CN1594337A - Process for preparing protoheme - Google Patents
Process for preparing protoheme Download PDFInfo
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- CN1594337A CN1594337A CNA200410040041XA CN200410040041A CN1594337A CN 1594337 A CN1594337 A CN 1594337A CN A200410040041X A CNA200410040041X A CN A200410040041XA CN 200410040041 A CN200410040041 A CN 200410040041A CN 1594337 A CN1594337 A CN 1594337A
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Abstract
The invention relates to a process for preparing protoheme which comprises the steps of, charging right amount of egg albumen precipitation reagent into animal blood or erythrocytes so as to obtain blood powder, extracting protoheme from the blood powder by chlorhydric acid acetone solution, charging alkaline liquor into the raffinate, adjusting pH=6.0-7.0, and evolution of the solid protoheme.
Description
Technical field
The present invention relates to protoheme preparation method's improvement, particularly exactly raw material carried out processing treatment, blood or red corpuscle are made blood meal with the protein precipitant precipitation after, prepare protoheme with acid acetone extracting again.
Background technology
Protoheme is a kind of good ferrous-fortifier and anti-anemic, is confirmed by pharmacology and clinical study.It can directly be absorbed by the body as mending chalybeate, and specific absorption is up to 10%～20%, and is evident in efficacy, few side effects.In addition, protoheme is the raw material of preparation liver function activator protoporphyrin disodium salt and laser haematoporphyrin diagnosis and treatment cancer drug, can be used as the pigment additive in the making food, and range of application is very extensive.
Usually protoheme separates preparation from animal's whole blood or its red corpuscle as raw material.Bibliographical information has many methods, such as ice acetic acid method, acetone method, base thing---organic solvent method, ion-exchange cellulose method and enzymolysis process etc.Ice acetic acid method is that the scholar Schalfeieff of Russia is used for a kind of method that the laboratory is extracted, separated protoheme the earliest.System utilizes Glacial acetic acid, sodium-chlor and blood to be total to the hot protoheme crystallization that gets, and still is used current industrial.But this method has a lot of shortcomings, and the Glacial acetic acid consumption is big, and production cost is higher; Produce the protein waste liquid that is dissolved in a large number in the acetic acid in the production, the recycling difficulty is extracted in the technology and is reclaimed and must use corrosion resistant device; Raw material should be fresh, otherwise yield and purity all can significantly descend.Base thing---organic solvent method is controlled man's invention for Japan assistant rattan, is with the organic solvent that contains base substance, and protoheme and globin in the oxyphorase are decomposed.Protoheme is dissolved state, and globin then gets off so that decorating film is precipitated, and therefore, both are more easily separated to come.Base substance is metal hydroxidess such as organic amines such as diethylamino, dichloroacetic acid, pyridine, ammonia, causticity yellow soda ash, caustic alkali.Organic solvent is lower alcohol and ketones such as acetone, butanone such as methyl alcohol, ethanol, propyl alcohol.This method is used a large amount of alkali, also can cause industrial pollution, yield also not high (3～4g/L red corpuscle).The ion-exchange cellulose method is nineteen eighty-three Finland scientist invention, utilizes carboxymethyl cellulose (CMC) absorption protoheme to be extracted, and what this method obtained is CMC-protoheme powder, obtain the pure product of protoheme, also need further to make with extra care, cost improves, and it is complicated that technology also becomes.Utilizing various proteolytic enzyme that oxyphorase is hydrolyzed into water soluble amino-acid and polypeptide, water-fast protoheme is separated, also is a kind of good method of not having chemical pollution, but hydrolysis time is long, the gained protoheme also is a crude product, still need make with extra care, and production cost improves.
Early stage acid acetone extraction legal system is equipped with protoheme, be directly to extract hemolysate with acid acetone, and the adding sodium-acetate, protoheme is separated out from extracting solution, the problem that this method exists is, if the acetone consumption is few, foreign protein is removed unclean, and consumption greatly then can cause the cost height, also brings a series of difficult problems such as equipment, cycle simultaneously to production, practicality is had a greatly reduced quality, and gained protoheme purity has only about 60%.At present also useful acid acetone extraction blood meal gets the report of high purity protoheme, but blood meal generally is to adopt low-temperature freezing, or spray-drying process makes, and needs specific installation, and cost is improved greatly.The product of more domestic producers is because of the not high price Jin of purity $6～10/g at present, and purity is high, and the about $90/g of price, and the high import like product of purity are about price Gao Da $300/g.
Summary of the invention
Comprehensive above operational path the present invention proposes and adopts protein precipitant precipitation whole blood or red corpuscle to prepare blood meal, and the production cycle shortens, and equipment requirements reduces, the product purity height, and the yield height, the low ， $3～5/g of cost is fit to suitability for industrialized production.
Find that through overtesting the multiple protein precipitation agent all has the effect that oxyphorase and whole blood albumen precipitation are got off, the protein precipitant that the present invention adopts is a kind of in the following solution:
1) 5%～30% trichloroacetic acid solution
2) 5%～20% sulphosalicylic acid solution
3) 10%～20% tannic acid solution
4) 5%～15% Tungstophosphoric acid, sodium salt solution
5) 0.1mol/L～1.0mol/L BaCl
2Solution
6) 0.8mol/L～1.5mol/L (NH
4)
2SO
4Solution
7) 0.5mol/L～1.2mol/L CuSO
4Solution
8) 0.1mol/L～1.5mol/L ZnSO
4Solution
9) 0.1mol/L～1.5mol/L Zn (Ac)
2Solution
10) 0.1mol/L～1.5mol/L CaCl
2Solution
11) 0.5mol/L～1.5mol/L Pb (Ac)
2Solution
12) 0.1mol/L～1.5mol/L MgSO
4Solution
13) acetone
14) 95% ethanol-chloroform (1: 2～2: 1)
15) 60%～100% ethanol
16) 60%～100% methyl alcohol
17) chloroform
18) propyl alcohol
The consumption of protein precipitant is 2～3 times of volume of whole blood, is 0.5～1 times of erythrocyte volume.Protoheme preparation method provided by the invention, technology is simple, and processing ease is with short production cycle, and equipment requirements is simple, and yield and purity height are fit to suitability for industrialized production, specify below in conjunction with example.
Embodiment
Embodiment 1: fresh pig blood 1000ml, add 0.8% trisodium citrate and make antithrombotics, trisodium citrate can be with a small amount of dissolved in distilled water and is added in advance in the container, adds acetone 1500ml under the vigorous stirring, treat that albumen precipitation fully after, suction filtration.Collecting precipitation pulverizes into powder after volatilizing solvent, adds hydrochloric acid acetone 2000ml, stirs extracting 10min, suction filtration, with hydrochloric acid acetone 500ml gradation washing precipitation to canescence, merging filtrate.Filtrate is transferred pH to 6～7 with 1mol/L NaOH solution, separates out precipitation, and the centrifugal 10min of 3000r/min gets precipitation.Precipitation is separated out a large amount of red-purple precipitations with 0.1mol/L NaOH solution 200ml dissolving, 1mol/L HCl accent pH to acid, and the centrifugal 10min of 3000r/min removes supernatant, and precipitation washes with water to neutrality, and 37 ℃ of dryings get protoheme 2.9g, product purity 99.5%.
Embodiment 2: fresh pig blood 1000ml, add 0.8% trisodium citrate and make antithrombotics, trisodium citrate can be with a small amount of dissolved in distilled water and is added in the container in advance, the centrifugal 15min of 3000r/min, incline and supernatant liquor, collect red corpuscle, use 0.9%NaCl solution washing red corpuscle 2 times again, get clean red corpuscle 420ml.In red corpuscle, add isopyknic distilled water, stir 30min and make haemolysis, add 30% trichloroacetic acid solution 200ml then, stir 10min, after treating that precipitation fully, the centrifugal 10min of 3000r/min gets the oxyphorase precipitation, add acid acetone (pH＜3) 2000ml, stir extracting 10min, suction filtration, precipitation is washed till canescence with acid acetone, merging filtrate, about 2500ml.Filtrate transfers pH to neutral with 1mol/L NaOH solution, separates out precipitation, the centrifugal 10min of 3000r/min, collecting precipitation.Precipitation is dissolved with 0.1mol/L NaOH solution 200ml, and 1mol/L HCl transfers pH to acid, separates out a large amount of precipitations, the centrifugal 10min of 3000r/min, and precipitation washes with water to neutrality, and 37 ℃ of dryings get protoheme 2.8g, product purity 99.8%.
Embodiment 3: fresh pig blood 1000ml, add 0.8% trisodium citrate and make antithrombotics, trisodium citrate can be with a small amount of dissolved in distilled water and is added in advance in the container, the centrifugal 15min of 3000r/min, and inclining supernatant liquor, collect red corpuscle, use 0.9%NaCl solution washing red corpuscle 2 times again, get clean red corpuscle 440ml, add acetone 500ml, stir 10min, after treating that precipitation fully, the centrifugal 10min of 3000r/min gets the oxyphorase precipitation, add acid acetone (pH＜3) 2000ml, stir extracting 10min, suction filtration, precipitation is washed till canescence with acid acetone, merging filtrate, about 2500ml.Filtrate transfers pH to neutral with 1mol/L NaOH solution, separate out precipitation, the centrifugal 10min of 3000r/min, collecting precipitation, after precipitation is dissolved with 0.1mol/L NaOH solution 200ml, 1mol/L HCl transfers pH to acid, separates out a large amount of precipitations, the centrifugal 10min of 3000r/min, precipitation washes with water to neutrality, 37 ℃ of dryings get protoheme 2.8g, product purity 99.7%.
Embodiment 4: fresh pig blood 1000ml, add 0.8% trisodium citrate and make antithrombotics, trisodium citrate can be with a small amount of dissolved in distilled water and is added in the container in advance, the centrifugal 15min of 3000r/min, incline and supernatant liquor, collect red corpuscle, use 0.9%NaCl solution washing red corpuscle 2 times again, get clean red corpuscle 420ml.In red corpuscle, add isopyknic distilled water, stir 30min and make haemolysis, add 0.5mol/L CuSO then
4Solution 200ml stirs 10min, treat that precipitation fully after, the centrifugal 10min of 3000r/min, the oxyphorase precipitation, add acid acetone (pH＜3) 2000ml, stir extracting 10min, suction filtration, precipitation is washed till canescence with acid acetone, merging filtrate, about 2500ml.Filtrate transfers pH to neutral with 1mol/L NaOH solution, separates out precipitation, the centrifugal 10min of 3000r/min, collecting precipitation.Precipitation is dissolved with 0.1mol/L NaOH solution 200ml, and 1mol/L HCl transfers pH to acid, separates out a large amount of precipitations, the centrifugal 10min of 3000r/min, and precipitation washes with water to neutrality, and 37 ℃ of dryings get protoheme 2.7g, product purity 99.8%.
Embodiment 5: fresh pig blood 1000ml, add 0.8% trisodium citrate and make antithrombotics, trisodium citrate can be with a small amount of dissolved in distilled water and is added in advance in the container, adds 15% sulphosalicylic acid solution 1000ml under the vigorous stirring, treat that albumen precipitation fully after, suction filtration.Collecting precipitation pulverizes into powder after volatilizing solvent, adds hydrochloric acid acetone 2000ml, stirs extracting 10min, suction filtration, with hydrochloric acid acetone 500ml gradation washing precipitation to canescence, merging filtrate.Filtrate is transferred pH to 6～7 with 1mol/L NaOH solution, separates out precipitation, and the centrifugal 10min of 3000r/min gets precipitation.Precipitation is separated out a large amount of red-purple precipitations with 0.1mol/L NaOH solution 200ml dissolving, 1mol/L HCl accent pH to acid, and the centrifugal 10min of 3000r/min removes supernatant, and precipitation washes with water to neutrality, and 37 ℃ of dryings get protoheme 2.9g, product purity 99.5%.
The protoheme qualitative test
1. proterties: this law prepares the gained protoheme, under reflected light, be chalybeate Powdered crystal, microscopically is observed the needle-like crystal that takes on a red color, be soluble in basic solution, as ammoniacal liquor and sodium hydroxide solution, be insoluble to hydrochloric acid, 70%～80% ethanol, ether and water, stable in water, identical with reference substance.
2. infrared spectra: get protoheme reference substance and sample respectively with pressing potassium bromide troche, Infrared spectrum scanning, result show, the infared spectrum of sample and reference substance basically identical.
3. UV spectrum: it is an amount of to take by weighing protoheme reference substance and sample respectively, is made into finite concentration, scans at wavelength 300～500nm place.The result shows that sample has maximum absorption at 385 ± 1nm wavelength place, and is identical with reference substance.
The protoheme quantitative assay
1. drawing standard curve
Precision takes by weighing protoheme reference substance 18mg, to the 100ml volumetric flask, adds and uses 0.1mol/L NaOH solution dilution to scale again after 0.1mol/L NaOH solution makes fully dissolving in right amount, shakes up.The above-mentioned solution 1.0,2.0,3.0,4.0 of accurate respectively absorption, 5.0ml puts in the 100ml volumetric flask,, shakes up to scale with 0.1mol/L NaOH solution dilution.Making blank with solvent, measure absorbance in wavelength 385nm place, is ordinate zou with the absorbance A, and concentration C is an X-coordinate drawing standard curve.
2. sample determination
Precision takes by weighing the about 20mg of protoheme sample, puts in the 100ml volumetric flask, after adding 0.1mol/L NaOH solution and making fully dissolving in right amount, uses 0.1mol/L NaOH solution dilution to scale again, shakes up.The accurate above-mentioned solution 3.0ml of absorption puts and uses 0.1mol/L NaOH solution dilution to scale in the 100ml volumetric flask, shakes up.Make blank with solvent, measure absorbance in wavelength 385nm place, and calculate content.
Claims (3)
1. a method of utilizing animal blood to extract protoheme is extracted the animal blood meal with acid acetone soln, and the preparation method who it is characterized in that blood meal adopts in whole blood or red corpuscle the adding protein precipitant to be precipitated and prepares.
2. method according to claim 1 is characterized in that the protein precipitant that adopts can be a kind of in the following solution:
1) 5%～30% trichloroacetic acid solution
2) 5%～20% sulphosalicylic acid solution
3) 10%～20% tannic acid solution
4) 5%～15% Tungstophosphoric acid, sodium salt solution
5) 0.1mol/L～1.0mol/L BaCl
2Solution
6) 0.8mol/L～1.5mol/L (NH
4)
2SO
4Solution
7) 0.5mol/L～1.2mol/L CuSO
4Solution
8) 0.1mol/L～1.5mol/L ZnSO
4Solution
9) 0.1mol/L～1.5mol/L Zn (Ac)
2Solution
10) 0.1mol/L～1.5mol/L CaCl
2Solution
11) 0.5mol/L～1.5mol/L Pb (Ac)
2Solution
12) 0.1mol/L～1.5mol/L MgSO
4Solution
13) acetone
14) 95% ethanol～chloroform (1: 2～2: 1)
15) 60%～100% ethanol
16) 60%～100% methyl alcohol
17) chloroform
18) propyl alcohol
3. method according to claim 1, the consumption that it is characterized in that protein precipitant are 2～3 times of volume of whole blood, are 0.5～1 times of erythrocyte volume.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200410040041XA CN1297282C (en) | 2004-06-21 | 2004-06-21 | Process for preparing protoheme |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200410040041XA CN1297282C (en) | 2004-06-21 | 2004-06-21 | Process for preparing protoheme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1594337A true CN1594337A (en) | 2005-03-16 |
| CN1297282C CN1297282C (en) | 2007-01-31 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB200410040041XA Expired - Fee Related CN1297282C (en) | 2004-06-21 | 2004-06-21 | Process for preparing protoheme |
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| Country | Link |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101941975A (en) * | 2010-08-19 | 2011-01-12 | 四川省德阳市生化制品有限公司 | Method for preparing heme from animal blood corpuscle powder serving as raw material |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1126653A (en) * | 1978-12-22 | 1982-06-29 | Jan H. Luijerink | Process of preparing blood cell protein and heme from hemoglobin |
| CN1037321C (en) * | 1993-05-06 | 1998-02-11 | 林贻箴 | Process for extracting heme and protein powder from animal blood |
-
2004
- 2004-06-21 CN CNB200410040041XA patent/CN1297282C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101941975A (en) * | 2010-08-19 | 2011-01-12 | 四川省德阳市生化制品有限公司 | Method for preparing heme from animal blood corpuscle powder serving as raw material |
| CN101941975B (en) * | 2010-08-19 | 2014-01-15 | 四川德博尔制药有限公司 | Method for preparing heme from animal blood corpuscle powder serving as raw material |
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| Publication number | Publication date |
|---|---|
| CN1297282C (en) | 2007-01-31 |
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