CN101020714A - Earthworm protein with function of lowering blood pressure and its prepn process - Google Patents
Earthworm protein with function of lowering blood pressure and its prepn process Download PDFInfo
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Abstract
The earthworm protein with function of lowering blood pressure is prepared with 40-100 deg.c hot water earthworm extracting liquid, and through gel filtering and chromatography, and has molecular weight of 2,000-24,000. The preparation process of the earthworm protein includes the following steps: preparing water extracting liquid of earthworm, extracting protein and purifying the protein. The earthworm protein with function of lowering blood pressure is selected with the effect on the activity of angiotensin converting enzyme (ACE) as the index, and has obvious blood pressure lowering effect and no toxic side effect.
Description
Technical field
The present invention relates to earthworm protein that has hypotensive activity and preparation method thereof.
Background technology
At present, the one line medicine for the treatment of essential hypertension clinically is an angiotensin converting enzyme inhibitor, belong to Puli (pril) series mostly, as captopril (captoril), exapril, lisinapril etc., be the dipeptidase derivant that contains proline(Pro) of synthetic, prepare the drawback that this class medicine has complex process.In China, Chinese traditional medicine sleeper (earthworm) is with a long history as Altace Ramipril, still constantly see relevant report in recent years, " the substantive hypertensive research of earth-worm extractive as raw material treatment kidney " literary composition as Chinese combination of Chinese tradiational and Western medicine ephrosis magazine volume the 2nd phase the 80th page of-82 pages of reports in February calendar year 2001 the 2nd, in these reports, what use all is the water decoction of earthworm, so far do not see the report that discloses hypotensive this difficult problem of composition chemical nature in the earthworm, this has hindered the application of earthworm as Altace Ramipril to a certain extent.
Summary of the invention
First purpose of the present invention provides the earthworm protein with hypotensive activity;
Second purpose of the present invention provides the method for preparing the earthworm protein with hypotensive activity from earthworm.
In order to realize first purpose, the earthworm protein with hypotensive activity that is provided is that 40 ℃-100 ℃ hot water leaching liquid with earthworm is the protein (peptide) of 2000-24000 through the molecular weight that gel-filtration, chromatography make.
In order to realize second purpose, preparation has the method for the earthworm protein of hypotensive activity from earthworm, comprises the steps: successively
1) aqueous extract of preparation earthworm: the weightmeasurement ratio that by unit is g/ml joins 1 part of earthworm in 4 parts of-20 parts of distilled water, in temperature is to soak under 100 ℃ the condition, and soak time is no less than 15 minutes, makes the aqueous extract of earthworm;
2) contain protein in the aqueous extract of checking earthworm: the aqueous extract of getting earthworm, be 12000g, 4 ℃ and went precipitation in centrifugal 4 minutes-6 minutes, the 200nm-800nm length scanning, there is absorption peak at 260nm place and 280nm place at scintigram, prove in the aqueous extract of this earthworm to contain nucleic acid and protein;
3) from the aqueous extract of earthworm, extract protein: learnt from else's experience 2) the centrifugal precipitation of going of step, checking contains the supernatant liquor of proteinic earthworm aqueous extract, last Sephadex G50 post, with flow velocity is the tri-distilled water wash-out of 0.6ml-1.2ml/min, collect elutriant with test tube, these elutriants are carried out the 200nm-800nm length scanning, with hippuryl-glycyl-glycine (HGG) or hippuryl-histidyl--leucine (HHL) is that substrate is done Zinc metallopeptidase Zace1 (ACE) active suppression test, then inhibited collection liquid is merged, be lower than freeze-drying under-15 ℃ the condition, obtain the earthworm protein primary extract, the earthworm protein primary extract is carried out the amino acid kind with high pressure liquid chromatography (HPLC) (HPLC) to be measured, result's proof contains Aspartic Acid, L-glutamic acid, Serine, glycine, Histidine, arginine, Threonine, L-Ala, proline(Pro), tyrosine, Xie Ansuan, methionine(Met), Gelucystine, Isoleucine, leucine, phenylalanine and Methionin, this earthworm protein primary extract promptly are angiotensin converting enzyme inhibitor (ACEI);
4) purification of protein: the earthworm protein primary extract through any or two kinds of chromatographies in anion-exchange chromatography, hydrophobic interaction chromatography, the affinity chromatography, is purified earthworm protein, and the step of going forward side by side carries out mass spectroscopy and amino-acid sequence is measured.
Above-mentioned 1) earthworm in the step is dried earthworm of cleaning or the fresh and alive earthworm of cleaning and stimulate with static the eliminating peritoneal fluid through tri-distilled water.
Above-mentioned 1) in the step, why earthworm is chosen in temperature and is under 100 ℃ the condition, soak time is no less than the aqueous extract that made earthworm in 15 minutes, be because soak time is longer, in the earthworm aqueous extract as 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes and 105 minutes, its protein concn did not have notable difference in 15 minutes with immersion after tested, ratio inhibition vigor to Zinc metallopeptidase Zace1 (ACE) is also closely similar, also can reach the sterilization requirement in 100 ℃, 15 minutes simultaneously.
The hypotensive principle of angiotensin converting enzyme inhibitor (ACEI) is:
(renin-angiotensin system RAS) have vital role aspect the fluid and electrolyte balance in blood pressure regulation and body, and Zinc metallopeptidase Zace1 (ACE) plays a crucial role in this system renin-angiotensin system.At first, Angiotensin generates the angiotensin I (AngI) of 10 amino-acid residues under the effect of feritin, AngI falls the histidyl leucine from the terminal hydrolysis of C-under the effect of ACE, generation has the Angiotensin II (Ang II) of 8 amino-acid residues of rising blood pressure effect, Ang II combines with receptor in target cell on the one hand, make arteriole smooth muscle contraction propagation, vessel wall thickening, Peripheral resistance increases, can also make retention of sodium and water in sympathetic nerve granting increase, aldosterone secretion increase, the body on the other hand, finally cause elevation of blood pressure.Studies show that in recent years, body local are organized Ang II excessive concentration, can cause myocardial cell's hypertrophy, left ventricular hypertrophy, glomerular sclerosis, play an important role to hypertensive.Another natural substrate of ACE is bradykinin (bridykinin), the effect that bradykinin has the blood vessel of releiving, brings high blood pressure down, but after ACE made bradykinin fall 2 amino-acid residues from the terminal hydrolysis of C-, product just no longer had the effect that brings high blood pressure down.Angiotensin converting enzyme inhibitor (ACEI) can suppress the hydrolytic activity of Zinc metallopeptidase Zace1 (ACE) on the one hand, reduced the generation of Ang II, thereby reduced blood pressure, suppressed the effect of ACE on the other hand again, made bradykinin can continue the performance hypotensive activity bradykinin.
The method of protein with hypotensive activity for preparing from earthworm provided by the present invention is that index is chosen the protein that is extracted with the activity influence to Zinc metallopeptidase Zace1 (ACE), and method is easy, quick.Has hypotensive activity in order to verify after prepared protein is in entering body, (SHR) tests to spontaneous hypertensive rat, the result has significant hypotensive activity, has no side effect, and lesion tissues such as the heart of suffering from Hypertensive Rats, kidney are partly had the obvious function of improving.
Embodiment
Example 1
Take by weighing the dried earthworm of 5g, clean, place in the triangular flask with cover, add tri-distilled water 100ml, continued to boil after being heated to 100 ℃ 20 minutes, get the aqueous extract of earthworm, get the aqueous extract of earthworm and be 12000g, 4 ℃ were gone precipitation in centrifugal 4 minutes-6 minutes, the 200nm-800nm length scanning, with hippuryl-glycyl-glycine (HGG) or hippuryl-histidyl--leucine (HHL) is substrate, get the supernatant liquor of 0.2ml earthworm aqueous extract and do the test of ACE activity influence, do the SDS-PAGE electrophoresis simultaneously, Coomassie brilliant blue dyeing, after confirming to have the ACEI effect, with Freeze Drying Equipment with this supernatant liquor freeze-drying, take by weighing 0.5g, add the 1ml tri-distilled water, with Sephadex G50 post (1.8 * 70cm) chromatographies on the CTA purifer chromatograph, with flow velocity is the tri-distilled water wash-out of 0.6ml-1.2ml/min, the 260nm wavelength, the 280nm length scanning, collect elutriant by every test tube 1ml, detect the ACEI activity of each test tube thereupon, and the ratio that calculates each test tube suppresses vigor, this more former supernatant liquor of step improve 20.1 times than inhibition vigor, to have active each the test tube elutriant of ACEI merges, with the Freeze Drying Equipment freeze-drying is white powder, this white powder promptly is the earthworm protein primary extract, through check, contain Aspartic Acid 4.21% in this earthworm protein primary extract, L-glutamic acid 7.70%, Serine 2.59%, glycine 2.49%, Histidine 1.10%, arginase 12 .49%, Threonine 1.94%, L-Ala 3.37%, proline(Pro) 2.09%, tyrosine 1.42%, Xie Ansuan 2.46%, methionine(Met) 0.62%, Gelucystine 0.35%, Isoleucine 2.14%, leucine 3.73%, phenylalanine 2.17%, Methionin 4.74% is done the hypotensive test of animal and is carried out the separation and purification of example 2 with this earthworm protein primary extract.
Method of calculation than inhibition vigor are:
When being substrate with hippuryl-glycyl-glycine (HGG):
Than inhibition vigor=107.2 * (standard pipe 228nm absorbancy-pipe 228nm absorbancy to be measured)/protein concentration;
When being substrate with hippuryl-histidyl--leucine (HHL), being taken at the every change of 228nm 0.01nm is a unit:
Than inhibition vigor=100 * (standard pipe 228nm absorbancy-pipe 228nm absorbancy to be measured)/protein concentration;
Wherein: protein concentration (g/ml)=1.5 * 280nm absorbancy-0.74 * 260nm absorbancy,
Or: protein concentration (g/ml)=0.75 * 280nm absorbancy.
The ACE activity test method is as follows:
1, solution preparation:
1), 0.05mol/L Tris-HCl damping fluid (pH7.1 includes 0.1mol/L NaCl);
2), 14m mol/L substrate buffer solution, take by weighing 41.06mg hippuryl-glycyl-glycine (HGG) and be dissolved in this damping fluid of 10ml;
3)、0.1mol/L HCl；
4), sample is for having the active earthworm elutriant of ACEI, concentration is 0.01mg/ml;
5), Zinc metallopeptidase Zace1 (ACE) produces: after the fresh pig lung is cleaned, reject great vessels, fat, be cut into fragment, adding temperature is 4 ℃, the Tris-HCl damping fluid that contains 0.25mol/L sucrose, the pH7.2 of this damping fluid, the weightmeasurement ratio of fresh pig lung and this damping fluid is by restraining: milliliter is counted 3: 10, homogenate 8 minutes, in temperature centrifugal 30 minutes of 9000g under 4 ℃ the condition, get supernatant liquor, under being 0 ℃ condition, temperature carries out the ammonium sulfate two-stage precipitation again, the concentration of first step sulfate precipitate ammonium is 1.6-2.0mol/L, placed after 2 hours 3000g-4500g centrifugal 1 hour, get supernatant liquor, the concentration of second stage sulfate precipitate ammonium is 2.1-2.6mol/L, placed after 10 hours 15000g-25500g centrifugal 2 hours, getting precipitation dialyses, remove ammonium sulfate and get enzyme liquid, with Sephadex G25 gel column on this enzyme liquid, get enzyme peak part and carry out Q HyperD ion exchange chromatography again, with obtaining than vigor after the Freeze Drying Equipment freeze-drying is the zymin of 1300u-1400u/mg, and this zymin is Zinc metallopeptidase Zace1 (ACE).
2, application of sample: establish pipe to be measured, control tube, the blank pipe of standard and standard control pipe, in pipe to be measured and control tube, add the substrate buffer solution of ACE, 0.1ml of 0.15ml and the sample of 0.2ml respectively, in blank pipe of standard and standard control pipe, add the substrate buffer solution of ACE, 0.1ml of 0.15ml and the Tris-HCl damping fluid of 0.2ml respectively, after each manages abundant mixing, be water-bath 60 minutes under 37 ℃ the condition in temperature, and then carry out following step:
3, in control tube and standard control pipe, add the HCl 0.25ml of 1mol/L, react termination in 5 minutes;
4, in each pipe, add ethyl acetate 1.5ml, with the oscillator powerful mixing after 15 seconds, centrifugal 5 minutes of 1250g, draw upper strata 0.5ml, move in four kinds of corresponding glass small test tubes, evaporation is 15 minutes under 120 ℃-130 ℃ condition, in four kinds of glass small test tubes, add 1mol/L NaCl 3.0ml more respectively, with oscillator powerful mixing 15 seconds, place after 15 minutes, survey absorbancy with the 1cm cuvette at 228nm wavelength place at the UV-2501 ultraviolet spectrophotometer again.
The detailed content that the earthworm protein primary extract that use-case 1 is purchased is done the hypotensive test of animal is:
Experimental animal: spontaneous hypertensive rat SHR that adult healthy is male and wister rat, body weight are 200 ± 10g, are provided by Shanghai Slac Experimental Animal Co., Ltd..
Content of the test, method and result:
One, acute toxicity test: sequential method is measured the medium lethal dose of earthworm protein, and the result does not have obvious toxic-side effects.
Two, acute hypotensive test:
1, anesthesia: get 8 of wister rats, intraperitoneal injection of anesthesia is carried out with 3% vetanarcol 1ml/kg in the back of weighing;
2, trachea cannula: making a kerf through the wister rat neck of anesthesia, exposing tracheae, between tracheae two cartilaginous rings, making a t shaped incision, with intubate by this t shaped incision in the thoracic cavity direction is inserted tracheae, use fine rule ligation intubate again;
3, arteria carotis communis intubate: isolate arteria carotis communis in that tracheae is other, with its distal end of fine rule ligation, clamp its proximal part, fixing with remaining needle and ligation that heparin washed near the insertion of distal end place with bulldog clamp;
4, with various dose tongue intravenously administrable, observe the variation and the record of blood pressure, result's dragon protein mass-energy provably obviously reduces the blood pressure of normal wister rat.
Three, chronic hypotensive test: get 24 of spontaneous hypertensive rat SHR, be divided into model group at random, high dose group and low dose group, getting 8 wister rats in addition is the normal control group, four groups of equal abdominal injections, once a day, successive administration 28 days, model group wherein and normal control group are given physiological saline, every day, dosage was 0.8g/kg, high dose group and low dose group are given earthworm protein, the former dosage every day is 0.8g/kg, latter's dosage every day is 0.4g/kg, every interval did not have wound caudal artery method measuring blood pressure in 7 days after the administration, and in the last administration after 10 hours, each group is got blood by arteria carotis communis respectively, (radioimmunoassay RIA) measures blood radio immunoassay, the nervous plain II (Ang II) of cardiac muscle and kidney local vascular, plasma aldosterone (Ald), endothelin level (ET), the content of blood plasma prostacyclin (PGI2), concrete data are as follows:
1, earthworm protein is to the influence of spontaneous hypertensive rat SHR blood pressure
Basic blood pressure before table 1 medication
| Group | Dosage (mg/kg) | Quantity (only) | SBP(mmHg) | DBP(mmHg) |
| The normal control group | 800 | 8 | 124.09±7.71 | 80.64±4.88 |
| High dose group | 800 | 8 | 165.39±8.91 ★★ | 114.42±8.63 ★★ |
| Low dose group | 400 | 8 | 163.65±8.60 ★★ | 115.9±10.00 ★★ |
| Model group | 800 | 8 | 161.53±9.32 ★★ | 116.75±7.98 ★★ |
Compare with the normal control group,
★ ★P＜0.01
14 days blood pressure of table 2 medication
| Group | Dosage (mg/kg) | Quantity (only) | SBP(mmHg) | DBP(mmHg) |
| The normal control group | 800 | 8 | 125.68±6.71 | 83.49±2.33 |
| High dose group | 800 | 8 | 140.35±8.13 ★★△▲ | 93.2±4.11 ★★△▲ |
| Low dose group | 400 | 8 | 150.71±3.17 ★★△ | 102.32±7.43 ★★△ |
| Model group | 800 | 8 | 160.79±8.03 ★★ | 114.41±7.55 ★★ |
Compare with the normal control group,
★ ★P＜0.01; Compare with model group,
△P＜0.05; Compare with low dose group,
▲P＜0.05.
28 days blood pressure of table 3 medication
| Group | Dosage (mg/kg) | Quantity (only) | SBP(mmHg) | DBP(mmHg) |
| The normal control group | 800 | 8 | 123.53±8.46 | 82.95±3.22 |
| High dose group | 800 | 8 | 125.40±6.72 △▲ | 86.48±3.46 △▲ |
| Low dose group | 400 | 8 | 140.86±3.74 ★★△ | 94.07±4.48 ★★△ |
| Model group | 800 | 8 | 161.04±8.09 ★★ | 114.48±8.47 ★★ |
Compare with the normal control group,
★ ★P＜0.01; Compare with model group,
△P＜0.05; Compare with low dose group,
▲P＜0.05.
The digital proof of table 1, table 2 and table 3, earthworm protein can obviously reduce the blood pressure of spontaneous hypertensive rat SHR, with model group significant difference is arranged relatively.
2, earthworm protein influence that rat serum angiotensin (Ang II) is expressed:
Table 4 earthworm protein is to the influence of rat plasma Angiotensin II (Ang II) content
| Group | Dosage (mg/kg) | Quantity (only) | Plasma A ng II (pg/ml) |
| The normal control group | 800 | 8 | 116.78±9.45 |
| High dose group | 800 | 8 | 146.17±3.18 ★★△▲ |
| Low dose group | 400 | 8 | 260.45±37.44 ★★△ |
| Model group | 800 | 8 | 303.11±41.65 ★★ |
Compare with the normal control group,
★ ★P＜0.01; Compare with model group,
△P＜0.05; Compare with low dose group,
▲P＜0.05.
Table 5 earthworm protein is to the influence of rat heart muscle Angiotensin II (Ang II) content
| Group | Dosage (mg/kg) | Quantity (only) | Ang II(ng/ml) |
| The normal control group | 800 | 8 | 3.69±0.62 |
| High dose group | 800 | 8 | 5.33±0.71 ★★△▲ |
| Low dose group | 400 | 8 | 6.74±0.83 ★★△ |
| Model group | 800 | 8 | 7.70±0.93 ★★ |
Compare with the normal control group,
★ ★P＜0.01; Compare with model group,
△P＜0.05; Compare with low dose group,
▲P＜0.05.
The digital proof of table 4, table 5, earthworm protein can obviously reduce the blood plasma of spontaneous hypertensive rat SHR, the level of Angiotensin (Ang II) of cardiac muscle, with model group significant difference are arranged relatively.
3, earthworm protein influence that the rat plasma prostacyclin is expressed:
Table 6 earthworm protein is to the influence of rat plasma prostacyclin content
| Group | Dosage (mg/kg) | Quantity (only) | Prostacyclin (pg/ml) |
| The normal control group | 800 | 8 | 94.27±11.37 |
| High dose group | 800 | 8 | 81.77±6.94 ★★△▲ |
| Low dose group | 400 | 8 | 70.53±10.67 ★★△ |
| Model group | 800 | 8 | 58.99±8.94 ★★ |
Compare with the normal control group,
★ ★P＜0.01; Compare with model group,
△P＜0.05; Compare with low dose group,
▲P＜0.05.
Table 7 earthworm protein is organized the influence of prostacyclin content to rat kidney
| Group | Dosage (mg/kg) | Quantity (only) | Prostacyclin (pg/ml) |
| The normal control group | 800 | 8 | 14.09±1.24 |
| High dose group | 800 | 8 | 14.39±0.94 △▲ |
| Low dose group | 400 | 8 | 12.65±1.56 ★★△ |
| Model group | 800 | 8 | 11.27±1.48 ★★ |
Compare with the normal control group,
★ ★P＜0.01; Compare with model group,
△P＜0.05; Compare with low dose group,
▲P＜0.05.
The digital proof of table 6, table 7, earthworm protein can improve the level of the prostacyclin of spontaneous hypertensive rat SHR blood plasma and renal tissue, with model group significant difference is arranged relatively.
4, earthworm protein influence that the rat aldosterone is expressed:
Table 8 earthworm protein is to the influence of rat plasma aldosterone content
| Group | Dosage (mg/kg) | Quantity (only) | Aldosterone (pg/ml) |
| The normal control group | 800 | 8 | 216.60±14.47 |
| High dose group | 800 | 8 | 239.53±15.15 △▲ |
| Low dose group | 400 | 8 | 277.65±27.25 ★★△ |
| Model group | 800 | 8 | 357.08±27.03 ★★ |
Compare with the normal control group,
★ ★P＜0.01; Compare with model group,
△P＜0.05; Compare with low dose group,
▲P＜0.05.
Table 9 earthworm protein is organized the influence of aldosterone content to rat kidney
| Group | Dosage (mg/kg) | Quantity (only) | Aldosterone (pg/ml) |
| The normal control group | 800 | 8 | 7.59±1.62 |
| High dose group | 800 | 8 | 7.28±1.51 △▲ |
| Low dose group | 400 | 8 | 9.58±1.10 ★★△ |
| Model group | 800 | 8 | 13.59±1.24 ★★ |
Compare with the normal control group,
★ ★P＜0.01; Compare with model group,
△P＜0.05; Compare with low dose group,
▲P＜0.05.
The digital proof of table 8, table 9, earthworm protein can reduce spontaneous hypertensive rat SHR blood plasma and renal tissue the aldosterone level, with model group significant difference is arranged relatively.
5, earthworm protein influence that the rat plasma endothelin is expressed:
Table 10 earthworm protein is to the influence of rat plasma content of ET
| Group | Dosage (mg/kg) | Quantity (only) | Endothelin (pg/ml) |
| The normal control group | 800 | 8 | 122.65±7.66 |
| High dose group | 800 | 8 | 142.13±9.73 ★★△▲ |
| Low dose group | 400 | 8 | 176.53±5.26 ★★△ |
| Model group | 800 | 8 | 204.38±9.50 ★★ |
Compare with the normal control group,
★ ★P＜0.01; Compare with model group,
△P＜0.05; Compare with low dose group,
▲P＜0.05.
The digital proof of table 10, earthworm protein can reduce the level of spontaneous hypertensive rat SHR endothelin level, with model group significant difference is arranged relatively.
6, influence by HE dyeing observation place dragon protein confrontation rat left compartment muscle cell size: the area of the left compartment muscle cell of spontaneous hypertensive rat SHR group, girth, the increase highly significant of longest diameter (LD) and the shortest diameter (SD), compare its difference with the wister group statistical significance (P＜0.01) is arranged, compare with high dose group, the area of the left compartment muscle cell of high dose group, girth, longest diameter (LD) and the shortest diameter (SD) then have obvious decline (P＜0.01), compare the area of the left compartment muscle cell of low dose group with low dose group, girth, longest diameter (LD) and the shortest diameter (SD) decline less (P＜0.05).
Table 11 earthworm protein is to the influence of rat left ventricular mass
| Group | Dosage (mg/kg) | Quantity (only) | Left ventricular mass (g) |
| The normal control group | 800 | 8 | 600.25±45.23 |
| High dose group | 800 | 8 | 665.27±32.60 ★△▲ |
| Low dose group | 400 | 8 | 741.50±16.94 ★★△ |
| Model group | 800 | 8 | 816.45±55.42 ★★ |
Compare with the normal control group,
★ ★P＜0.01,
★P＜0.05; Compare with model group,
△P＜0.05; Compare with low dose group,
▲P＜0.05.
Table 12 earthworm protein influences rat left ventricular mass exponential
| Group | Dosage (mg/kg) | Quantity (only) | Left ventricular mass index (mg/g) |
| The normal control group | 800 | 8 | 2.09±0.10 |
| High dose group | 800 | 8 | 2.52±0.12 ★★△▲ |
| Low dose group | 400 | 8 | 2.91±0.11 ★★△ |
| Model group | 800 | 8 | 2.92±0.16 ★★ |
Compare with the normal control group,
★ ★P＜0.01; Compare with model group,
△P＜0.05; Compare with low dose group,
▲P＜0.05.
7, respectively organize the change of myocardial ultrastructure: wister group myocardium myo protofibril marshalling, clear by electron microscopic observation, structure of mitochondria, intercalated disc structure are normal; It is sparse, disorderly that SHR control group cardiac muscle fibre is arranged, and mitochondrial membrane is imperfect, structure of mitochondria swelling, destroys, is cavity shape interstitial fibers and do not have hyperplasia; It is sparse, fuzzy, disorderly slightly that the high dose group cardiac muscle fibre is arranged, but plastosome, endoplasmic reticulum structure are all normal; Low dose group part structure of mitochondria is normal, the mitochondrial vacuolar degeneration that has, and ridge fracture endoplasmic reticulum structure is normal.
Example 2
The earthworm protein primary extract that use-case 1 is produced carries out separation and purification:
1, (Anion exchange chromatography, IEX): chromatography column is the DEAE-Sepharose that manually loads to anion-exchange chromatography
TMFast Flow; Buffer A: select 50mmol/L Tris damping fluid for use, pH8.8; Buffer B: select 50mmol/L Tris damping fluid+1.0mol/LNaCl for use, pH8.8; Gradient: 0 → 1mol/L NaCl; Elution flow rate: 5ml/min; Applied sample amount: 2.0ml; Gathering speed: 1min/ pipe; Detect wavelength: 280nm, 260nm; The white powder that collection post use-case 1 is produced is a sample, with the buffer A dissolving, regulates pH8.8; Chromatography column, continues behind the last sample to wash to baseline stability linear gradient elution 100%A to 100%B with buffer A to baseline stability with the buffer A balance; The component that obtains after the collection post separates is carried out ACE and is suppressed active mensuration, this step goes up the step and purifies 4.1 times, and further observe its purity with SDS-PAGE, collection has ACE and suppresses active protein elution peak, concentrated freeze-dried is the white powder of purifying for the first time, is stored in-20 ℃ the environment standby.
2, hydrophobic interaction chromatography (Hydrophobic interaction chromatography, HIC): chromatography column is HiTrap Phenyl FF (high sub); Buffer A: select 50mmol/L Tris damping fluid for use, pH7.0; Buffer B: select 50mmol/L Tris damping fluid+1.0mol/L (NH4) for use
2SO
4, pH7.0; Gradient: 0.4mol/L → 0 (NH4)
2SO
4Elution flow rate: 1ml/min; Applied sample amount: 1.0ml; Gathering speed: 1min/ pipe; Detect wavelength: 280nm, 260nm; The collection post is a sample with the white powder of the purifying first time, with the buffer B dissolving, regulates pH7.0; Chromatography column, continues behind the last sample to wash to baseline stability linear gradient elution 100%B to 100%A with buffer A to baseline stability with the buffer A balance; The component that obtains after the collection post separates is carried out ACE and is suppressed active mensuration, this step goes up the step and purifies 3.2 times, and further observe its purity with SDS-PAGE, collection has ACE and suppresses active protein elution peak, concentrated freeze-dried is the white powder of purifying for the second time, is stored in-20 ℃ the environment standby.
Example 3
Taking by weighing fresh and alive compatriot likes to win earthworm and (purchases in Beijing Agricultural University, artificial breeding) 15g, tri-distilled water is cleaned, and utilizes static to stimulate and gets rid of peritoneal fluid, places in the tool plug triangular flask, add tri-distilled water 100ml, continued to boil after being heated to 100 ℃ 15 minutes, the aqueous extract of earthworm, the aqueous extract of getting earthworm is 12000g, 4 ℃ and was gone precipitation in centrifugal 6 minutes, the 200nm-800nm length scanning, following step is with example 1.
Example 4
The earthworm protein primary extract that example 1 is produced carries out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), manually gets near the band of molecular weight 12000 and carries out ground substance assistant laser desorption ionization mass spectrum (maldi-tof-ms) analysis.With trypsin hydrolyzing, and its peptide spectrum is carried out homology sequence compare, find its sequence and gi 456321, g-crystallin (clarias fuscus) has certain similarity.
Example 5
Take by weighing dried earthworm 50g, add the 200ml tri-distilled water, homogenate, getting equivalent places respectively in the tool plug triangular flask, in temperature immersion corresponding successively under 4 ℃, 24 ℃, 40 ℃, 60 ℃, 80 ℃ the condition after 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, centrifugal through 12000g, get supernatant liquor at the 200nm-800nm length scanning, do Zinc metallopeptidase Zace1 (ACE) active suppression test respectively, prove that can obtain higher ratio in time more than 40 ℃ suppresses the vigor supernatant liquor.
Claims (3)
1, a kind of earthworm protein with hypotensive activity is characterized in that 40 ℃-100 ℃ hot water leaching liquid with earthworm is the protein (peptide) of 2000-24000 through the molecular weight that gel-filtration, chromatography make.
2, a kind of method for preparing the earthworm protein with hypotensive activity from earthworm is characterized in that comprising the steps: successively
1) aqueous extract of preparation earthworm: the weightmeasurement ratio that by unit is g/ml joins 1 part of earthworm in 4 parts of-20 parts of distilled water, in temperature is to soak under 100 ℃ the condition, and soak time is no less than 15 minutes, makes the aqueous extract of earthworm;
2) contain protein in the aqueous extract of checking earthworm: the aqueous extract of getting earthworm, be 12000g, 4 ℃ and went precipitation in centrifugal 4 minutes-6 minutes, the 200nm-800nm length scanning, there is absorption peak at 260nm place and 280nm place at scintigram, prove in the aqueous extract of this earthworm to contain nucleic acid and protein;
3) from the aqueous extract of earthworm, extract protein: learnt from else's experience 2) the centrifugal precipitation of going of step, checking contains the supernatant liquor of proteinic earthworm aqueous extract, last Sephadex G50 post, with flow velocity is the tri-distilled water wash-out of 0.6ml-1.2ml/min, collect elutriant with test tube, these elutriants are carried out the 200nm-800nm length scanning, with hippuryl-glycyl-glycine (HGG) or hippuryl-histidyl--leucine (HHL) is that substrate is done Zinc metallopeptidase Zace1 (ACE) active suppression test, then inhibited collection liquid is merged, be lower than freeze-drying under-15 ℃ the condition, obtain the earthworm protein primary extract, the earthworm protein primary extract is carried out the amino acid kind with high pressure liquid chromatography (HPLC) (HPLC) to be measured, result's proof contains Aspartic Acid, L-glutamic acid, Serine, glycine, Histidine, arginine, Threonine, L-Ala, proline(Pro), tyrosine, Xie Ansuan, methionine(Met), Gelucystine, Isoleucine, leucine, phenylalanine and Methionin, this earthworm protein primary extract promptly are angiotensin converting enzyme inhibitor (ACEI);
4) purification of protein: the earthworm protein primary extract through any or two kinds of chromatographies in anion-exchange chromatography, hydrophobic interaction chromatography, the affinity chromatography, is purified earthworm protein, and the step of going forward side by side carries out mass spectroscopy and amino-acid sequence is measured.
3, according to claim 2 from earthworm preparation have the method for earthworm protein of hypotensive activity, it is characterized in that 1) earthworm in the step, be the dried earthworm cleaned or clean and stimulate the fresh and alive earthworm that gets rid of peritoneal fluid with static through tri-distilled water.
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| CNA2007100137860A CN101020714A (en) | 2007-03-12 | 2007-03-12 | Earthworm protein with function of lowering blood pressure and its prepn process |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103845273A (en) * | 2014-03-14 | 2014-06-11 | 江苏隆力奇生物科技股份有限公司 | Lumbricus peptide capable of resisting skin aging as well as preparation method and application thereof |
| CN113663821A (en) * | 2021-08-20 | 2021-11-19 | 中国人民解放军陆军军医大学第一附属医院 | Separation method of platelet-rich fibrin gel |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103845273A (en) * | 2014-03-14 | 2014-06-11 | 江苏隆力奇生物科技股份有限公司 | Lumbricus peptide capable of resisting skin aging as well as preparation method and application thereof |
| CN103845273B (en) * | 2014-03-14 | 2017-06-30 | 江苏隆力奇生物科技股份有限公司 | A kind of earthworm peptide of resisting age of skin and its preparation method and application |
| CN113663821A (en) * | 2021-08-20 | 2021-11-19 | 中国人民解放军陆军军医大学第一附属医院 | Separation method of platelet-rich fibrin gel |
| CN113663821B (en) * | 2021-08-20 | 2023-08-29 | 中国人民解放军陆军军医大学第一附属医院 | Separation method of platelet-rich fibrin gel |
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