on complement activation and can be further used as an active ingredient to prepare novel anticomplement medicines.
Drawings
FIG. 1 shows a process for separating homogeneous polysaccharides from Hedyotis diffusa.
FIG. 2, HPGPC chromatograms of five homopolysaccharides, HDP-3(A), HDP-4(B), HDP-5(C), HDP-6(D), HDP-7(E), were prepared, wherein TSK-GELGMPWXL gel column (300X 7.6 mm); eluent: distilled water; flow rate: 0.8 ml/min.
Detailed Description
Example 1 preparation of spreading hedyotis herb polysaccharide
Pulverizing herba Hedyotidis Diffusae 10kg, 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; removing free protein from the compound solution with 10% trichloroacetic acid, centrifuging, adjusting supernatant to neutral, dialyzing, concentrating, freeze drying to obtain crude polysaccharide, dissolving 120g of crude polysaccharide in distilled water, centrifuging, and subjecting supernatant to preliminary separation by DEAE-cellulose column chromatography. Eluting with distilled water, 0.1, 0.2, 0.4 and 0.8mol/L NaCl solution, eluting with volume more than 2 times column volume (about 10L), and flow rate of 25mL/min, collecting fractions, and detecting absorbance value at 490nm (after sulfuric acid-phenol method color development) with separate tubes. According to the chromogenic reaction of sugar and the result of HPGPC detection, the fractions are combined, concentrated, dialyzed and freeze-dried to obtain 6 secondary components: Water-HDP, 0.1M-HDP, 0.2M-HDP, 0.4M-HDP, 0.8M-HDP and 1.6M-HDP;
dissolving 0.1M-HDP (8.26g) in distilled water, centrifuging, and subjecting the supernatant to SephacrylTMSeparating with S-200 gel chromatographic column, eluting with distilled water solution at flow rate of 0.5mL/min, collecting each fraction, detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with a separating tube, mixing the same fractions according to HPGPC detection result, concentrating, dialyzing, and freeze drying to obtain homogeneous polysaccharide HDP-3(60 mg);
dissolving 0.2M-HDP (5.93g) in distilled water, centrifuging, and collecting supernatantSephacryl for liquid fractionationTMSeparating by S-200 chromatography, eluting with distilled water at flow rate of 0.5mL/min, collecting each fraction, detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with a separation tube, combining the same fractions according to HPGPC detection result, concentrating, dialyzing, and freeze-drying to obtain homogeneous polysaccharide HDP-4(500 mg);
dissolving 0.4M-HDP (6.92g) in distilled water, centrifuging, and fractionating the supernatant with SephacrylTMPerforming S-200 chromatographic separation, eluting with distilled water at flow rate of 0.5mL/min, collecting each fraction, detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with a separating tube, combining the same fractions according to HPGPC detection result, concentrating, dialyzing, and freeze-drying to obtain homogeneous polysaccharide HDP-5(1500 mg);
dissolving 0.8M-HDP (3.32g) in distilled water, centrifuging, and fractionating the supernatant with SephacrylTMPerforming S-300 chromatographic separation, eluting with distilled water at flow rate of 0.5mL/min, collecting each fraction, detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with a separating tube, combining the same fractions according to HPGPC detection result, concentrating, dialyzing, and freeze-drying to obtain homogeneous polysaccharide HDP-6(60 mg);
dissolving 1.6M-HDP (2.04g) in distilled water, centrifuging, and fractionating the supernatant with SephacrylTMPerforming S-300 chromatographic separation, eluting with distilled water at flow rate of 0.5mL/min, collecting each fraction, detecting absorbance value at 490nm (after color development by sulfuric acid-phenol method) with a separating tube, combining the same fractions according to HPGPC detection result, concentrating, dialyzing, and freeze-drying to obtain homogeneous polysaccharide HDP-7(60 mg);
the prepared herba Hedyotidis Diffusae polysaccharides HDP-3, HDP-4, HDP-5, HDP-6 and HDP-7 are homogeneous components, which are detected by High Performance Gel Permeation Chromatography (HPGPC).
Example 2 structural characterization of spreading hedyotis herb polysaccharides (HDP-3, HDP-4, HDP-5, HDP-6 and HDP-7)
(1) Determination of molecular weight
Measuring relative molecular force of polysaccharide sample by High Performance Gel Permeation Chromatography (HPGPC), based on the basic principle that homogeneous polysaccharide passes through gel permeation chromatography to form symmetrical chromatographic peak, wherein the peak-off time is related to molecular weight, and is calculated according to standard curve obtained from known molecular weight;
chromatographic conditions are as follows: by TSK GMPWXLSeparating with gel column at flow rate of 0.8mg/mL and sample volume of 20 μ L, using ultrapure water as mobile phase, column temperature of 25 deg.C, and Evaporative Light Scattering Detector (ELSD);
the experimental method comprises the following steps: accurately weighing 2.0mg of each of homogeneous polysaccharide and dextran series standard, preparing into 2.0mg/mL solution with distilled water, detecting after passing through a 0.45-micron microporous filter membrane before sample injection, recording retention time, drawing a standard curve by taking logarithmic value (Lg) of standard polysaccharide molecular weight as ordinate and retention time as abscissa, obtaining corresponding linear regression equation, and calculating the relative molecular weight of homogeneous polysaccharide. The relative molecular weights of HDP-3, HDP-4, HDP-5, HDP-6 and HDP-7 are 208.1kDa, 194.5kDa, 742.2kDa, 238.7kDa and 166.2kDa, respectively;
(2) determination of total sugar, uronic acid, protein and sulfate group content
The HDP-3 total sugar content is 90.00 +/-3.60 percent, the HDP-4 total sugar content is 92.99 +/-2.79 percent, the HDP-5 total sugar content is 90.61 +/-2.72 percent, the HDP-6 total sugar content is 93.13 +/-2.79 percent and the HDP-7 total sugar content is 91.35 +/-2.74 percent through a sulfuric acid-phenol method;
the content of the uronic acid is detected by an m-hydroxyl biphenyl method, the content of HDP-3 uronic acid is 28.45 +/-0.85%, the content of HDP-4 uronic acid is 10.80 +/-0.32%, and the content of HDP-5 uronic acid is 6.51 +/-0.20%;
protein content determination by coomassie brilliant blue method: the content of HDP-3 protein is 0.54 plus or minus 0.02 percent, the content of HDP-4 protein is 0.79 plus or minus 0.02 percent, the content of HDP-5 protein is 1.01 plus or minus 0.03 percent, the content of HDP-6 protein is 0.97 plus or minus 0.03 percent, and the content of HDP-7 protein is 0.68 plus or minus 0.02 percent;
(3) monosaccharide composition analysis
HDP-3, HDP-4, HDP-5, HDP-6 and HDP-7 are respectively subjected to complete acid hydrolysis by reacting 2mol/L TFA at 110 ℃ for 5h, the obtained complete acid hydrolysis product is subjected to derivatization by reacting with 2mol/L PMP methanol solution for 150min under the alkaline condition of concentrated ammonia water, and the derivatization product is subjected to LC-MS analysis;
HDP-3 is a polysaccharide composed of six monosaccharides with a molar ratio of arabinose: rhamnose: mannose: glucose: galactose: galacturonic acid 1.1:1.8:1.1:0.9:1.4: 2.5;
HDP-4 is a polysaccharide composed of five monosaccharides, the molar ratio of monosaccharide mannose: rhamnose: glucuronic acid: glucose: galactose ═ 2.1:1.4:1.1:2.7: 2.8;
HDP-5 is a polysaccharide composed of six monosaccharides in a molar ratio of mannose: rhamnose: glucuronic acid: glucose: galactose: arabinose 6.8:3.5:2.7:15.2:9.9: 3.4;
HDP-6 is a polysaccharide composed of five monosaccharides, the molar ratio of the monosaccharides being mannose: rhamnose: glucose: galactose: arabinose is 3.6:2.4:9.0:5.3: 5.7;
HDP-7 is a polysaccharide composed of five monosaccharides, the molar ratio of the monosaccharides being mannose: rhamnose: glucose: galactose: arabinose 2.8:2.5:14.1:4.4: 3.0;
(4) methylation analysis
Methylation of polysaccharides by modified Hakomori procedure (HDP-3, HDP-4 and HDP-5 uronic acid polysaccharide containing CMC-NaBH, first)4Reduced and then methylated), the methylated product was perhydrolyzed with 0.2mol/L TFA, NaBD4Reducing and acetylating acetic anhydride to produce a partially methylated alditol acetate derivative, and performing GC-MS analysis;
HDP-3 methylation results show structures containing end-linked galacturonic acid, 1, 3-linked rhamnose, 1, 4-linked galacturonic acid, end-linked rhamnose, 1, 3-linked arabinose, 1, 4-linked galactose, 1,3, 5-linked arabinose, 1,3, 4-linked glucose, 1,3, 4-linked mannose, 1,4, 6-mannose, 1,3, 6-linked glucose, 1, 4-linked mannose, in a molar ratio of 2.8:1.0:2.8:2.8:1.0:3.9:1.0:0.9:0.9:0.9:0.9: 0.9:0.8: 1.0. Wherein, the main components are end group galacturonic acid, 1, 4-linked galactose and end group rhamnose;
HDP-4 methylation results show that the structure contains: 1,4, 6-linked mannose, 1,3, 4-linked galactose, 1, 3-linked glucuronic acid, 1, 4-linked glucose, 1,3, 6-linked galactose, 1, 3-linked rhamnose, terminal-linked galactose and terminal-linked mannose in a molar ratio of 2.0:2.2:2.0:5.7:1.0:1.2:1.1:2.2:2.0, wherein the 1, 4-linked galactose, 1,4, 6-linked mannose, 1,3, 4-linked galactose, 1, 3-linked glucose, terminal galactose and terminal mannose are mainly used;
HDP-5 methylation results show that the structure contains: the end group is connected with arabinose, the end group is connected with rhamnose, 1, 5-connected arabinose, the end group is connected with mannose, the end group is connected with glucose, 1, 4-connected mannose, 1, 4-connected glucose, 1, 3-connected mannose, 1,3, 4-connected glucose, 1,4, 6-connected galactose and 1,4, 6-connected glucose, the molar ratio is 1.0:1.8:0.8:2.8:1.0: 4.0:1.0:0.9:3.9:2.0, wherein the end group is mainly composed of mannose, 1, 4-connected glucose and 1,4, 6-connected galactose;
HDP-6 methylation results show that the structure contains: the end group is connected with arabinose, the end group is connected with rhamnose, 1, 5-connected arabinose, the end group is connected with mannose, the end group is connected with glucose, the end group is connected with galactose, 1, 3-connected galactose, 1, 4-connected mannose, 1, 4-connected glucose, 1,3, 5-connected arabinose, 1,3, 4-glucose, 1,3,4, 6-connected glucose, 1,3, 6-connected galactose and 1,2,4, 6-connected glucose, the molar ratio is 1.7:1.8:0.9:2.0:1.0:1.0:2.0:0.8:3.0:1.8:1.0:1.0:1.0:0.9, wherein the end group is mainly composed of 1, 4-connected glucose, end group mannose and 1, 3-connected galactose;
HDP-7 methylation results show that the structure contains: the terminal group is connected with arabinose, the terminal group is connected with rhamnose, 1, 3-is connected with rhamnose, the terminal group is connected with glucose, 1, 2-is connected with mannose, 1, 3-is connected with galactose, 1, 4-is connected with glucose, 1, 6-is connected with galactose, 1,2, 4-is connected with arabinose, 1,2, 6-is connected with mannose, 1,2,3, 4-is connected with glucose, 1,3, 6-is connected with galactose and 1,3,4, 6-is connected with galactose, the molar ratio is 0.9:1.0:1.0:5.1:1.0:1.1:2.9:0.9:1.1:0.9:1.0:1.0:1.1:1.0, wherein the terminal group glucose and the 1, 4-is mainly used.
Example 3 classical pathway complement inhibition assay
Collecting serum of 3-month-old guinea pig, sensitizing with 2% sheep red blood cell, diluting with barbital buffer solution to 1:100, and using as the present classical pathwayThe source of the complement of (1), diluting rabbit anti-sheep erythrocyte antibody with Barbiturate Buffer Solution (BBS) to 1:1000 as hemolysin; sheep Red Blood Cells (SRBC) preserved in Alsever's fluid were configured into 2% SRBC. Polysaccharide 3mg is precisely weighed, added with BBS buffer solution for dissolution, and diluted into 8 concentrations in a double way. 200. mu.L of polysaccharide solutions of different concentrations were mixed with a solution which had been diluted to 1:100 mu L complement was preincubated at 37 ℃ for 10min, then 100 mu L hemolysin (1:1000) and 100 mu L2% SRBC were added in sequence, placed in a low temperature high speed centrifuge after 30min in 37 ℃ water bath, and centrifuged at 5000rpm and 4 ℃ for 10 min. Taking 200 mu L of supernatant from each tube, placing the supernatant in a 96-well plate, and measuring the absorbance at 405 nm; the experiment is carried out by setting polysaccharide control group (200 μ L polysaccharide with corresponding concentration added with 400 μ LBBS buffer solution), complement control group (200 μ L BBS buffer solution instead of polysaccharide) and whole blood dissolving group (100 μ L2% SRBC dissolved in 500 μ L triple distilled water), subtracting absorbance value of polysaccharide control group from absorbance value of polysaccharide with each concentration, calculating hemolysis inhibition rate, plotting by using logarithm of polysaccharide concentration as X-axis and hemolysis inhibition rate as Y-axis, and calculating Concentration (CH) of sample required for 50% hemolysis inhibition by obtained fitting curve50Values), heparin was used as a positive control, and the results showed that the homopolysaccharide had significant inhibitory activity on classical complement pathway activation (as shown in table 1).
Example 4 alternative pathway complement inhibition assay
Serum of healthy adult male volunteers was extracted with EGTA-BBS buffer (barbital buffer, pH 7.4, containing 5mM Mg)2+And 8mM EGTA) diluted to 1:10 as a complement source of the alternative pathway, preparing 0.5% rabbit red blood cells from rabbit red blood cells stored in 3.8% sodium citrate solution by EGTA-BBS buffer solution, precisely weighing polysaccharide about 3mg, adding EGTA-BBS buffer solution, diluting to 8 concentrations, pre-incubating polysaccharide solution 150. mu.L and complement 150. mu.L of 1:8 at 37 deg.C for 10min, adding 200. mu.L of 0.5% rabbit red blood cells, placing in a water bath at 37 deg.C for 30min, centrifuging at 5000rpm and 4 deg.C for 10min, collecting 200. mu.L supernatant from each tube in a 96-well plate, measuring absorbance at 405nm, and setting polysaccharide control group (150. mu.L of polysaccharide solution with corresponding concentration and 350. mu.L EGTA-BBS buffer solution) and complement control group (150. mu.L EGBBS buffer solution is substituted for EGBBS buffer solution)Replacing;
polysaccharide solution and whole hemolyzed group (200 μ L of 0.5% rabbit red blood cell dissolved in 300 μ L of triple distilled water), calculating hemolysis inhibition ratio by subtracting absorbance value of polysaccharide group at each concentration from absorbance value of corresponding polysaccharide control group, plotting with logarithm of polysaccharide concentration as X axis and hemolysis inhibition ratio as Y axis, and calculating the concentration of sample (AP) required for inhibiting hemolysis by 50% (by fitting curve obtained50Values) were obtained using heparin as a positive control, and the results are shown in table 1.
TABLE 1 inhibition of complement activation by Oldenlandia diffusa homopolysaccharide
CH50And AP50The values are expressed as: mean ± SD (n ═ 3); NA: no active.
Claims (1)
1. The use of herba Hedyotidis Diffusae polysaccharides HDP-3, HDP-4, HDP-5, HDP-6 and HDP-7 with the following structural characteristics in preparing body inhibiting medicine;
(1) the structure characteristics of spreading hedyotis herb polysaccharide HDP-3 are as follows: polysaccharides composed of six monosaccharides and having a molecular weight of 208 kDa; the total sugar content is 90%; the protein content is lower than 1%; the uronic acid content is 28%; the monosaccharide molar ratio of arabinose to rhamnose to mannose to glucose to galactose to galacturonic acid is 1.1:1.8:1.1:0.9:1.4: 2.5; the connection mode mainly comprises terminal galacturonic acid, 1, 4-linked galactose and terminal rhamnose;
(2) the structure characteristics of spreading hedyotis herb polysaccharide HDP-4 are as follows: polysaccharides composed of five monosaccharides and having a molecular weight of 194 kDa; the total sugar content was 93%; the protein content is lower than 1%; the uronic acid content is 11%; the molar ratio of the monosaccharides mannose rhamnose glucuronic acid glucose galactose 2.1:1.4:1.1:2.7: 2.8; the connection mode mainly comprises 1, 4-connecting galactose, 1,4, 6-connecting mannose, 1,3, 4-connecting galactose, 1, 3-connecting glucose, terminal galactose and terminal mannose;
(3) the structure characteristics of spreading hedyotis herb polysaccharide HDP-5 are as follows: polysaccharides composed of six monosaccharides and having a molecular weight of 742 kDa; the total sugar content was 91%; the protein content is 1%; the uronic acid content is 6%; the monosaccharide molar ratio is mannose: rhamnose: glucuronic acid: glucose: galactose: arabinose 6.8:3.5:2.7:15.2:9.9: 3.4; the connection mode mainly comprises terminal mannose, 1, 4-connecting glucose and 1,4, 6-connecting galactose;
(4) the structure characteristics of spreading hedyotis herb polysaccharide HDP-6 are as follows: polysaccharides composed of five monosaccharides and having a molecular weight of 238 kDa; the total sugar content was 93%; the protein content is lower than 1%; the monosaccharide molar ratio is mannose to rhamnose to glucose to galactose to arabinose which is 3.6:2.4:9.0:5.3: 5.7; the connection mode mainly comprises 1, 4-connecting glucose, terminal mannose and 1, 3-connecting galactose;
(5) the structure characteristics of spreading hedyotis herb polysaccharide HDP-7 are as follows: polysaccharides composed of five monosaccharides and having a molecular weight of 166 kDa; the total sugar content was 91%; the protein content is lower than 1%; the monosaccharide molar ratio is 2.8:2.5:14.1:4.4: 3.0; the connection mode is mainly terminal glucose and 1, 4-connection glucose.
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