Extraction method of atractylodes macrocephala polysaccharide with anticoagulant activity

Abstract

The invention discloses an extraction and separation method of atractylodes macrocephala polysaccharide with anticoagulant activity, which comprises the following steps: by taking atractylodes macrocephala rhizome as a raw material, performing ethanol degreasing, hot water extraction and ethanol precipitation to obtain crude polysaccharide, then performing Sevage method deproteinization and dialysis purification, and finally performing ethanol fractional precipitation to obtain a plurality of polysaccharide components. Wherein the 80% ethanol precipitation component (AMP-80) has optimal homogeneity and concentrated molecular weight distribution, the total sugar content is 92.18%, the uronic acid content is 24.65%, and the main monosaccharide composition is glucose (45.13%), galacturonic acid (25.46%) and arabinose (21.60%). In-vitro coagulation experiments show that the AMP-80 can significantly prolong PT, APTT and TT in a concentration range of 2.5-10 mg / mL, reduce the content of FIB and show good anticoagulant activity. The atractylodes macrocephala polysaccharide component provided by the invention can be used for preparing anticoagulant drugs, and has the advantages of easily available raw materials, controllable process, definite activity and the like.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction technology, specifically relating to a method for extracting and separating Atractylodes macrocephala polysaccharides with anticoagulant activity. Background Technology

[0002] Atractylodes macrocephala ( Atractylodes macrocephala Koidz. Atractylodes macrocephala (Bai Zhu) is a perennial herb belonging to the genus Atractylodes of the Asteraceae family. It is used to treat various ailments including weak spleen and stomach, loss of appetite, fatigue, shortness of breath, abdominal distension, diarrhea, phlegm retention, edema, jaundice, dampness-related arthralgia, dysuria, dizziness, spontaneous sweating, and threatened miscarriage. The rhizome of Atractylodes macrocephala, after being dried and ground, is the main ingredient in traditional Chinese medicine. The chemical components of Atractylodes macrocephala mainly include volatile oils, polysaccharides, flavonoids, saponins, and other components such as amino acids, vitamins, and trace elements. It possesses various biological activities such as anti-inflammatory, antioxidant, and antitumor effects, but its hemostatic activity has not been reported.

[0003] The human circulatory system is a complex homeostatic system. One of its core functions is to maintain blood flow and to rapidly and locally initiate hemostasis when blood vessels are damaged. Blood coagulation, or clotting for short, occurs through intrinsic or extrinsic coagulation pathways, producing prothrombin activator (PT). PT, under the influence of coagulation factors, produces thrombin, which ultimately converts fibrinogen into fibrin, transforming the blood from a sol to a gel state. Clinically, prothrombin time (PT) is used to reflect the status of the extrinsic coagulation pathway. The intrinsic coagulation pathway refers to the process from factor XII activation to factor X activation; clinically, activated partial thromboplastin time (APTT) is used to reflect the status of the intrinsic coagulation pathway. From factor X activation to fibrin formation, the coagulation pathway involves both intrinsic and extrinsic pathways, primarily including two stages: thrombin generation and fibrin formation.

[0004] Bleeding is common in clinical practice due to various reasons. Traditional anticoagulant drugs, such as heparin, have many side effects (gingival bleeding, epistaxis, skin ecchymosis, and even severe gastrointestinal bleeding and intracranial hemorrhage), and the yield of raw materials is low. Therefore, finding a natural drug is particularly important. Based on this, this application was developed. Summary of the Invention

[0005] The purpose of this invention is to overcome the deficiencies of existing technologies and provide a method for extracting and separating Atractylodes macrocephala polysaccharides with anticoagulant activity. This polysaccharide significantly prolongs PT, APTT, and TT, and reduces FIB content, exhibiting good anticoagulant activity and can be used to prepare anticoagulant drugs. To achieve the above objectives, the present invention adopts the following technical solution: An extraction method for Atractylodes macrocephala polysaccharides with anticoagulant activity, comprising the following steps: (1) Crush the rhizome of Atractylodes macrocephala, extract it with an ethanol aqueous solution, filter it, and collect the residue; (2) Add distilled water to the residue, boil to extract, filter, concentrate the extract and add anhydrous ethanol to a final concentration of 70-80% (v / v), let stand, collect the precipitate, reconstitute and freeze dry to obtain crude polysaccharide; (3) The protein in the crude polysaccharide was removed by the Sevage method, followed by dialysis with a dialysis bag and freeze-drying to obtain Atractylodes macrocephala polysaccharide; (4) Dissolve Atractylodes macrocephala polysaccharide in distilled water, centrifuge and take the supernatant, and add anhydrous ethanol in sequence to make the final concentration reach 10%, 20%, 40%, 60%, 70% and 80% (v / v), collect the precipitates respectively, and obtain 10%, 20%, 40%, 60%, 70% and 80% ethanol precipitate components; the Atractylodes macrocephala polysaccharide with anticoagulant activity is at least one of the 10%, 20%, 40%, 60%, 70% and 80% ethanol precipitate components.

[0006] More preferably, the Atractylodes macrocephala polysaccharide with anticoagulant activity mentioned in step (4) is the 80% ethanol precipitate component. The 80% ethanol precipitate component (AMP-80) is the anticoagulant active component with the best homogeneity.

[0007] Furthermore, the 80% ethanol precipitate (AMP-80) has a neutral sugar content of 92.18% and a uronic acid content of 24.65%. The monosaccharide composition of the 80% ethanol precipitate (AMP-80) includes glucose (45.13%), galacturonic acid (25.46%), arabinose (21.60%), mannose (3.22%), rhamnose (1.15%), and glucuronic acid (3.44%).

[0008] Specifically, in step (1), the sample is extracted with 70-85% ethanol aqueous solution for 36-48 hours, 2-4 times, to remove fat-soluble components, filtered, and the residue is collected.

[0009] Specifically, in step (2), distilled water is added to the residue at a material-to-liquid ratio of 1g:10-30mL, and the residue is boiled and extracted for 1-3 hours, and the extraction is repeated 2-4 times.

[0010] Specifically, in step (3), dialysis is performed for 2-4 days using a dialysis bag with a molecular weight cutoff of 7000-9000 Da.

[0011] This invention provides Atractylodes macrocephala polysaccharide with anticoagulant activity obtained by the above extraction method.

[0012] The present invention also provides the application of the above-mentioned Atractylodes macrocephala polysaccharide with anticoagulant activity in the preparation of anticoagulant drugs.

[0013] Furthermore, in the above-mentioned applications, the Atractylodes macrocephala polysaccharide with anticoagulant activity can prolong prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) in the concentration range of 2.5–10 mg / mL, and reduce fibrinogen (FIB) content.

[0014] The present invention also provides an anticoagulant drug comprising the aforementioned Atractylodes macrocephala polysaccharide with anticoagulant activity.

[0015] The present invention describes an extraction and separation method for Atractylodes macrocephala polysaccharides with anticoagulant activity. Using Atractylodes macrocephala rhizomes as raw material, crude polysaccharides are obtained through ethanol defatting, hot water extraction, ethanol precipitation, and freeze-drying. These crude polysaccharides are then purified by Sevage method to remove proteins and by dialysis. Finally, multiple polysaccharide fractions are obtained through ethanol fractionation precipitation. The 80% ethanol precipitate fraction (AMP-80) exhibits the best homogeneity, with a concentrated molecular weight distribution, a neutral total sugar content of 92.18%, a uronic acid content of 24.65%, and its main monosaccharide components being glucose, galacturonic acid, and arabinose.

[0016] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention provides a method for extracting and separating the effective components of Atractylodes macrocephala polysaccharides, which are used as raw materials. The polysaccharides are separated and purified using ethanol fractional precipitation. The 80% ethanol precipitate fraction (AMP-80) exhibits the best homogeneity, with a concentrated molecular weight distribution, a neutral total sugar content of 92.18%, a uronic acid content of 24.65%, and the main monosaccharide composition being glucose (45.13%), galacturonic acid (25.46%), and arabinose (21.60%). In vitro coagulation experiments show that the 80% ethanol precipitate fraction (AMP-80) significantly prolongs PT, APTT, and TT and reduces FIB content within a concentration range of 2.5–10 mg / mL, demonstrating good anticoagulant activity and suitable for preparing anticoagulant drugs. Furthermore, the extraction process of this invention uses inexpensive and readily available raw materials, and offers advantages such as process controllability and well-defined activity. Attached Figure Description

[0017] Figure 1 HPGPC spectra of each component of Atractylodes macrocephala polysaccharide by ethanol fractionation precipitation method; Figure 2 This study aims to determine the in vitro anticoagulant activity of Atractylodes macrocephala polysaccharides. Detailed Implementation

[0018] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0019] In the following examples, all raw materials used are common commercially available products that can be purchased directly, or can be prepared using conventional techniques in the art.

[0020] The Sevage method described in the examples for removing proteins from crude polysaccharides can be carried out using conventional techniques in the art. For example, it can be referred to Chinese patent application CN202311003571.6 "A coarse trichomoniasis polysaccharide and its preparation method and its application in the preparation of immunomodulatory drugs" (CN117088991A).

[0021] Room temperature refers to 25±5℃. Example 1

[0022] Source plant: Rhizome of Atractylodes macrocephala.

[0023] An extraction method for Atractylodes macrocephala polysaccharides with anticoagulant activity includes the following steps: (1) Using the rhizome of Atractylodes macrocephala as raw material, the rhizome was crushed and then soaked in 80% ethanol aqueous solution at room temperature for 48 h (the amount of 80% ethanol aqueous solution added should be enough to submerge the Atractylodes macrocephala powder). After filtration, the extraction was repeated 3 times to remove fat-soluble components. The residue was collected. (2) Add distilled water to the residue according to the material-liquid ratio (1:20, g / mL), boil and extract for 2 h, filter, repeat the extraction 3 times, combine the extracts, concentrate and add anhydrous ethanol to the final ethanol concentration of 80%, stand at 4°C for 24 h, filter, collect the precipitate, redissolve in ultrapure water, freeze dry to obtain crude polysaccharide. (3) The protein in the crude polysaccharide was removed using the Sevage method (solution:chloroform:n-butanol = 15:4:1, volume ratio). It was then reconstituted with ultrapure water, dialyzed for 3 days using a dialysis bag (molecular weight cutoff 8000 kDa), and then freeze-dried to obtain Atractylodes macrocephala polysaccharide; (4) The freeze-dried Atractylodes macrocephala polysaccharide was dissolved in distilled water to prepare a 5 mg / mL polysaccharide solution. Centrifugation (10000 r / min, 15 min) yielded a clear supernatant. Anhydrous ethanol was slowly added to the solution while stirring continuously until the final concentration reached 10% (v / v). The mixture was placed in a 4 ℃ refrigerator for at least 12 h, and then centrifuged (10000 r / min, 10 min) to obtain a precipitate. The first separated fraction was named AMP-10. Then, the supernatant was further precipitated with anhydrous ethanol until the final concentrations reached 20%, 40%, 60%, 70%, and 80% (v / v) respectively. The obtained precipitate fractions were named AMP-20, AMP-40, AMP-60, AMP-70, and AMP-80, respectively. To obtain better activity, the above components were reconstituted with distilled water, concentrated by rotary evaporation (to remove ethanol for subsequent freeze-drying), and then freeze-dried to obtain purified Atractylodes macrocephala polysaccharide components AMP-20, AMP-40, AMP-60, AMP-70, and AMP-80. They were weighed and set aside for subsequent related tests and coagulation activity experiments.

[0024] In steps (2), (3) and (4), freeze drying is carried out in a gradient, specifically -50℃ for 150 min; -40℃ for 180 min; -30℃ for 180 min; -20℃ for 180 min; -10℃ for 180 min; 0℃ for 150 min; 10℃ for 150 min; 20℃ for 120 min; 30℃ for 120 min; and 40℃ for 120 min.

[0025] Figure 1 HPGPC spectra of each component obtained by the ethanol fractionation method of Atractylodes macrocephala polysaccharide are presented. The results show that: Figure 1 As shown, the peaks of ethanol-precipitated fractions AMP-20, AMP-40, AMP-60, and AMP-70 are broad, with multiple extraneous peaks and poor symmetry, indicating poor homogeneity of these fractions. In contrast, the peak of ethanol-precipitated fraction AMP-80 is single and symmetrical with a narrow peak width, indicating better homogeneity of the polysaccharide. Therefore, further research will be conducted on ethanol-precipitated fraction AMP-80. Example 2

[0026] Determination of basic content of Atractylodes macrocephala polysaccharides in 80% ethanol precipitate (AMP-80).

[0027] Neutral sugar content was detected using the phenol-sulfuric acid method. A glucose standard solution was prepared and serially diluted to concentrations of 0, 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL. The solutions were thoroughly mixed at a volume ratio of concentrated sulfuric acid: 5% phenol solution: serially diluted glucose solution = 5:1:1, allowed to stand at room temperature for 10 min, and the absorbance was measured at 490 nm using a microplate reader. A standard curve was plotted with glucose solution concentration on the x-axis and absorbance (OD) on the y-axis. Atractylodes macrocephala polysaccharide samples were prepared at an appropriate concentration (0.1 g / mL) and tested using the same method. The neutral sugar content was calculated from the standard curve. The uronic acid content in the polysaccharides was determined using the m-hydroxybiphenyl method, with glucuronic acid as the standard reference.

[0028] The results showed that the total sugar content of Atractylodes macrocephala polysaccharide in the 80% ethanol precipitate fraction (AMP-80) was 92.18%, and the uronic acid content was 24.65%. Example 3

[0029] The 80% ethanol precipitate (AMP-80) is composed of Atractylodes macrocephala polysaccharide monosaccharides.

[0030] The monosaccharide composition of Atractylodes macrocephala polysaccharides was determined by PMP-HPLC, with some adjustments made to the determination method. 5 mg of polysaccharide was accurately weighed into a pistol flask, and 1 mL of trifluoroacetic acid solution (4 mol / L) was added to dissolve it completely. The flask was sealed and reacted at 120°C for 5 h. After natural cooling, an appropriate amount of methanol was added, and the mixture was evaporated to dryness under reduced pressure. This process was repeated several times to remove residual reagents. 0.2 mL of ultrapure water was added to the hydrolysis product to dissolve it. 0.2 mL of a mixed standard solution (each monosaccharide standard, concentration 0.5 mg / mL) was placed in another pistol flask. 0.5 mL of internal standard solution (0.1 mol / L lythose solution), 0.2 mL of NaOH solution (0.23 mol / L), and 0.5 mL of PMP methanol solution (PMP refers to 1-phenyl-3-methyl-5-pyrazolone, 0.1 mol / L) were added to each pistol flask, respectively. The flasks were sealed and reacted at 70°C for 100 min, followed by natural cooling. Add 0.3 mL of hydrochloric acid solution (0.15 mol / L), mix well, add 1 mL of chloroform, vortex for 30 s to remove excess PMP, retain the upper aqueous phase, repeat twice, and filter through a 0.22 μm microporous membrane for later use.

[0031] Chromatographic conditions: Agilent TC-C18 column (250 mm × 4.6 mm, 5 μm). Mobile phase: acetonitrile, 0.1 mol / L phosphate buffer (pH = 6.7); gradient elution, flow rate 1 mL / min, detection wavelength 254 nm, column temperature 35 °C, injection volume 10 μL.

[0032] Calculation method: The internal standard method was used, with lysine (Lyx) as the internal standard. The result is expressed as a mass fraction, as shown in the following formula:

[0033] Where m1 is the mass of monosaccharide in the sample, αr and A represent the peak area of ​​the internal standard and the corresponding peak area of ​​each monosaccharide in the mixed standard, respectively, and A1 and αs are the peak areas of the corresponding monosaccharide and internal standard in the sample, respectively. ms is the amount of internal standard added, and mr and M are the mass of the internal standard and the corresponding monosaccharide in the mixed standard, respectively.

[0034] The results showed that the polysaccharide of Atractylodes macrocephala precipitate (AMP-80) in 80% ethanol was mainly composed of glucose, galacturonic acid and arabinose, with glucose (45.13%), galacturonic acid (25.46%), arabinose (21.60%), mannose (3.22%), rhamnose (1.15%) and glucuronic acid (3.44%).

[0035] Example 4

[0036] Four in vitro coagulation tests.

[0037] Instruments and materials: RAC-030 fully automated coagulation analyzer (Rayto Corporation, USA), scutellaria baicalensis extract (BioValley), prothrombin time (PT) assay kit, thrombin time (TT) assay kit, activated partial thromboplastin time (APTT) assay kit, fibrinogen (FIB) content assay kit.

[0038] Experimental animals: Healthy male Sprague-Dawley (SD) rats aged 6 - 8 weeks at the SPF level, purchased from Henan Skbes Biotechnology Co., Ltd. (SCXK Yu 2020-0005). The rats were housed in individually ventilated cages with constant temperature and humidity. Before the experiment, the rats were allowed to freely eat and drink, with a constant temperature of (23 ± 1°C) and humidity of 60%. The rats were adaptively raised for one week according to a 12 h light / dark cycle (light time: 08:30 - 20:30). The above pre-experiment feeding methods for the rats and subsequent experimental treatments of the rats were in line with the "Guide for the Care and Use of Laboratory Animals" issued by the National Institutes of Health and were approved by the Biomedical Research Ethics Sub-committee of Henan University (No.: HUSOM2022-267).

[0039] Preparation of plasma: After anesthetizing the SPF rats, abdominal aortic blood was collected using a blood collection needle into a blood collection tube containing 3.2% sodium citrate (blood:sodium citrate anticoagulant = 1:9). After standing for 30 min, it was placed in a 4°C centrifuge and centrifuged at a speed of 3500 r / min for 10 min. The supernatant was collected into a new EP tube for use.

[0040] Preparation of sample solutions: Preparation of the solvent: DMSO and 1,2-propanediol were mixed in a volume ratio of 3:2. Preparation of the breviscapine solution: 40 mg of breviscapine (positive control) was accurately weighed and dissolved in 2000 μL of the solvent to obtain a solution with a concentration of 20 mg / mL. Preparation of the atractylodes polysaccharide solution: 2.5 mg, 5 mg, and 10 mg of atractylodes polysaccharide were accurately weighed and dissolved in 1000 μL of the solvent to obtain solutions with concentrations of 2.5 mg / mL, 5 mg / mL, and 10 mg / mL.

[0041] Test method for PT: Add 25 μL of the sample solution to the test cup, and then add 50 μL of plasma.

[0042] Test method for APTT: Add 25 μL of the sample solution to the test cup, and then add 50 μL of plasma.

[0043] Test method for FIB: Add 25 μL of the sample solution to the test cup, and then add 50 μL of plasma.

[0044] Test method for TT: Add 25 μL of the sample solution to the test cup, and then add 100 μL of plasma.

[0045] Data statistics.

[0046] Data were statistically processed using GraphPad Prism 10.0 software. One-way ANOVA was used for comparisons among multiple groups, and results are expressed as (X ± SD). A p-value < 0.05 was considered statistically significant between groups.

[0047] Figure 2 The in vitro anticoagulant activity of Atractylodes macrocephala polysaccharides was determined. Results showed that: (See attached...) Figure 2 As shown, Atractylodes macrocephala polysaccharides at concentrations of 2.5 mg / mL, 5 mg / mL, and 10 mg / mL significantly prolonged prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) while reducing fibrin (FIB) levels. The PT prolongation effect was consistent with that of scutellaria baicalensis extract, and the TT effect was greater than that of the scutellaria baicalensis extract group, demonstrating significant anticoagulant activity. This indicates that it can be used to prepare anticoagulant drugs.

Claims

1. A method for extracting white turmeric polysaccharide having anticoagulant activity, characterized in that, The method comprises the following steps: (1) crushing the rootstock of Atractylodes macrocephala Koidz and extracting with ethanol water solution, filtering and collecting the residue; (2) adding distilled water to the residue, boiling and extracting, filtering, concentrating the extract, adding anhydrous ethanol to a final concentration of 70-80%, standing, collecting the precipitate, redissolving and freeze-drying to obtain crude polysaccharide; (3) removing the protein in the crude polysaccharide by Sevage method, then dialyzing with a dialysis bag and freeze-drying to obtain Atractylodes macrocephala Koidz polysaccharide; (4) dissolving the Atractylodes macrocephala Koidz polysaccharide in distilled water, centrifuging to obtain the supernatant, adding anhydrous ethanol to a final concentration of 10%, 20%, 40%, 60%, 70% and 80% (v / v) in sequence, respectively collecting the precipitate to obtain 10%, 20%, 40%, 60%, 70% and 80% ethanol precipitate components; the Atractylodes macrocephala Koidz polysaccharide with anticoagulant activity is at least one of the 10%, 20%, 40%, 60%, 70% and 80% ethanol precipitate components.

2. The extraction method according to claim 1, characterized in that, The Atractylodes macrocephala Koidz polysaccharide with anticoagulant activity in step (4) is the 80% ethanol precipitate component.

3. The extraction method according to claim 2, characterized in that, The monosaccharide composition of the 80% ethanol precipitate component comprises glucose, galacturonic acid, arabinose, mannose, rhamnose and glucuronic acid.

4. The extraction method of claim 1, wherein, In step (1), 70-85% ethanol water solution is used for extraction for 36-48 hours, and the extraction is performed 2-4 times.

5. The extraction method of claim 1, wherein, In step (2), distilled water is added to the residue according to the solid-liquid ratio of 1g:10-30mL, boiling and extracting for 1-3 hours, and the extraction is performed 2-4 times.

6. The extraction method of claim 1, wherein, In step (3), the dialysis bag with a molecular weight cut-off of 7000-9000Da is used for dialysis.

7. The Atractylodes macrocephala Koidz polysaccharide with anticoagulant activity obtained by the extraction method according to any one of claims 1 to 6.

8. The use of the Atractylodes macrocephala Koidz polysaccharide with anticoagulant activity according to claim 7 in the preparation of an anticoagulant drug.

9. Use according to claim 8, characterized in that, The Atractylodes macrocephala Koidz polysaccharide with anticoagulant activity can prolong the prothrombin time, activated partial thromboplastin time and thrombin time and reduce the fibrinogen content in the concentration range of 2.5-10mg / mL.

10. An anticoagulant medicament, characterized in that, The Atractylodes macrocephala Koidz polysaccharide with anticoagulant activity according to claim 7 is contained.

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