CN104774243A - Eucheuma polypeptide extract, as well as preparation method and application thereof - Google Patents
Eucheuma polypeptide extract, as well as preparation method and application thereof Download PDFInfo
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- CN104774243A CN104774243A CN201510163788.2A CN201510163788A CN104774243A CN 104774243 A CN104774243 A CN 104774243A CN 201510163788 A CN201510163788 A CN 201510163788A CN 104774243 A CN104774243 A CN 104774243A
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Abstract
The invention discloses an eucheuma polypeptide extract, as well as a preparation method and application thereof. A polypeptide extract is prepared from an eucheuma raw material. Experiments prove that the extract has good new performances of resisting platelet aggregation, prolonging bleeding time and coagulation time and inhibiting thrombosis, and can be applied to preparation of medicines, medicinal components, prodrugs or health products for resisting platelet aggregation or thrombus.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract and its preparation method and application.
Background technology
Eucheuma muricatum (Gmel.) Web. Van Bos. (Eucheuma), is also Acanthopeltis japonica Okamura, chicken glue dish, and belong to the plump fleshiness of frond of algae, cylindric, flat pressure or flat, radiation or two lateral ramifications, this genus about has 20 kinds, and China about has 5 kinds, is tropical marine alga, has very high pharmaceutical use.Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide component sources is in Eucheuma muricatum (Gmel.) Web. Van Bos..
Thrombotic diseases is that a class has a strong impact on healthy disease, often shows as myocardial infarction, Ischemic Cerebral Infarction, venous thromboembolism.The drug main of current clinical treatment thrombotic diseases will be divided into anti-platelet drug, anticoagulation medicine and thrombolytic agent.Anti-platelet drug has the hematoblastic adhesion of suppression, gathering and release function, thus prevents thrombosis.Antiplatelet drug can prevent the generation of cardiovascular disorder effectively, and can extend the lifetime of patient, and antiplatelet drug is increasingly extensive in clinical application.The Critical policies that new antiplatelet drug is the novel medicament for resisting platelet aggregation of screening or prodrug is found from natural active matter.
And at present temporarily without the antithrombotic function of bibliographical information Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide component, all do not find the report doing antithrombotic reagent or healthcare products use with Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide component at home and abroad.
Summary of the invention
The object of the invention is to overcome above-mentioned the deficiencies in the prior art, a kind of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract and preparation method thereof is provided.
Another object of the present invention is the novelty teabag of openly a kind of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract, and changing Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract can anticoagulant significantly, prevents thrombosis.
Above-mentioned purpose of the present invention is achieved by following technical solution:
A preparation method for Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract, concrete steps are as follows:
S1. get fresh Eucheuma muricatum (Gmel.) Web. Van Bos. to pulverize, adding concentration is that the HCl solution homogenate of 15 ~ 25 mmol/L obtains rough liquid, and the add-on of described HCl solution adds 8 ~ 12 L by every kilogram of Eucheuma muricatum (Gmel.) Web. Van Bos.;
S2. add appropriate saturated potassium chloride solution (under 20 DEG C of conditions, the mass concentration of saturated potassium chloride solution is about 25%) in rough liquid, the concentration to Repone K is 1.3 ~ 1.8%, the viscosity of rough liquid appropriateness is reduced, is convenient to subsequent disposal;
S3. by the rough liquid centrifugation of step S2, get supernatant liquor, adopt gel filtration chromatography to be separated after supernatant liquor coarse filtration, working concentration is the HCl eluant solution of 15 ~ 25 mmol/L, and elution peak has two groups;
S4. second group of eluted product is collected, after regulator solution potential of hydrogen to pH value 6.0 ~ 7.0, centrifugation, the obtained required extract of supernatant liquor lyophilize.
Preferably, the gel column that described gel filtration chromatography adopts is dextran G-50 gel column (Sephadex G-50).
More preferably, the post of described dextran G-50 gel column is high is 28 ~ 32 cm, and internal diameter width is 1.8 ~ 2.2cm.
Preferably, when described gel filtration chromatography is separated, the loading height of rough liquid be gel column post high 5 ~ 10%.
Preferably, when described gel filtration chromatography is separated, the flow rate control of elutriant is 0.8 ~ 1.2 mL/min.
Described supernatant liquor coarse filtration is specially employing 2 ~ 4 layers of filtered through gauze, and described gauze is that conventional gauze is filtered in this area.
Centrifugation described in step S1 or S3 is that low-speed centrifugal is separated, and specifically centrifugal rotating speed is 8000 ~ 12000 turns/min, and centrifugation time is 13 ~ 18 min.
A kind of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract obtained by preparation method of the present invention.The present invention is by simple experiment, and as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirms that extract is single electrophoretic band, main component is the polypeptide component of molecular weight lower than 10 KDa.
Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract is preparing the application in platelet aggregation-against or antithrombotic reagent.
Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract is preparing the application in platelet aggregation-against or antithrombotic healthcare products.
The present invention tests by experiment, find that Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract has anticoagulant, extend bleeding time and clotting time, the new capability of inhibition thrombosis, can be applied to and prepare antithrombotic reagent or healthcare products, for myocardial infarction, Ischemic Cerebral Infarction, venous thromboembolism etc. that anti-hemostasis suppository causes.
The concrete type of service of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract of the present invention comprises following several: 1, can be made into pulvis, oral with physiological saline solution; 2, can be made into injection, use by injection requirement; 3, can interpolation sugar beverage processed, make oral liquid product.
The present invention to the working conditions of described Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract without particular requirement, can use at ambient temperature, dosage is about: 10 ~ 400 μ g/kg/ every days, make by the condition of protein polypeptide component based food or medicine, simultaneously with reference to the antithrombotic reagent of current market and the scope of application of healthcare products.
Compared with prior art, the present invention has following beneficial effect:
The present invention's success is obtained polypeptide extract from Eucheuma muricatum (Gmel.) Web. Van Bos. raw material, prove that this extract has good platelet aggregation-against by experiment, extend bleeding time and clotting time, the new capability of inhibition thrombosis, can be applied to this extract and prepare platelet aggregation-against or antithrombotic medicine, drug component, prodrug or healthcare products.
Embodiment
Below in conjunction with specific embodiment, the present invention is further explained, but embodiments of the present invention is not limited in any way.Unless stated otherwise, involved in embodiment reagent, method are the conventional reagent in this area and method.
embodiment 1the preparation of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract
(1) take 20g Eucheuma muricatum (Gmel.) Web. Van Bos. mechanical disintegration, obtain rough liquid with the HCl solution homogenate of 160 mL 25 mmol/L.
(2) rough liquid comparatively thickness, adds appropriate saturated potassium chloride solution (under 20 DEG C of conditions, the mass concentration of saturated potassium chloride solution is about 25%) in rough liquid, makes the concentration of Repone K be 1.3%.
(3) by the rough liquid centrifugation (8000 turns/min of step (2), centrifugal 18 min), collect supernatant liquor, after three layers of filtered through gauze supernatant liquor, dextran G-50 gel filtration chromatography is adopted to be separated, the post of gel column is high is 28 cm, and internal diameter width is 1.8 cm, loading height be gel column post high 5%.After ready to balance, working concentration is the HCl eluant solution of 25 mmol/L, and the flow rate control of elutriant is 0.8 mL/min, obtains two groups of elution peaks.
(4) collect second group of eluted product, after regulator solution potential of hydrogen to pH value 6.0 ~ 7.0, centrifugation (8000 turns/min, centrifugal 18 min), gets supernatant liquor lyophilize, obtained Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract.
The Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract adopting sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) proved embodiment obtained is single electrophoretic band, and main component is the polypeptide component of molecular weight lower than 10 KDa.
embodiment 2the preparation of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract
(1) take 200g Eucheuma muricatum (Gmel.) Web. Van Bos. mechanical disintegration, obtain rough liquid with the HCl solution homogenate of 2200 mL 15 mmol/L.
(2) rough liquid comparatively thickness, adds appropriate saturated potassium chloride solution in rough liquid, makes the concentration of Repone K be 1.8%.
(3) by the rough liquid centrifugation (12000 turns/min of step (2), centrifugal 13 min), collect supernatant liquor, after three layers of filtered through gauze supernatant liquor, dextran G-50 gel filtration chromatography is adopted to be separated, the post of gel column is high is 32 cm, and internal diameter width is 2.2 cm, loading height be gel column post high 7%.After ready to balance, working concentration is the HCl eluant solution of 15 mmol/L, and the flow rate control of elutriant is 1.2 mL/min, obtains two groups of elution peaks.
(4) collect second group of eluted product, after regulator solution potential of hydrogen to pH value 6.0 ~ 7.0, centrifugation (12000 turns/min, centrifugal 13 min), gets supernatant liquor lyophilize, obtained Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract.
The Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract adopting sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) proved embodiment obtained is single electrophoretic band, and main component is the polypeptide component of molecular weight lower than 10 KDa.
embodiment 3the preparation of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract
(1) take 1000g Eucheuma muricatum (Gmel.) Web. Van Bos. mechanical disintegration, obtain rough liquid with the HCl solution homogenate of 10 L 20 mmol/L.
(2) rough liquid comparatively thickness, adds appropriate saturated potassium chloride solution in rough liquid, makes the concentration of Repone K be 1.5%.
(3) by the rough liquid centrifugation (10000 turns/min of step (2), centrifugal 15 min), collect supernatant liquor, after three layers of filtered through gauze supernatant liquor, dextran G-50 gel filtration chromatography is adopted to be separated, the post of gel column is high is 30 cm, and internal diameter width is 2.0 cm, loading height be gel column post high 10%.After ready to balance, working concentration is the HCl eluant solution of 20 mmol/L, and the flow rate control of elutriant is 1.0 mL/min, obtains two groups of elution peaks.
(4) collect second group of eluted product, after regulator solution potential of hydrogen to pH value 6.0 ~ 7.0, centrifugation (10000 turns/min, centrifugal 15 min), gets supernatant liquor lyophilize, obtained Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract.
The Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract adopting sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) proved embodiment obtained is single electrophoretic band, and main component is the polypeptide component of molecular weight lower than 10 KDa.
embodiment 4eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract is tested the impact of whole-blood platelet aggregation
Adopt prior art to obtain rabbit whole blood thrombocyte, be specially: collect rabbit blood with plastics tubing and add the ACD(86mmol/L Trisodium Citrate of rabbit blood volume 15%, 111mmol/L glucose, 53mmol/L citric acid) anti-freezing.Get whole blood 0.5ml and medicine (the Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract that embodiment 1 is obtained) and hatch 5min, then zymoplasm (500 U/L) or 20 μm of ol/L adenosine diphosphate (ADP) (ADP) process are used, by whole blood aggregometer record aggregate situation, record data, experimental data carries out t inspection.Concrete drug dose and test result are as shown in following table 1 and table 2.
Table 1 Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract is on the impact (n=4) of the rabbit whole blood platelet aggregation of thrombin induction
Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract (μ g/ml) | Maximum platelet aggregation rate (Ω) | Inhibiting rate (%) | |
1 | 0 | 18.75±1.88 | -- |
2 | 1.25 | 9.37±1.28 | 50** |
3 | 2.5 | 8.25±2.36 | 56** |
4 | 5 | 5.62±3.23 | 70** |
*P<0.05,
**P<0.01。
Table 2 Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract is on the impact (n=4) of the rabbit whole blood platelet aggregation that ADP induces
Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract (μ g/ml) | Maximum platelet aggregation rate (Ω) | Inhibiting rate (%) | |
1 | 0 | 11.25±2.65 | -- |
2 | 1.25 | 6.00±3.20 | 53.3** |
3 | 2.5 | 3.86±1.46 | 65.7** |
4 | 5 | 3.13±1.07 | 72.2** |
*P<0.05,
**P<0.01。
As can be seen from the experimental result of table 1 and table 2, Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract can effective anticoagulant.
embodiment 5eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract is on the thrombotic impact experiment of rat carotid artery
With 10% Chloral Hydrate 4.5ml/kg abdominal injection by rat anesthesia, be separated a bilateral common carotid artery and vein, through intravenous (IV) drug (the Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract that embodiment 2 is obtained, employing Normal Saline configuration concentration is the mother liquid medicine of 2 μ g/ μ l) after, carotid artery (electric current 1mA is stimulated with YLS-14B instrument for generating thrombus in small animal, stimulation time is 5 minutes), observe carotid thrombosis congestion situations, SPSS10.0 statistical analysis software is adopted to experimental data, adopt q method of inspection, P < 0.01 thinks there is statistical significance.Concrete drug dose and test result as shown in table 3 below.
The impact (n=6) that table 3 Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract is formed rat suppository
Group | Dosage | 5 minutes average plugging rates (%) |
Control group | -- | 79±8 |
Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract group | 12.5 μg/kg | 30±26* |
Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract group | 25 μg/kg | 11±13* |
Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract group | 50 μg/kg | 3±6* |
XUESHUANTONG group | 30 mg/kg | 8±9* |
Heparin group | 1600 U/kg | 16±20* |
Compare with control group:
*p<0.01.
As can be seen from the experimental result of table 3, Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract effectively can suppress the formation of rat carotid artery thrombus.
embodiment 6the impact of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract on mouse bleeding time and clotting time is tested
(1) mensuration (cutting tail method) in bleeding time
50 Kun Ming mice, body weight 18 ~ 22g, male and female half and half, be divided into 5 groups at random, give high dosage (50 μ g/kg), middle dosage (25 μ g/kg), the medicine (the Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract that embodiment 3 is obtained adopts Normal Saline configuration concentration to be the mother liquid medicine of 2 μ g/ μ l) of low dosage (12.5 μ g/kg), XUESHUANTONG (30mg/kg) and physiological saline respectively.30 min after mice by intraperitoneal injection administration, cut off in Mouse Tail-tip 0.5cm place with scissors, from hemorrhage time manual time-keeping, the tail point hemorrhage place of the broken ends of fractured bone is pasted gently every 30s filter paper, to when filter paper loseing completely red bloodstain, be designated as this mouse bleeding time, bleeding time the longest observation 60min, still hemorrhagely more than 60min calculate by 60min.Recorded the time, each group is carried out significant difference and compares.
(2) mensuration (slide method) in clotting time
With reference to above-mentioned steps (1) to 1 h after mice by intraperitoneal injection administration, pluck eyeball blood sampling, measure the clotting time: after mouse gets eye with slide method, at slide two ends, each 2 diameter are about the drop of blood of 5mm, use manual time-keeping immediately.Every 30s pin, drop of blood ecto-entad being provoked once gently, bleeding till liquid all chooses the trace of blood until observing two.To only provoking the trace of blood from blood sampling, be the clotting time, clotting time the longest observation 30min, calculating by 30min more than 30min not blood coagulation.Recorded the time, each group is carried out significant difference and compares.
The measurement result in bleeding time and the measurement result in clotting time as shown in table 4, as can be seen from Table 3, Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract can effectively extend mouse bleeding time and clotting time, and then effectively can suppress the formation of thrombus.
Table 4 Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract is on the impact (n=10) in mouse bleeding time (BT) and clotting time (CT)
Group | Dosage | BT(min) | CT(min) |
Control group | -- | 11.2±4.4 | 1.3±0.4 |
Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract group | 12.5 μg/kg | 21.3±8** | 3.3±2.1** |
Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract group | 25 μg/kg | 18.7±3.7* | 3.2±1.1** |
Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract group | 50 μg/kg | 20.4±6.3** | 3.4±1.5** |
XUESHUANTONG group | 30 mg/kg | 19.2±4.7* | 3.0±1.3* |
Compare with control group:
*p<0.05,
*p<0.01.
Claims (10)
1. a preparation method for Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract, is characterized in that, concrete steps are as follows:
S1. get fresh Eucheuma muricatum (Gmel.) Web. Van Bos. to pulverize, adding concentration is that the HCl solution homogenate of 15 ~ 25 mmol/L obtains rough liquid, and the add-on of described HCl solution adds 8 ~ 12 L by every kilogram of Eucheuma muricatum (Gmel.) Web. Van Bos.;
S2. adding appropriate saturated potassium chloride solution to the concentration of Repone K in rough liquid is 1.3 ~ 1.8%;
S3. by the rough liquid centrifugation of step S2, get supernatant liquor, adopt gel filtration chromatography to be separated after supernatant liquor coarse filtration, working concentration is the HCl eluant solution of 15 ~ 25 mmol/L, and elution peak has two groups;
S3. second group of eluted product is collected, after regulator solution potential of hydrogen to pH value 6.0 ~ 7.0, centrifugation, the obtained required extract of supernatant liquor lyophilize.
2. the preparation method of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract according to claim 1, is characterized in that, the gel column that described gel filtration chromatography adopts is dextran G-50 gel column.
3. the preparation method of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract according to claim 2, it is characterized in that, the post of described dextran G-50 gel column is high is 28 ~ 32 cm, and internal diameter width is 1.8 ~ 2.2cm.
4. the preparation method of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract according to claim 1, is characterized in that, when described gel filtration chromatography is separated, the loading height of rough liquid be gel column post high 5 ~ 10%.
5. the preparation method of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract according to claim 1, is characterized in that, when described gel filtration chromatography is separated, the flow rate control of elutriant is 0.8 ~ 1.2 mL/min.
6. the preparation method of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract according to claim 1, it is characterized in that, described supernatant liquor coarse filtration is specially employing 2 ~ 4 layers of filtered through gauze.
7. the preparation method of Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract according to claim 1, it is characterized in that, the rotating speed of centrifugation described in step S1 or S3 is 8000 ~ 12000 turns/min, and centrifugation time is 13 ~ 18 min.
8. the Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract that preparation method obtains according to any one of claim 1 to 7.
9. Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract described in claim 8 is preparing the application in platelet aggregation-against or antithrombotic reagent.
10. Eucheuma muricatum (Gmel.) Web. Van Bos. polypeptide extract described in claim 8 is preparing the application in platelet aggregation-against or antithrombotic healthcare products.
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CN105012932A (en) * | 2015-07-29 | 2015-11-04 | 高秀婷 | Spleen tonifying oral liquid and preparation method thereof |
CN106913858A (en) * | 2017-05-02 | 2017-07-04 | 广东医科大学 | Eucheuma polypeptide prevents and treats the application of pulmonary fibrosis |
CN109369781A (en) * | 2018-11-22 | 2019-02-22 | 浙江海洋大学 | A kind of anti-oxidant tetrapeptide of Eucheuma and its application |
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CN110464742A (en) * | 2019-09-08 | 2019-11-19 | 广东医科大学 | A kind of Extraction of Eucheuma gelatinae and its preparing the application in treating organs fibrosis medicine |
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CN106913858A (en) * | 2017-05-02 | 2017-07-04 | 广东医科大学 | Eucheuma polypeptide prevents and treats the application of pulmonary fibrosis |
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