CN115073620B

CN115073620B - Two homogeneous blackberry lily polysaccharides with anticomplementary activity and preparation method and application thereof

Info

Publication number: CN115073620B
Application number: CN202210509297.9A
Authority: CN
Inventors: 胡政宇; 孙金凤; 李镐; 周微; 金龙; 段元祺
Original assignee: Yanbian University
Current assignee: Yanbian University
Priority date: 2022-05-10
Filing date: 2022-05-10
Publication date: 2023-03-28
Anticipated expiration: 2042-05-10
Also published as: CN115073620A

Abstract

The invention discloses two homogeneous blackberry lily polysaccharides with anticomplementary activity and a preparation method and application thereof, belonging to the technical field of polysaccharide medicines. The invention separates two homogeneous polysaccharides BCP-A1 and BCP-B1 from belamcanda chinensis through the processes of extraction, deproteinization, DEAE-52 cellulose chromatographic separation, sephadexG-100 chromatographic separation and the like, and experiments prove that the homogeneous belamcanda chinensis polysaccharides have obvious inhibition effect on complement activation and can be further used as active ingredients to prepare novel anticomplement medicines.

Description

Two homogeneous blackberry lily polysaccharides with anticomplementary activity and preparation method and application thereof

Technical Field

The invention belongs to the technical field of polysaccharide medicines, and particularly relates to two homogeneous blackberry lily polysaccharides with anticomplementary activity, a preparation method thereof and application thereof in preparing anticomplementary medicines.

Background

The prior art discloses that a complement system is the first defense line of body natural immune defense, plays important roles of monitoring, defense and elimination in eliminating exogenous pathogenic microorganisms and apoptotic cell metabolism, and has a profound influence on activation and correct guidance of adaptive immunity. The complement system is a 'double-edged sword', and excessive activation can cause damage to normal tissues of a human body to cause various diseases, such as rheumatoid arthritis, senile dementia, systemic lupus erythematosus and Acute Respiratory Distress Syndrome (ARDS). 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 of the drugs can reduce the body defense function, cause the defects of reduced anti-infection capability, easy secondary infection, diffusion of potential focuses, generation of various complications, side effects and the like, and thus, a novel complement inhibitor with high efficiency, low toxicity and specificity is urgently needed clinically.

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. The polysaccharide component of the traditional Chinese medicine is safe, reliable and low in toxicity, and has a unique immune regulation mechanism and potential anticomplementary activity. Belamcanda chinensis is the dried rhizome of Belamcanda chinensis (L.) Redout of Belamcanda of Iridaceae, and is mainly distributed in Jilin, liaoning, shanxi, etc. It is bitter in taste and cold in nature, enters lung meridian, has effects of clearing heat and detoxicating, eliminating phlegm, and relieving sore throat, and can be used for treating diseases such as heat-toxin phlegm-fire stagnation, and sore throat. However, no report on the extraction, separation and preparation of homogeneous polysaccharides with anticomplementary activity from Belamcanda chinensis has been found so far.

Disclosure of Invention

In view of the above, the present invention aims to provide two homogeneous blackberry lily polysaccharides with anticomplement activity, a preparation method thereof and an application thereof in the preparation of anticomplement drugs.

The purpose of the invention is realized by the following technical scheme:

the invention provides a preparation method of homogeneous blackberry lily polysaccharide, which mainly comprises the following steps:

(1) Taking dried blackberry lily rhizome, crushing, sieving with a 20-80-mesh sieve, adding water for extraction at the liquid-material ratio of 20;

(2) Taking the blackberry lily crude polysaccharide obtained in the step (1), adding distilled water for redissolution, adding Sevag reagent according to the volume ratio of 3-10 to 1, oscillating, centrifuging, taking the upper layer polysaccharide solution, concentrating and drying to obtain the blackberry lily polysaccharide after deproteinization;

(3) Taking the deproteinized blackberry lily polysaccharide obtained in the step (2), adding deionized water for redissolving, loading the sample to a DEAE-52 cellulose chromatographic column, carrying out gradient elution by sequentially using NaCl solution with the concentration of 0 gradient increased to 1.0mol/L, combining the same polysaccharide components according to the color reaction of a phenol-sulfuric acid method, concentrating, dialyzing, and freeze-drying to obtain two secondary components of BCP-A and BCP-B;

(4) And (3) respectively re-dissolving the secondary component BCP-A and the secondary component BCP-B obtained in the step (3) by using deionized water, respectively loading the secondary component BCP-A and the secondary component BCP-B to a Sephadex G-100 chromatographic column, taking distilled water as eluent, combining the same polysaccharide components according to a phenol-sulfuric acid method color reaction, concentrating, dialyzing, and freeze-drying to obtain the homogeneous blackberry lily polysaccharides BCP-A1 and BCP-B1.

Further, in the step (1), the liquid-material ratio is 35-40 (mL/g), the extraction temperature is 80-90 ℃, and the extraction time is 1-3 h.

Further, the Sevag reagent in the step (2) is a mixed solution of chloroform and n-butanol with the volume ratio of 4:1.

Further, in the step (2), sevag reagent is added according to the volume ratio of 3-10 to 1, the operation process of oscillating, centrifuging and taking the upper polysaccharide solution is repeated for 2-5 times.

Further, in step (3), naCl solutions of 0, 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.4mol/L and 0.5mol/L in this order were eluted at a flow rate of 1mL/min for 125min per gradient.

Further, step (4) was performed by eluting with distilled water at a flow rate of 0.4mL/min for 700min.

The invention also provides the homogeneous blackberry lily polysaccharide prepared by the preparation method, and the homogeneous blackberry lily polysaccharide comprises homogeneous blackberry lily polysaccharide BCP-A1 and homogeneous blackberry lily polysaccharide BCP-B1.

Further, BCP-A1 is homogeneousA neutral polysaccharide having a molecular weight of about 6.0820X 10 ⁴ kDa; the total sugar content is 94.07 +/-1.62%; no uronic acid, monosaccharide composition showing that it consists of mannose, glucose and galactose, in a molar ratio of 1.00; the results of methylation analysis show that the structure contains: manp- (1 →, glcp- (1 →, → 4) -Galp- (1 → and → 3,4) -Galp- (1 → four glycosidic linkages in mole percent 9.63%,13.63%,25.85% and 50.89%.

Further, BCP-B1 is a homogeneous acidic polysaccharide having a molecular weight of about 2.2744X 10 ⁴ kDa; the total sugar content is 91.46 ± 1.37%; the uronic acid content is 33.92 ± 2.74%; monosaccharide composition it is shown to consist of mannose, galacturonic acid, glucose, galactose and arabinose, at a molar ratio of 1.00; the results of methylation analysis show that the structure contains: → 5) -Araf- (1 →, manp- (1 →, glcp- (1 →, → 4) -Glcp, → 4) -GalpA/Galp- (1 → and → 3,4) -Galp- (1 →, the molar ratio percentages being 19.61%, 4.93%, 15.71%, 6.16%, 32.27% and 21.32%.

The invention also provides application of the homogeneous blackberry lily polysaccharide in preparation of anticomplement medicines.

Furthermore, the homogeneous blackberry lily polysaccharide BCP-A1 and BCP-B1 can obviously inhibit cell hemolysis caused by activation of the classical pathway and the alternative pathway of complement, namely, the homogeneous blackberry lily polysaccharide BCP-A1 and BCP-B1 have obvious anticomplementary effect.

Further, the anticomplementary activity of BCP-A1 is CH ₅₀ ＝0.009±0.003mg/mL，AP ₅₀ =0.015 ± 0.003mg/mL, able to interact with target C2, C4 and Factor B to inhibit complement activation;

BCP-B1 anticomplementary Activity CH ₅₀ ＝0.004±0.001mg/mL，AP ₅₀ =0.028 ± 0.005mg/m, can interact with target C2, C4 and Factor B to inhibit complement activation.

Compared with the prior art, the invention has the following beneficial effects:

the invention extracts and separates the rhizome of belamcanda chinensis to obtain two homogeneous belamcanda chinensis polysaccharides (BCP-A1 and BCP-B1), and the in vitro experiment proves that the two belamcanda chinensis polysaccharides have obvious complement inhibition activity, can be used as anticomplement drugs for development and development, and provides a material basis for the treatment of complement-related diseases.

Drawings

In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described below.

FIG. 1 is the separation and elution curve of blackberry lily polysaccharide on DEAE-52 chromatographic column;

FIG. 2 is the separation and elution curve of blackberry lily polysaccharide on Sephadex G-100 chromatographic column;

FIG. 3 is a BCP-A1 (a) and BCP-B1 (B) gel permeation chromatogram;

FIG. 4 is a standard monosaccharide (a), BCP-A1 (B), and BCP-B1 (c) monosaccharide composition chromatogram.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and experimental equipment, materials, reagents and the like used in the experimental method can be purchased from chemical companies.

Belamcanda chinensis (L.) Redout é used in the examples was obtained from the university of Yanbian medicinal plantations orchard.

EXAMPLE 1 preparation of Belamcanda chinensis polysaccharides BCP-A1 and BCP-B1

Taking 1.0kg of dried blackberry lily rhizome, crushing by a crusher, sieving by a 20-mesh sieve, adding water for extraction at the liquid-material ratio of 40 (mL/g) and the extraction temperature of 85 ℃, extracting for 1.5h, filtering to obtain filtrate, concentrating the filtrate to 1/5 of the original volume, adding 95% ethanol to the final concentration of 80%, refrigerating at 4 ℃, standing for 24h, centrifuging to remove supernatant, taking precipitate, drying to constant weight to obtain crude blackberry lily polysaccharide, adding distilled water for redissolving, adding Sevag reagent (chloroform: n-butyl alcohol = 4:1) in the proportion of 5:1, oscillating, centrifuging, removing middle layer denatured protein, taking upper layer polysaccharide solution, repeating the operation for 3-5 times, concentrating and drying, adding deionized water for redissolving, separating by using a DEAE-52 cellulose chromatographic column, sequentially using deionized water, 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.4mol/L and 0.5mol/L NaCl solution at the flow rate of 1mL/min, collecting and eluting according to the sequence of a gradient of a sodium chloride-sodium chloride (NaCl), collecting eluate, collecting fractions according to the same number of each bottle, collecting and collecting eluate, and collecting the elution fractions, wherein each fraction is shown in a secondary reaction curve of a secondary reaction chart as a, and the drawing, and the number of a, and the elution is as a secondary drawing, and the code of a light absorption curve of a, and the elution component, and the elution is shown in a, and the code of a color of a BCP, and the code of a BCP, and the code of a. And then respectively adding deionized water to dissolve two secondary components BCP-A and BCP-B again, separating by Sephadex G-100 chromatography, taking distilled water as eluent, collecting the eluent according to 5 mL/bottle with the flow rate of 0.4mL/min, eluting for about 700min, merging the same polysaccharide components according to a phenol-sulfuric acid method color reaction, and concentrating, dialyzing and freeze-drying to obtain the homogeneous polysaccharide, wherein the elution curve is shown as the attached figure 2: BCP-A1 and BCP-B1.

Example 2 structural characterization of Belamcanda chinensis polysaccharides (BCP-A1 and BCP-B1)

(1) Determination of molecular weight

The homogeneity and molecular weight of the polysaccharide of Belamcanda chinensis is determined by high performance gel permeation chromatography combined with refractive index detector (HPGPC-RID). A2.0 mg/mL blackberry lily polysaccharide solution was filtered through a 0.45 μm microfiltration membrane and injected into a Shodex sugar KS-804 sugar column (8.0 mm. Times.300 mm). The chromatographic conditions are as follows: the sample amount is 20 mu L, ultrapure water is used as a mobile phase, the flow rate is 1.0mL/min, the column temperature is 50 ℃, and the temperature of the differential refractive index detector is 35 ℃. Data processing was recorded on the N2000 GPC chromatography workstation. The molecular weight of the polysaccharide was calculated using a series of dextrans of different weight average molecular weight as standards for calibration curves.

The experimental result shows that the molecular weights of BCP-A1 and BCP-B1 are 6.0820X 10 respectively ⁴ kDa and 2.2744X 10 ⁴ kDa. As shown in figure 3, the HPGPC chromatogram of the blackberry lily polysaccharide is homogeneous and unique. Therefore, BCP-A1 and BCP-B1 are both homogeneous belamcanda chinensis polysaccharide.

(2) Total sugar and uronic acid content determination

Determination of total sugar content by sulfuric acid-phenol method: the total sugar content of BCP-A1 and BCP-B1 is 94.07 + -1.62% and 91.46 + -1.37%, respectively.

The content of uronic acid is determined and detected by an m-hydroxy biphenyl method: no uronic acid was detected in BCP-A1, uronic acid saccharide of BCP-B1 was 23.92 ± 2.74%.

(3) Monosaccharide composition determination

The monosaccharide component is determined by a 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization method. 2mg of blackberry lily polysaccharide is precisely weighed and mixed with 1mL of 2mol/L trifluoroacetic acid (TFA), and the mixture is subjected to sealing reaction and hydrolysis for 2 hours. Adding 200 mu L of 0.5mol/L PMP methanol solution and 0.3mol/L NaOH solution into the hydrolysate in sequence, carrying out water bath at 70 ℃ for 1h, adding 200 mu L of 0.3mol/L HCl, extracting with chloroform for three times, and collecting a water layer. Derivatization was performed as above with mannose (Man), glucosamine (GlcN), ribose (Rib), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara) and fucose (Fuc) as standard controls. And (3) passing the pretreated sample and the standard substance through a 0.45-micron microporous filter membrane, and injecting a sample. Chromatographic conditions are as follows: supersil ODS2 column (5 μm,4.6 mm. Times.250 mm), column temperature 30 ℃, detector wavelength 245nm, flow rate 0.8mL/min, sample size 20 μ L, mobile phase acetonitrile: 0.1mol/mL phosphate buffer solution (pH 6.8) =82 (v/v).

The measurement results show that as shown in fig. 4, BCP-A1 consists of three monosaccharides, mannose, glucose and galactose, in a molar ratio of 1.00. BCP-B1 consists of five monosaccharides, mannose, galacturonic acid, glucose, galactose and arabinose, in a molar ratio of 1.00.

(4) Methylation analysis

Methylation: adding 20mg polysaccharide into round bottom flask, adding 12mL dimethyl sulfoxide, performing ultrasonic treatment under nitrogen protection (until polysaccharide sample is dissolved), adding sodium hydroxide powder (240 mg, and grinding for use), and performing ultrasonic treatment under nitrogen protection to dissolve. Then, the solution is placed in an ice-water bath to be solidified, 3.6mL of methyl iodide is slowly added, ultrasonic reaction is carried out for 30min at the normal temperature, nitrogen is introduced to drive the residual methyl iodide, the ice-water bath is carried out again to solidify the methyl iodide, and the steps are repeated for many times to carry out methylation again. After the methylation reaction was completed, 2mL of distilled water was added to decompose excess methyl iodide, and the solution was dialyzed for 48h, during which time the distilled water was changed several times. After dialysis, chloroform extraction was performed 3 times, and chloroform layers were combined and washed with distilled water 3 times, and anhydrous Na was added ₂ SO ₄ Drying for 24 hr, concentrating the chloroform layer under reduced pressure, drying, and detecting by infrared spectroscopyMeasuring 3200-3400 cm ^-1 The broad absorption peak in the range disappears, indicating that complete methylation has occurred and is available for further hydrolysis.

Hydrolysis: dissolving methylated polysaccharide into 5mL of 90% formic acid solution, sealing and hydrolyzing at 100 ℃ for 4h, decompressing and evaporating to dryness, washing with 3mL of methanol for 3 times, and evaporating to dryness. Then adding 6mL of 2mol/L prepared TFA, hydrolyzing at 110 ℃ for 3h, decompressing and drying, washing with methanol for 3 times, and drying to remove TFA completely.

Reduction: 6mL of fresh 10mg/mL NaBH was added ₄ Reacting at room temperature for 3 hours, shaking for several times during the reaction, then carrying out reduction reaction at room temperature overnight, dripping 0.1mol/L acetic acid after the reaction is finished to decompose excessive borax and adjusting the pH value of the solution to 5.5-7.0, carrying out reduced pressure spin drying on the reaction solution at 50 ℃, adding 2mL methanol, evaporating to dryness, repeating for three times, and finally evaporating to dryness.

Acetylation: adding 2mL of pyridine and 2mL of acetic anhydride, carrying out closed reaction at 100 ℃ for 2h, carrying out reduced pressure evaporation after complete reaction, adding chloroform for extraction, taking a chloroform layer, evaporating to dryness, redissolving chloroform for later use, and analyzing by GC-MS.

Temperature programming: the initial temperature is maintained at 140 deg.C for 2min, increased to 200 deg.C at 2 deg.C/min for 2min, and increased to 280 deg.C at 10 deg.C/min for 5min. The injection port temperature is 250 ℃, the carrier gas is helium, the flow rate of a capillary column is 1mL/min, and the capillary chromatographic column is DM-5MS (30 m multiplied by 0.32mm multiplied by 0.25 mu m). The results of the methylation analysis are shown in Table 1.

TABLE 1 results of methylation analysis of BCP-A1 and BCP-B1

Example 3 classical pathway complement inhibition assay

GVB-Ca is taken from 6% sheep red blood cells ²⁺ /Mg ²⁺ (Beijing Ku Lai Bo technology Limited, cat # DZSL0356-500 ML) buffer to 2X 10 ⁹ cells/mL, and hemolysin (Nanjing Sen Bei Ga Biotech Co., ltd., product number: SBJ-RXS 10) 1:1 are mixed uniformly in proportion, and after being subjected to water bath at 37 ℃ for 30min, unbound hemolysin is removed by centrifugation, so that sensitized sheep red blood cells (EAs) are obtained for later use. Normal healthy adult human serum (NHSP) as the meridianComplement sources of classical pathways. Accurately weighing rhizoma Belamcandae polysaccharide, and adding GVB-Ca ²⁺ /Mg ²⁺ Preparing buffer solutions into different concentration gradients, adding NHSP diluted by 1. The inhibition of hemolysis was calculated for different concentration gradients of belamcanda chinensis and the concentration of 50% of the components required for inhibition of hemolysis (CH) was calculated using GraphPad prism 6.0 ₅₀ Value).

The components and the addition amount in each group are as follows:

blank group: 40 μ L of EAs +260 μ L of GVB-Ca ²⁺ /Mg ²⁺ ；

Total hemolysis group: 40 μ L EAs +260 μ L DDW;

NHSP group: 150 μ L NHSP +40 μ L EAs +110 μ L GVB-Ca ²⁺ /Mg ²⁺ ；

Negative control group: 30 μ L of 1mmol/L glucose +150 μ L NHSP +40 μ L EAs +80 μ L GVB-Ca ²⁺ /Mg ² ⁺ ；

Positive control group: 12 μ L of different concentrations of heparin +150 μ L of NHSP +40 μ L of EAs +98 μ L of GVB-Ca ²⁺ /Mg ²⁺ ；

Administration group: 6 μ L samples at different concentrations +150 μ L NHSP +104 μ L GVB-Ca ²⁺ /Mg ²⁺ +40μL EAs。

The results show that CH of BCP-A1 and BCP-B1 ₅₀ The values are 0.015 +/-0.003 mg/mL and 0.028 +/-0.005 mg/mL respectively, and both homogeneous belamcanda chinensis polysaccharides have remarkable inhibitory activity on the activation of the classical complement pathway (as shown in Table 2).

Example 4 alternative pathway complement inhibition assay

Normal healthy human serum passes GVB-Mg ²⁺ EGTA buffer incubation for 15min on ice was used as the complement source for this alternative pathway. Taking GVB-Ca ²⁺ /Mg ²⁺ Mixing the buffer solution with fresh rabbit blood, centrifuging in low temperature high speed centrifuge for 5min, discarding supernatant, and concocting with Ashi solution to obtain 2%Rabbit red blood cells (Erab). GVB-Mg for 2% rabbit erythrocyte ²⁺ /EGTA buffer to 5 × 10 ⁸ cells/mL; NHSP and diluted belamcanda chinensis polysaccharide with different concentrations are pre-incubated for 15min, rabbit red blood cells (Erab) are added to be incubated for 30min in a water bath at 37 ℃, cooled on ice, stopped reacting and centrifuged. And (3) respectively taking the supernatant of each tube to dilute in a 96-well plate, measuring the absorbance value at 412nm, and simultaneously and respectively setting a blank group, a full hemolysis group, an NHSP group, a negative medicine control group (glucose) and a positive medicine control group (heparin) in the experiment. Calculating the hemolysis inhibition rate of the blackberry lily polysaccharide under different concentration gradients. And the concentration of the polysaccharide required to inhibit hemolysis at 50% (AP) was calculated for each fraction using GraphPad prism 6.0 ₅₀ Value).

The components and the addition amount in each group are as follows:

blank group: 25 μ L Erab +75 μ L GVB-Mg ²⁺ /EGTA；

Total hemolysis group: 25 μ L Erab +75 μ L DDW;

NHSP group: 10 μ L NHSP +25 μ L Erab +65 μ L GVB-Mg ²⁺ /EGTA；

Negative control group: 1 μ L of 1mmol/L glucose +10 μ L NHSP +25 μ L Erab +64 μ L GVB-Mg ²⁺ /EGTA；

Positive control group: 11 μ L of heparin of different concentrations +54 μ L NHSP +25 μ L Erab +54 μ L GVB-Mg ²⁺ /EGTA；

Administration group: 1 μ L samples at different concentrations +10 μ L NHSP +25 μ L Erab +64 μ L GVB-Mg ²⁺ /EGTA。

The results show that AP of BCP-A1 and BCP-B1 ₅₀ The values were 0.015. + -. 0.003mg/mL and 0.028. + -. 0.005 mg/mL, respectively, and both homogeneous belamcanda chinensis polysaccharides had significant inhibitory activity on activation of the alternative complement pathway (as shown in Table 2).

TABLE 2 inhibition of complement activation by Belamcanda chinensis polysaccharide

Remarking: all data are expressed as mean ± SD (n = 3);

CH ₅₀ and AP ₅₀ Represents the 50% hemolysis inhibitory concentration by the classical and alternative pathways, respectively;

heparin was the positive control.

Example 5 complement System target assay

The classical pathway: selecting the lowest concentration of belamcanda chinensis polysaccharide required for inhibiting hemolysis to be close to 100% in the classical pathway as the critical concentration of anticomplementary action target spot, and sequentially adding deleted serum (C2, C3, C4, C5, C9) and sensitized sheep red blood cell (2.0 × 10) ⁹ cells/mL), mixing, incubating in a 37 ℃ water bath for 30min, and centrifuging at 4 ℃ for 5min with a centrifuge of 2000 Xg. Each group was provided with a sample group, a serum-deficient group and a positive control group. Taking 0.2mL of supernatant of each tube to a 96-well plate, measuring the absorbance value at 540nm, subtracting the absorbance value of the corresponding control group, and calculating the hemolysis rate.

The alternative pathway: selecting the lowest concentration of belamcanda chinensis polysaccharide required for inhibiting hemolysis to be close to 100% under the bypass pathway as the critical concentration of anticomplementary action target spot, and sequentially adding deletion serum (C3, C5, C9, factor B, factor D, factor P) and rabbit erythrocyte (5.0 × 10) ⁸ cells/mL) for 30min, incubated on ice for 5min to stop the reaction, and centrifuged at 1000 Xg for 3min at 4 ℃. A sample group, a serum-deficient group and a positive control group were set, respectively. Transfer 30. Mu.L of supernatant to a 96-well plate, add 270. Mu.L of GVB-Mg to each well ²⁺ Diluting with EGTA buffer solution, measuring absorbance at 412nm, and calculating hemolysis rate by subtracting absorbance of corresponding control group.

The experimental results are shown in Table 3, and the hemolysis rates of BCP-A1 and BCP-B1 to target C2, C4 and Factor B are all lower than 20%, which indicates that BCP-A1 and BCP-B1 interact with target C2, C4 and Factor B to inhibit complement activation.

TABLE 3 hemolytic rate of complement target

The embodiments described above are presented to enable those skilled in the art to make and use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims

1. The preparation method of the homogeneous blackberry lily polysaccharide is characterized by mainly comprising the following steps:

(1) Taking dried rhizome of blackberry lily, crushing, sieving with a 20-80-mesh sieve, adding water for extraction at the liquid-material ratio of 20-40 (1 mL/g) to 1mL/g, wherein the extraction temperature is 70-90 ℃, the extraction time is 1-10 h, filtering, taking filtrate, concentrating to 1/3-1/10 of the original volume, adding 95% ethanol until the final concentration is 70-85%, standing at 0-4 ℃ for 12-48 h, centrifuging, and drying the obtained precipitate to obtain crude blackberry lily polysaccharide;

(3) Taking the deproteinized blackberry lily polysaccharide obtained in the step (2), adding deionized water for redissolving, loading the sample to a DEAE-52 cellulose chromatographic column, carrying out gradient elution, combining the same polysaccharide components according to a phenol-sulfuric acid method color reaction, concentrating, dialyzing, and freeze-drying to obtain two secondary components of BCP-A and BCP-B;

(4) Respectively re-dissolving the secondary component BCP-A and the secondary component BCP-B obtained in the step (3) by using deionized water, respectively loading the secondary component BCP-A and the secondary component BCP-B to a Sephadex G-100 chromatographic column, carrying out constant elution, taking distilled water as an eluent, combining the same polysaccharide components according to a phenol-sulfuric acid method color reaction, concentrating, dialyzing, and freeze-drying to obtain homogeneous blackberry lily polysaccharide BCP-A1 and BCP-B1;

the specific process of gradient elution in the step (3) is that NaCl solutions of 0, 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.4mol/L and 0.5mol/L are used for elution in sequence at the flow rate of 1mL/min, and each gradient elution is 100-150 min;

the specific process of constant elution in the step (4) is to use distilled water to elute at the flow rate of 0.4mL/min for 500-800 min.

2. The preparation method according to claim 1, wherein the liquid-material ratio in the step (1) is 35-40.

3. The method according to claim 1, wherein the Sevag reagent in step (2) is a mixed solution of chloroform and n-butanol at a volume ratio of 4:1.

4. The preparation method according to claim 1, wherein the Sevag reagent is added to the solution in step (2) in a volume ratio of 3 to 10:1, the solution is shaken and centrifuged, and the operation of taking the upper polysaccharide solution is repeated 2 to 5 times.

5. The homogeneous blackberry lily polysaccharide obtained by the preparation method of any one of claims 1 to 4, wherein the homogeneous blackberry lily polysaccharide comprises homogeneous blackberry lily polysaccharide BCP-A1 and homogeneous blackberry lily polysaccharide BCP-B1.

6. The homopolysaccharide of claim 5, wherein homopolysaccharide BCP-A1 is a neutral polysaccharide having a molecular weight of about 6.0820 x 10 ⁴ kDa; the total sugar content is 94.07 ± 1.62%; does not contain uronic acid, consists of mannose, glucose and galactose, and has a molar ratio of 1.00; the structure of the utility model comprises: manp- (1 →, glcp- (1 →, → 4) -Galp- (1 → and → 3,4) -Galp- (1 → four glycosidic linkages in mole percent 9.63%,13.63%,25.85% and 50.89%.

7. The homopolymeric blackberry lily polysaccharide of claim 5, wherein the homopolymeric blackberry lily polysaccharide BCP-B1 is an acidic polysaccharide having a molecular weight of about 2.2744 x 10 ⁴ kDa; the total sugar content is 91.46 ± 1.37%; candyThe content of aldehyde acid is 33.92 plus or minus 2.74 percent; consists of mannose, galacturonic acid, glucose, galactose and arabinose in a molar ratio of 1.00; the structure of the utility model comprises: → 5) -Araf- (1 →, manp- (1 →, glcp- (1 →, → 4) -Glcp, → 4) -GalpA/Galp- (1 → and → 3,4) -Galp- (1 →, the molar percentages being 19.61%, 4.93%, 15.71%, 6.16%, 32.27% and 21.32%.

8. Use of a homogeneous blackberry lily polysaccharide according to any one of claims 5 to 7 in the preparation of an anticomplementary medicament.