CN112159484B - Anticoagulant fructus polygoni multiflori polysaccharide and extraction and separation method and application thereof - Google Patents
Anticoagulant fructus polygoni multiflori polysaccharide and extraction and separation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of plant extraction, and particularly relates to anticoagulant polysaccharide of perilla frutescens, which is obtained by degreasing the perilla frutescens, removing small molecular polar components, extracting with water, precipitating with ethanol to obtain crude polysaccharide, and further purifying the refined polysaccharide after protein removal and decoloration by using a DEAE-52 cellulose chromatographic column and a Sephadex G-100 gel chromatographic column. The polysaccharide is inspected for the in vitro coagulation effect, and the result shows that the polysaccharide has good anticoagulation effect and can be used for preparing anticoagulation medicaments.
Description
Technical Field
The invention belongs to the technical field of plant extraction, and particularly relates to anticoagulant perilla frutescens polysaccharide, and an extraction and separation method and application thereof.
Background
Herba Stachydis Japonicae belonging to genus OleaceaeChionanthuThe plant is produced in Gansu, Shaanxi, Shanxi, Hebei, Henan, from south to Yunnan, Sichuan, Guangdong, Fujian and Taiwan. Growing in forests or bushes in mountains or hills at altitude 100-. There are distributions in korea and japan. At present, the research on the tassels is mainly on the cultivation technology and the garden greening, and the research on the medicinal value and the chemical components of the tassels is mainly concentrated on the tassels and the root bark parts of the tassels. The chemical components mainly comprise flavonoid, lignanoid, triterpenes, sterols and the like. At present, the study on the perillaseed polysaccharide is not seen, and the pharmacological active ingredient of the perillaseed polysaccharide is not clear.
Coagulation is essentially the process of converting water-soluble fibrinogen into water-insoluble solid fibrin by producing prothrombin activator in the intrinsic or (and) extrinsic pathway of coagulation, producing thrombin by the action of coagulation factors, and finally converting fibrinogen into fibrin by the action of thrombin. Plasma Prothrombin Time (PT) primarily reflects the activity of clotting factors I, II, V, VII, X in the extrinsic coagulation pathway; the partial activation thromboplastin time (APTT) mainly reflects the condition of an intrinsic coagulation system and is related to the activity of intrinsic coagulation factors such as VIII, X, XI, XII and the like; the Thrombin Time (TT) value is an important index which mainly reflects the degree of conversion of fibrinogen into fibrin; plasma Fibrinogen (FIB) reflects primarily the fibrinogen content.
Thrombus is a common disease and frequently encountered disease seriously harming public health, is the main pathological basis of cardiovascular and cerebrovascular diseases with disability and high fatality rate such as cerebral apoplexy, coronary heart disease, atherosclerosis and the like, seriously harms human health, reduces the life quality of patients and family members, and has great influence on social development, human health, economy and the like. Anticoagulants can be used for preventing and treating intravascular embolism or thrombosis diseases, and preventing apoplexy or other thrombotic diseases, and are drugs for preventing coagulation process by influencing certain coagulation factors in the coagulation process. At present, effective anticoagulant drugs are urgently needed, the life quality of patients is improved, and the economic burden is reduced.
Disclosure of Invention
The invention aims to provide a polysaccharide of perilla frutescens with anticoagulant effect, which is extracted and separated from the perilla frutescens, and also provides an extraction and separation method and application of the polysaccharide.
In order to achieve the purpose, the invention adopts the following technical scheme:
an extraction and separation method of anticoagulant fructus perillae polysaccharide comprises the following steps:
(1) pulverizing Chinese fringetree seed, soaking in petroleum ether for defatting, volatilizing petroleum ether from residue, soaking in ethanol for extraction, and volatilizing the residue to remove ethanol smell; heating and extracting with pure water as extraction solvent, concentrating the filtrate under reduced pressure, adding anhydrous ethanol into the concentrated solution, standing, centrifuging, collecting precipitate, redissolving the precipitate, and freeze drying to obtain crude polysaccharide of fructus Perillae Frutescentis;
(2) removing protein and decolorizing with Sevage method to obtain refined polysaccharide of fructus Perillae;
(3) dissolving the perillaseed refined polysaccharide with ultrapure water, centrifuging to remove insoluble substances, adding the solution into a DEAE-52 cellulose chromatographic column, performing gradient elution by using NaCl solutions with concentrations of 0.00mol/L, 0.05mol/L, 0.15 mol/L and 0.25mol/L in sequence, detecting eluent by using a phenol-sulfuric acid method, determining the absorbance at 490 nm, drawing an elution curve by using the number of elution tubes as a horizontal coordinate and the absorbance as a vertical coordinate, combining polysaccharide samples with the same elution peak, concentrating, dialyzing, and freeze-drying; wherein, the elution peak of 0.00mol/L NaCl solution is the component CR-1;
(4) dissolving the component CR-1 with ultrapure water, centrifuging to remove insoluble substances, adding into a Sephadex G-100 gel chromatographic column, eluting with ultrapure water, detecting by using a phenol-sulfuric acid method, measuring the absorbance of the component CR-1 at the wavelength of 490 nm, drawing an elution curve by taking the number of elution tubes as a horizontal coordinate and the absorbance as a vertical coordinate to obtain 1 single main peak, and naming the concentrated, dialyzed and freeze-dried polysaccharide as CRp-1; the CRp-1 is anticoagulant fructus Perillae Frutescentis polysaccharide.
Further, the step (1) is specifically as follows: pulverizing herba Stachydis Japonicae seed, soaking in petroleum ether for 2-3 times (each time for 2-3 days) to defat, extracting with 70 + -5% ethanol for 2-3 times (each time for 2-3 days) to volatilize the residue to no alcohol smell; heating and extracting with pure water as extraction solvent at 80 + -5 deg.C for 2-3 times, each for 3-5 hr, mixing filtrates, concentrating under reduced pressure, adding anhydrous ethanol into the concentrated solution to adjust ethanol concentration to 70 + -2%, standing at 4 deg.C, centrifuging, collecting precipitate, re-dissolving the precipitate, and freeze drying to obtain crude polysaccharide of fructus Perillae Frutescentis.
Further, the step (2) is specifically as follows: dissolving the perillaseed crude polysaccharide with a proper amount of ultrapure water, adding a Sevage reagent according to the volume of 1/3 crude polysaccharide aqueous solution for protein removal, adding absolute ethyl alcohol into the obtained supernatant until the final concentration is 70%, standing at 4 ℃, centrifuging, and collecting precipitates to obtain the perillaseed crude polysaccharide without protein; dissolving the protein-removed fructus Perillae Frutescentis crude polysaccharide with appropriate amount of ultrapure water, adding anhydrous alcohol to final concentration of 70 + -2%, filtering, washing with anhydrous alcohol for decolorizing, and volatilizing to obtain fructus Perillae Frutescentis refined polysaccharide.
The invention provides anticoagulant polysaccharide of perilla frutescens prepared by the extraction and separation method.
The invention also provides application of the anticoagulant fructus perillae polysaccharide in preparation of anticoagulant medicaments.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts a special extraction and separation method to separate polysaccharide CRp-1 from the perilla frutescens, and inspects the in vitro coagulation activity of the polysaccharide, and the result shows that the polysaccharide has good anticoagulation activity and can be used for preparing anticoagulation medicaments, such as anticoagulant.
Drawings
FIG. 1 is a chromatographic elution curve of a polysaccharide DEAE-52 from the refining of Perilla frutescens by column chromatography;
FIG. 2 is a chromatographic elution curve of a fraction CR-1 on a SephadexG-100 gel column;
FIG. 3 is a UV-Vis spectral full wavelength scan of Poncirus trifoliata polysaccharide CRp-1;
FIG. 4 is a graph of the elution of Perilla frutescens polysaccharide CRp-1 by high performance gel exclusion chromatography;
in FIG. 5, A is a GC chromatogram of a standard monosaccharide mixture; b is GC chromatogram of hydrolysate of polysaccharide CRp-1 of fructus Perillae Frutescentis; in the figure, 1, 2, 3, 4, 5 and 6 respectively refer to rhamnose, arabinose, xylose, mannose, glucose and galactose.
Detailed Description
The technical content of the present invention will be described in further detail below with reference to specific embodiments, but the scope of the present invention is not limited thereto.
In the following examples, Perilla frutescens, which is collected from the Taihang mountain area, is the plant Perilla frutescens of the genus Perilla of the family Oleaceae (Chionanthus retusus) The seed of (1).
The ethanol concentrations herein are volume concentrations; NaCl as molar concentration.
An extraction and separation method of anticoagulant perilla frutescens polysaccharide specifically comprises the following steps:
(1) pulverizing Stachys Japonica Makino seed 2000 g, soaking in petroleum ether at room temperature for defatting for 2 times (preferably, the petroleum ether is added to submerge Stachys Japonica Makino seed 6-8 cm), recovering petroleum ether each time for 2 days, and volatilizing the residue until there is no ether smell (i.e. volatilizing petroleum ether). Soaking and extracting with 70% ethanol (ethanol is added at a solid-to-liquid ratio of 1g:5 mL) for 2 times, each for 2 d, recovering ethanol, and volatilizing the residue to remove ethanol smell (i.e. volatilizing ethanol); taking pure water (adding pure water according to a solid-to-liquid ratio of 1g:6 mL) as an extraction solvent, heating and extracting for 2 times at 80 ℃, each time for 4 hours, combining the filtrates, concentrating under reduced pressure to about one tenth of the initial volume of the filtrate to obtain a concentrated solution, then adding absolute ethyl alcohol into the concentrated solution to adjust the final concentration of the ethyl alcohol to 70%, standing overnight at 4 ℃ (12 hours), centrifuging at 4000 r/min for 10 min, collecting precipitates, redissolving the precipitates with ultrapure water, and freeze-drying in a freeze dryer to obtain crude polysaccharide of fructus Perillae Frutescentis;
(2) removing protein and decoloring by using a Sevage method to obtain the refined polysaccharide of the perilla frutescens, which specifically comprises the following steps: dissolving the crude polysaccharide of the perilla frutescens obtained in the step (1) with a proper amount of ultrapure water to obtain a crude polysaccharide aqueous solution, adding a Sevage reagent (chloroform: n-butanol =4:1, volume ratio) according to the volume of 1/3 of the crude polysaccharide aqueous solution, magnetically stirring and shaking for 20 min, centrifuging at 4000 r/min for 6 min, removing intermediate denatured protein and lower organic solvent, and repeating the operation of the supernatant according to the steps for 5-7 times until the protein is basically removed (namely no denatured protein appears). Adding anhydrous ethanol into the supernatant to a final concentration of 70%, standing at 4 deg.C for 6 h, centrifuging at 4000 r/min for 6 min, and collecting precipitate to obtain the protein-removed crude polysaccharide of fructus Perillae Frutescentis. Dissolving the protein-removed crude polysaccharide of the fructus Perillae Frutescentis with appropriate amount of ultrapure water, adding anhydrous ethanol to final concentration of 70%, filtering with Buchner funnel, washing with anhydrous ethanol for decolorizing, and volatilizing to obtain refined polysaccharide of fructus Perillae Frutescentis;
(3) dissolving perillaseed refined polysaccharide with ultrapure water, centrifuging at 12000 r/min for 5 min to remove insoluble substances, adding into DEAE-52 cellulose chromatographic column, gradient eluting with NaCl solutions with concentrations of 0.00mol/L, 0.05mol/L, 0.15 mol/L and 0.25mol/L in sequence at a flow rate of 0.8 mL/min, collecting one tube every 10 min, detecting the eluate with phenol-sulfuric acid method, measuring the absorbance at 490 nm, drawing an elution curve (shown in figure 1) with the number of elution tubes as abscissa and the absorbance as ordinate, combining polysaccharide samples with the same elution peak, concentrating at 55 deg.C under reduced pressure, dialyzing at room temperature (molecular weight cut-off of dialysis bag 8000) for 48 h to remove small molecular impurities, and freeze-drying in a freeze-drying machine; wherein, the elution peak of 0.00mol/L NaCl solution is the component CR-1;
(4) dissolving a component CR-1 with ultrapure water, centrifuging at 12000 r/min for 5 min to remove insoluble substances, adding into a Sephadex G-100 gel chromatographic column, eluting with ultrapure water, controlling the flow rate at 0.5 mL/min, collecting one tube every 6 min, detecting by a phenol-sulfuric acid method, measuring the absorbance at the wavelength of 490 nm, drawing an elution curve (shown in figure 2) by taking the number of elution tubes as a horizontal coordinate and the absorbance as a vertical coordinate, obtaining 1 single elution peak (with high content), concentrating under reduced pressure at 55 ℃, dialyzing at room temperature (molecular weight cut-off of a dialysis bag 8000) for 48 h, and freeze-drying in a freeze dryer to obtain white polysaccharide powder which is named as CRp-1, wherein CRp-1 is the anticoagulant perillaseed polysaccharide.
2. General physicochemical Properties, UV-Vis Spectroscopy, molecular weight determination and monosaccharide analysis of anticoagulated Perilla polysaccharide CRp-1
2.1 general physicochemical Properties
Taking glucose as a standard substance, and determining the total sugar content of the polysaccharide CRp-1 by adopting a phenol-concentrated sulfuric acid method; measuring the protein content by adopting a Coomassie brilliant blue dyeing method; measuring the solubility of polysaccharide CRp-1 in water and organic solvent such as ethanol, n-butanol, acetone, chloroform, petroleum ether, etc., and observing the solubility; carrying out Fehling reagent reaction, ferric trichloride reaction and iodine-potassium iodide reaction. The physicochemical properties of polysaccharide CRp-1 are shown in Table 1.
Table 1 general physicochemical properties of perillaseed polysaccharide CRp-1;
the results in Table 1 show that CRp-1 is a white powder, is easily soluble in water, and is insoluble in organic solvents such as methanol, ethanol, n-butanol, and acetone. CRp-1 had a total sugar content of 97.12% and a protein content of 1.65%. Both the Fehling reagent reaction and the ferric chloride reaction are negative, which shows that CRp-1 does not contain monosaccharide and polyphenol; the I-KI reaction was negative and CRp-1 was not a starch polysaccharide.
2.2 UV-Vis Spectroscopy
The UV-visible full-wavelength scanning spectrum of polysaccharide CRp-1 is shown in FIG. 3. As can be seen in FIG. 3, the characteristic absorption of the polysaccharide is shown at 190-200 nm, and no significant absorption is observed at both 260 nm and 280 nm, indicating that the nucleic acid and protein in polysaccharide CRp-1 are substantially eliminated.
2.3 determination of molecular weight
The molecular weight of polysaccharide CRp-1 was determined by high performance gel exclusion chromatography, and the results are shown in FIG. 4. As can be seen from FIG. 4, CRp-1 has a substantially symmetrical peak shape, CRP-1 has a relative molecular weight (Mw) of 223200 g/mol and a distribution coefficient (Mw/Mn) of 1.774.
2.4 monosaccharide composition analysis
2.4.1 hydrolysis of polysaccharides
CRp-110 mg of polysaccharide is added into a microwave tube, 3 mL of 4 mol/L trifluoroacetic acid is added, and the tube is sealed by nitrogen. Hydrolyzing in oil bath at 110 deg.C for 3 h, adding methanol, removing trifluoroacetic acid solution under reduced pressure, and repeating the above steps for 5 times to obtain hydrolysate.
2.4.2 derivatization of monosaccharides
To the hydrolyzate, 10 mg of hydroxylamine hydrochloride and 0.5 mL of pyridine were added, and the mixture was heated in an oil bath at 90 ℃ for 30 minutes. Taking out, cooling to room temperature, adding 0.4 mL of acetic anhydride, continuing to react in an oil bath at 90 ℃ for 30 min for acetylation, filtering a reaction product by a 0.22-micrometer filter membrane, and injecting the filtered reaction product into a gas chromatography for analysis; standard monosaccharides were treated in the same manner and a standard monosaccharide derivative mixture was prepared.
2.4.3 gas chromatography conditions
A chromatographic column: HP (30 m.times.0.35 mm, 0.25 μm); sample introduction temperature: 250 ℃; detector temperature: 280 ℃; temperature program of chromatographic column: maintaining the initial temperature at 100 deg.C for 1 min, and then increasing the temperature from 100 deg.C to 240 deg.C at 4 deg.C/min for 10 min; carrier gas: high-purity nitrogen with the flow rate of 2 mL/min; the sample size is 2 muL.
2.4.4 monosaccharide composition analysis results
CRp-1 is hydrolyzed and acetylated to obtain acetylated derivative, and GC analysis is performed to compare with GC spectrum of acetylated standard mixed monosaccharide, wherein the result of standard monosaccharide is shown as A in FIG. 5, and the result of polysaccharide CRp-1 is shown as B in FIG. 5. The results in fig. 5 show that from CRp-1, 6 monosaccharides, rhamnose, arabinose, xylose, mannose, glucose and galactose were identified in a molar ratio of 1: 5.381: 1.146: 10.804: 6.953: 11.908.
3. activity analysis of anticoagulant Perilla polysaccharide CRp-1
The method comprises the following steps: detection of blood coagulation activity of perillaseed polysaccharide CRp-1 by in vitro blood coagulation experiment
3.1 instruments and materials
TGL-16gR high speed table refrigerated centrifuge (Shanghai' an Tingning scientific Instrument plant);
RAC-030 full-automatic coagulation analyzer (Shenzhen Redu Life sciences GmbH);
yunnan Baiyao (Yunnan Baiyao group GmbH, ZJA 1708);
prothrombin Time (PT) assay kit (20191008M); activated Partial Thrombin Time (APTT) assay kit (20200319M); thrombin Time (TT) assay kit (20190821M); fibrinogen (FIB) content determination kits (20200310M) were purchased from Shenzhen Redu Life sciences GmbH.
3.2 Experimental animals
Rats, males, weight 180 ~ 200 g.
3.3 sample solution preparation
The solvent is physiological saline, CRp-1 is prepared into 2 mg/mL solution; the Yunnan Baiyao is prepared into 40 mg/mL.
3.4 Experimental methods
Preparation of plasma: taking blood from abdominal artery of rat, placing in blood taking tube containing sodium citrate, slightly inverting, mixing, centrifuging at 3000 rpm for 15 min, and collecting supernatant for use.
The detection method comprises the following steps: PT, APTT, TT and FIB are measured by a full-automatic coagulation analyzer according to the operation instructions of a Prothrombin Time (PT) measuring kit, an Activated Partial Thrombin Time (APTT) measuring kit, a Thrombin Time (TT) measuring kit (20190821M) and a Fibrinogen (FIB) content measuring kit, and the operation is carried out for three times in parallel.
3.5 data processing
The results are expressed as arithmetic mean and standard deviation, and the statistical values are compared with the significance difference by SPSS19.0 software One-Way ANOVA (One-Way ANOVA). The results are shown in Table 2.
TABLE 2 in vitro coagulation results of perillaseed polysaccharide CRp-1
As shown in Table 2, Yunnan white drug powder and polysaccharide CRp-1 significantly shortened APTT, PT and TT (:, as compared with the normal saline group, as shown in the table 2, the Yunnan white drug powder and polysaccharide CRp-1 can significantly shorten APTT, PT and polysaccharidep<0.05); compared with Yunnan white drug powder, polysaccharide CRp-1 can significantly reduce APTT and PT: (A)p<0.05）。
The analysis of the data shows that the polysaccharide CRp-1 has certain anticoagulant effect in vitro and can be used for preparing anticoagulant drugs, such as anticoagulant.
Claims (4)
1. The extraction and separation method of the polysaccharide of the perilla frutescens is characterized by comprising the following steps:
(1) pulverizing Chinese fringetree seed, soaking in petroleum ether for defatting, volatilizing petroleum ether from residue, soaking in ethanol for extraction, and volatilizing the residue to remove ethanol smell; heating and extracting with pure water as extraction solvent, concentrating the filtrate under reduced pressure, adding anhydrous ethanol into the concentrated solution, standing, centrifuging, collecting precipitate, redissolving the precipitate, and freeze drying to obtain crude polysaccharide of fructus Perillae Frutescentis;
(2) removing protein and decolorizing with Sevage method to obtain refined polysaccharide of fructus Perillae;
(3) dissolving the perillaseed refined polysaccharide with ultrapure water, centrifuging, adding the solution into a DEAE-52 cellulose chromatographic column, performing gradient elution by using NaCl solutions with concentrations of 0.00mol/L, 0.05mol/L, 0.15 mol/L and 0.25mol/L in sequence, detecting eluent by using a phenol-sulfuric acid method, determining the absorbance at 490 nm, drawing an elution curve by using the number of elution tubes as a horizontal coordinate and the absorbance as a vertical coordinate, combining polysaccharide samples with the same elution peak, concentrating, dialyzing, and freeze-drying; wherein, the elution peak of 0.00mol/L NaCl solution is the component CR-1;
(4) dissolving the component CR-1 with ultrapure water, centrifuging, adding into a Sephadex G-100 gel chromatographic column, eluting with ultrapure water, detecting by a phenol-sulfuric acid method, measuring the absorbance of the component CR-1 at the wavelength of 490 nm, drawing an elution curve by taking the number of elution tubes as a horizontal coordinate and the absorbance as a vertical coordinate to obtain 1 single elution peak, and naming the polysaccharide after concentration, dialysis and freeze drying as CRp-1; CRp-1 is polysaccharide of fructus Perillae Frutescentis.
2. The method for extracting and separating polysaccharide from perilla frutescens as claimed in claim 1, wherein the step (1) comprises: pulverizing herba Stachydis Japonicae seed, soaking in petroleum ether for 2-3 times (each time for 2-3 days) to defat, extracting with 70 + -5% ethanol for 2-3 times (each time for 2-3 days) to volatilize the residue to no alcohol smell; heating and extracting with pure water as extraction solvent at 80 + -5 deg.C for 2-3 times, each for 3-5 hr, mixing filtrates, concentrating under reduced pressure, adding anhydrous ethanol into the concentrated solution to adjust ethanol concentration to 70 + -2%, standing at 4 deg.C, centrifuging, collecting precipitate, re-dissolving the precipitate, and freeze drying to obtain crude polysaccharide of fructus Perillae Frutescentis.
3. The method for extracting and separating polysaccharide from perilla frutescens as claimed in claim 1, wherein the step (2) comprises: dissolving the perillaseed crude polysaccharide with a proper amount of ultrapure water, adding a Sevage reagent according to the volume of 1/3 crude polysaccharide aqueous solution for protein removal, adding absolute ethyl alcohol into the obtained supernatant until the final concentration is 70%, standing at 4 ℃, centrifuging, and collecting precipitates to obtain the perillaseed crude polysaccharide without protein; dissolving the protein-removed fructus Perillae Frutescentis crude polysaccharide with appropriate amount of ultrapure water, adding anhydrous alcohol to final concentration of 70 + -2%, filtering, washing with anhydrous alcohol for decolorizing, and volatilizing to obtain fructus Perillae Frutescentis refined polysaccharide.
4. The polysaccharide of the perilla frutescens prepared by the extraction and separation method of any one of claims 1 to 3.
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