KR100340095B1 - Process for Preparing Iron-Transferrin Extract - Google Patents

Abstract

The present invention relates to a method for preparing iron-transferrin extract. The present invention comprises the steps of performing ion exchange chromatography on plasma of slaughtered blood to obtain a transferrin-containing fraction; Filtering and lyophilizing the transferrin-containing fraction to obtain a concentrated transferrin extract powder; And dialysis and sterilization by adding iron to a diluted solution of transferrin extract powder, and then lyophilizing to obtain an iron-transferrin extract in powder form. It provides an iron preparation comprising the iron-transferrin extract as an active ingredient. According to the present invention, the iron solubilization ability in the duodenum is superior to the conventional iron preparation, and the bioavailability is excellent, and the raw materials used are also manufactured using iron-transferrin extract through a simple process using slaughtered blood which is mostly disposed of in the slaughterhouse. can do. In addition, the iron-transferrin extract of the present invention is stable in heat and can be subjected to sterilization process, so it is safe in hygienic aspects, and can be included as an active ingredient in various forms of medicinal iron preparations and iron-reinforced foods.

Description

Process for Preparing Iron-Transferrin Extract

The present invention relates to a method for preparing iron-transferrin extract. More specifically, the present invention is to prepare a transferrin extract using a batch type ion exchange chromatography from the slaughtered blood to increase the bioavailability of iron, by binding iron to the transferrin extract, The present invention relates to a method for economically manufacturing iron-transferrin extracts which are hygienic and has excellent bioavailability and thermal stability, and to an iron preparation comprising iron-transferrin extracts prepared by the above-mentioned methods as an active ingredient.

Iron is an essential nutrient for all types of organisms, most of which are associated with proteins and are involved in many important physiological processes. As a representative example, iron plays an important role in the production of adenosine triphosphate by participating in the role of transporting electrons from the substrate to oxygen in an electron transport system. In relation to this function, below-normal iron production decreases energy production, which leads to rapid fatigue and reduced work efficiency, especially iron deficiency in infancy may damage the brain.

Iron is present in the form of ferrous or ferric, depending on the oxidation state, and almost all forms complex in vivo, forming very low solubility ferric hydroxides at pH above 3.0 As the solubility of ferric iron decreases, it is necessary to form any complex or chelate that can prevent the formation of hydroxide or phosphate complexes during digestion and absorption. Do. In this case, the bioavailability of the iron-bonded complex depends on the stability of the complex, and the form of a soluble salt that is stable upon exposure to various conditions during passage through the intestine is most useful for absorption. In addition, when the stability of the complex is low, iron is easily separated from the complex, and when it is high, the utilization rate of the body varies depending on how much iron is separated in the intestine. It is an important determinant.

Iron deficiency is a common nutrient deficiency phenomenon, and many studies have been conducted to overcome it by strengthening iron, and when selecting an iron preparation to be used for iron enhancement, 1) relative bioavailability, 2) discoloration or odor causing Consideration should be given to the degree of reactivity, 3) preparation of iron preparations, stability during storage, and 4) coordination with other nutrients. In reality, finding an iron preparation that can satisfy all of these factors is a very difficult problem. Therefore, it has been required to develop an iron carrier that can strongly bind to iron and promote their absorption in the body.

Mainly used iron preparations are those using ferrous (Fe ²⁺ ) or ferric (Fe ³⁺ ) ions in the form of inorganic or organic salts, and ferritin or heme, iron proteins. Iron-based iron preparations are sold for medicine. In addition, an anemia treatment agent containing iron and difractose (Korean Patent Publication No. 96-013376), an iron preparation containing iron stabilized in a liposome using iron liposomes as iron carrier (Republic of Korea Patent Publication No. 95-013500) ) And a method for producing iron derivatives using globin or acetyl globin (Korean Patent No. 87-004706) have been developed to improve the bioavailability of iron, but the manufacturing process is not complicated and economical and the bioavailability of iron is excellent. There were some disadvantages. In particular, when the protein is to be used as an iron carrier, sufficient heat treatment cannot be performed due to the denaturation of the protein, thereby raising a hygienic safety problem due to incomplete heat treatment. Accordingly, efforts have been made to develop iron preparations that can be economically produced by a simple manufacturing process, can exhibit efficient bioavailability, and are hygienically safe.

On the other hand, the blood that is mostly discarded during the slaughter of cows or pigs contains about 16 to 18% of protein, and studies to use them as medicines and food materials have been actively conducted. Blood plasma protein in the blood contains a variety of biologically active factors, of which plasma transferrin is a glycoprotein that is responsible for the transport of iron through the blood to each organ, molecular weight of about 77,000 It is known to bind two ferric ions per molecule at early and normal blood pH. Transferrin plays an important role in transporting iron from the blood for the synthesis of hemoglobin in red blood cells, myoglobin in muscle organs, cytochrome or cytochrom oxidase, and per liter of plasma. Since it exists in high concentration of 2-3 g, it has been evaluated as the material which has the conditions of an ideal iron carrier. Until now, lactoferrin or egg white ovotransferrin has been actively developed as a functional food material having a specific iron-binding ability, but anemia treatment for plasma transferrin, which is the same family, As a functional food material that can be used for pharmaceutical and iron fortification has not yet been made.

Therefore, it is possible to produce economically by a simple manufacturing process, exhibit efficient bioavailability, and at the same time use a technology capable of producing a hygienic safe iron preparation, in particular plasma transferrin as an iron carrier, There is a constant need to develop techniques for manufacturing iron formulations.

Accordingly, the present inventors have made efforts to establish a technique that can use plasma transferrin as an iron carrier and a technique for effectively producing an iron preparation using the same as an active ingredient. The iron-transferrin extract obtained when the transferrin extract consisting mainly of transferrin and immunoglobulin was separated by ion exchange chromatography method, and the iron-transferrin extract obtained by using affinity between transferrin and iron was obtained. This hygienic and excellent bioavailability and heat resistant to confirm that it can be used as an active ingredient of a stable iron preparation, and completed the present invention.

After all, the main object of the present invention is to provide a method for preparing an iron-transferrin extract by separating the transferrin extract from the slaughtered blood by an ion exchange method and combining it with iron.

It is another object of the present invention to provide an iron preparation comprising an iron-transferrin extract prepared by the electrical preparation method as an active ingredient.

1 is a protein fractionation device used for the separation of transferrin extract.

The method for preparing the iron-transferrin extract of the present invention comprises the steps of: performing batch ion exchange chromatography using a protein fractionation device designed in the present invention with plasma of slaughtered blood to obtain a transferrin-containing fraction; Filtering and lyophilizing the transferrin fraction to obtain a concentrated transferrin extract powder; Iron-transferrin extract is prepared by dialysis and sterilization by adding iron to a diluted solution of transferrin extract powder, followed by lyophilization to obtain an iron-transferrin extract in powder form.

Hereinafter, the preparation method of the iron-transferrin extract of the present invention will be described in more detail by dividing the process.

First Step : Obtaining Transferrin-Containing Fraction

Plasma-containing fractions are obtained from blood by separating plasma from the slaughtered blood and performing batch ion exchange chromatography using electric plasma. At this time, the plasma of the slaughtered blood can be obtained by centrifugation in refrigerated state by treating the blood or pig blood collected immediately after the slaughter with anticoagulant and taking the supernatant.The ion exchange resin used for ion exchange chromatography is transferrin. As a property of adsorbing proteins, Sepharose (Sepharose), Sephadex (Sephadex), Sepacel (Sephacel) anion exchange resin can be used. In addition, in performing the batch ion exchange chromatography, a solvent which can be used to fractionate the transferrin extract from the plasma protein is a phosphate or tris having a pH of 6.0 to 11.0, preferably pH 8.0 to 10.0. Buffers may be used.

Second Process : Concentration

The transferrin-containing fraction obtained in the first step is filtered and lyophilized to give a concentrated transferrin extract powder. In this case, the transferrin-containing fraction is concentrated by filtration using a membrane having a molecular weight cut off (MWCO) of 30 kDa or an ultrafiltration unit (Millipore, U.S.A.). In addition, lyophilization is used to obtain a transferrin extract in powder form using a commonly used lyophilizer.

Third Step : Obtaining Iron-Transferrin Extract

Iron was added to the diluted solution of the transferrin extract powder obtained in the second step, dialyzed, sterilized, and lyophilized to obtain an iron-transferrin extract in powder form. At this time, the transferrin extract powder is generally preferably diluted to 1 to 30% concentration in distilled water. In addition, as the iron chelating to the transferrin diluted in the solution (chelating), a compound that produces ferric ions or trivalent ions can be used, such as iron chloride (FeCl ₃ ), iron sulfide (FeSO ₄ ), ferric citrate (ferric citrate). ), Ferric lactate, ferrous citrate, or ferrous lactate, preferably in the form of 0.5-100%, preferably 20-80% of the amount of transferrin extract. It can add and use in a range. In addition, dialysis is performed for 1 to 5 days, preferably 1 day in distilled water using a dialysis membrane of MWCO 2 to 10 kDa, sterilization is carried out by a conventional method for 15 minutes at 1.5 at 121 ℃.

On the other hand, the protein fractionation apparatus used to obtain the transferrin-containing fraction in the present invention, a method using a column (column) that is subject to a lot of constraints, such as the amount of sample and separation time to efficiently fractionate the plasma protein from the plasma of the slaughtered blood Is a batch protein fractionator configured to fractionate plasma proteins rapidly in large quantities (see FIG. 1). As shown in Figure 1, the protein fractionation device is made of glass and divided into three parts, ① is a reaction vessel (reaction vessel); ② is a glass filter unit (glass filter unit); And ③ represent an extraction vessel. In this device, a certain amount of ion exchange resin and plasma, and a primary eluate that can elute plasma proteins adsorbed on the ion exchange resin are placed in a reactor and stirred using a magnetic stirrer. After that, the glass filter and the extraction tank are installed on the reaction tank, and then turned upside down so that the ion exchange resin is blocked by the glass filter and not filtered by the extraction tank. The protein eluted by the primary elution solution can be separated through the glass filter by the extraction tank It is designed to be. In addition, it is designed to enable rapid extraction by decompression by installing an aspirator connector in the extraction tank for quick and efficient extraction. When protein in plasma is separated by using a protein fractionation device, the protein that is not eluted from the ion exchange resin by the primary elution solution is prepared in the same manner by preparing secondary and tertiary elution solutions under different conditions. It may be fractionated.

As a result, according to the method for preparing the iron-transferrin extract of the present invention, it is designed to easily and quickly separate the transferrin extract from the plasma of the slaughtered blood, the iron-transferrin extract obtained by combining with iron is easily heat-treated It can be used as an active ingredient in hygienic, bioavailable and stable iron preparations. In particular, the iron-transferrin extract of the present invention has a property that can be easily heat-treated or sterilized by the characteristics of transferrin having a high thermal stability, compared to other iron preparations having a disadvantage in that sufficient heat treatment is not possible due to protein denaturation. It can be included as an active ingredient in iron preparations that can ensure excellent safety.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Isolation of Transferrin Extract from Slaughter Blood

Batch ion exchange chromatography was performed from the slaughtered blood to obtain transferrin extracts: Swine blood collected immediately after the slaughter was centrifuged at 5,000 x g for 30 minutes at 4 ° C to obtain a plasma solution separated as a supernatant. Q Sepharose ion flow resin (Q Sepharose fast flow gel, PharmaciaBiotech, Sweden) was used as the ion exchange resin, 600 ml of Q Sepharose ion exchange resin and 1.5 liters of distilled water were added to the reactor and washed twice. The ion exchange resin was equilibrated with 10 mM Na ₂ HPO ₄ (pH 10.0), an adsorption solution. Plasma protein was adsorbed onto the Q Sepharose Ion Exchange Resin by adding 300 ml of plasma solution and 1.2 liters of the Adsorption Solution to the Q Sepharose Ion Exchange Resin equilibrated by the Adsorption Solution and stirring for 1 hour on a magnetic stirrer. Some non-adsorbed protein was recovered in the extraction tank along with the adsorption solution through a glass filter. Plasma proteins bound to Q Sepharose Ion Exchange Resin were fractionated from Q Sepharose Ion Exchange Resin by sequentially adding 0.1-0.5 M NaCl eluate. Plasma protein composition in each fraction was examined by SDS-PAGE, and the concentration of each plasma protein was measured using a densitometer on a polyacrylamide gel (see Table 1).

As shown in Table 1, most plasma transferrin (90.4%) was not adsorbed to the Q Sepharose ion exchange resin, but fractionated into the extraction tank by the adsorption solution 10mM Na ₂ HPO ₄ (pH 10.0), 0.1M NaCl The remaining amount of transferrin (9.6%) was isolated from the eluent. At this time, it was confirmed that the fraction extracted by the adsorption solution mainly consists of transferrin (30.4%) and immunoglobulin IgG (47.0%). Therefore, this fraction was called 'transferrin extract', and the fractionated transferrin extract was filtered and concentrated using membrane and MWCO 30kDa membrane and ultrafiltration unit, Millipore, USA, and then lyophilized to 4 ° C. Stored in.

Example 2 Preparation of Iron-Transferrin Extract for Oral Administration

In order to bind ferric ion as iron to the transferrin extract isolated in Example 1, 10 g of the lyophilized transferrin extract was diluted in 100 ml of tertiary distilled water to a 10% concentration, and then the amount of the transferrin extract 10 2.9 g of trivalent iron ion corresponding to% (w / w) was added in the form of iron chloride (FeCl ₃ ), followed by mixing with stirring. The mixture was then dialyzed in distilled water to remove excess iron and induce bond formation of a stable iron-transferrin extract: in order to yield a dialysis condition that would most effectively form an iron-transferrin bond, trivalent ion The transferrin mixture was placed in a dialysis membrane (Spectrum, USA) with MWCO 6-8 kDa and dialyzed with distilled water for 1, 2, 3, and 4 days, and the concentration and pH change of the trivalent ions were measured (see Table 2).

As shown in Table 2, 37% (3,314ppm) of the total concentration of trivalent ions (8,948ppm) administered to the 10% transferrin extract solution remained in the transferrin extract solution dialyzed in distilled water for 24 hours. Based on the dialysis time, the amount of remaining trivalent ions gradually decreased as the dialysis time elapsed, and this could be confirmed by the pH change of the solution. Therefore, the trivalent ion-transferrin extract which was dialyzed for 24 hours was the most effective iron. -Was considered to have formed transferrin chelating.

As a result, a red trivalent ion-transferrin extract solution dialyzed in distilled water for 24 hours was prepared, and sterilized at a pressure of 1.5 psi for 15 minutes at 121 ° C. to give hygienic safety by heat treatment, and then sterilized solution. Lyophilization gave a powdered iron-transferrin extract.

Example 3 : Iron solubilization of iron-transferrin extract in duodenal conditions

In order to evaluate the degree of absorption in the body of the trivalent ion-transferrin extract prepared in Example 2, the laboratory conditions (in vitro) of the duodenum where most iron absorption is made to pH 6.0 and 37 ℃, trivalent ion-transferrin The iron solubilization ability was assessed by the addition of the extract: 2 g of tri-ion-transferrin extract (containing 66.3 mg of tri-ion) was dissolved in tertiary distilled water, followed by 20 ml, and pH 6.0 using 1 N sodium hydroxide. The mixture was then shaken-incubated at 37 ° C. for 1 hour, and 20 ml of a FeCl ₃ solution containing trivalent ions corresponding to 66.3 mg was prepared as a control and turbid under the same conditions. Next, the solution separated from the upper layer was filtered through a filter paper without leaving the electric solution at 37 ° C. for 1 hour, and the filtrate was readjusted to pH 2.0 using 1 N hydrochloric acid, and then the amount of iron could be measured. A ferrozine assay was performed to determine the amount of iron remaining in the filtrate without precipitation and sinking.

As a result, all of the ferric ions precipitated in the control group incubated in the duodenal condition with only FeCl ₃ , and the residual iron in the filtrate was not detected by the ferrogene measurement method, but in the trivalent ion-transferrin extract group, 38% (25.19 mg) remained solubilized under duodenal conditions. Most of the absorption of iron in the body is made in the duodenum, so that iron is absorbed in the duodenum, it must be solubilized. Therefore, the bioavailability of iron can be inferred by the degree of iron solubilization in duodenal conditions (pH 6.0, 37 ℃). As can be seen, the iron solubilization ability of trivalent ion-transferrin extract in duodenal condition was excellent compared to the control group, and the iron bioavailability of trivalent ion-transferrin extract was also considered to be excellent.

Example 4 Iron Bioavailability of Iron-Transferrin Extract

The iron-availability-improving effect of iron-transferrin extract in vivo was evaluated by the following animal experiments: 40-week-old rats with iron deficiency diet for 4 weeks After iron-deficiency anemia was induced, ferric ion-transferrin extract was administered daily by 5 mg to 20 mg orally for 21 days. As a control, mice with iron deficiency anemia were induced. Only ferric ions were administered. Next, the iron bioavailability improvement effect of ferric ion-transferrin extract according to the period was administered only the ferric ion under the same conditions through the change of blood hemoglobin concentration, change of% hematocrit, weight change, etc. Comparative study with control (see Table 3).

As shown in Table 3, the rat group treated with the ferric ion-transferrin extract compared with the control rat group, the concentration of hemoglobin in blood,% hematocrit, total hemoglobin amount, and hemoglobin for iron dose. All of the increases were high. As a result, the iron-transferrin extract prepared according to the present invention has excellent properties of binding or solubilization of trivalent ions or ferric ions in laboratory conditions as well as in vivo conditions, and greatly improves the body absorption rate of iron. It was confirmed that.

Meanwhile, the iron-transferrin extract of the present invention may be included as an active ingredient in various forms of medicinal iron preparations as well as iron-enriched foods.

At this time, the electric iron agent is an iron-transferrin extract as an active ingredient, solid, semi-solid or by appropriately adding a surfactant, excipient, colorant, stabilizer, buffer, suspending agent, isotonic agent and other compatible additives according to a conventional method It may be formulated as an oral dosage form in the form of a liquid. In addition, examples of the electric oral dosage agent include tablets, pills, granules, soft-light capsules, powders, fine granules, powders, emulsions, suspensions, emulsions and suppositories.

The dosage of the iron preparation containing the iron-transferrin extract of the present invention as an active ingredient varies depending on the type of the disease to be treated or prevented, the method of administration, the age, the treatment time, and the like. Based on the recommended iron nutritional amount of 10 to 20 mg per day, the daily dose per adult should be determined within the range of 50 to 400 mg based on the iron-transferrin extract as an active ingredient.

Example 5 Acute Toxicity Test

The powdered trivalent ion-transferrin extract prepared in Example 2 was administered to 10 three-week-old rats (SD rats) based on the recommended iron nutritional amount of 10 mg / day of adult (60 kg). An effective amount of iron in consideration of (300 g) was set to 50 µg, and 2 g, which is about 2,000 times of 1 mg of iron-transferrin extract containing iron, was orally administered for 7 days, but no case was recognized.

As described above in detail the specific parts of the present invention, for those skilled in the art, these specific descriptions are only preferred embodiments, which are not intended to limit the scope of the present invention. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

As described and demonstrated in detail above, the present invention is to separate the transferrin extract from the slaughtered blood by an ion exchange method, combined with iron to produce an iron-transferrin extract and the iron-transferrin extract prepared by the electrical method Provided an iron preparation containing as an active ingredient. According to the present invention, the iron solubilization ability in the duodenum is superior to the conventional iron preparation, and the bioavailability is excellent, and the raw materials used are also manufactured using iron-transferrin extract through a simple process using slaughtered blood which is mostly disposed of in the slaughterhouse. can do. In addition, the iron-transferrin extract of the present invention is stable in heat and can be subjected to sterilization process, so it is safe in hygienic aspects, and can be included as an active ingredient in various forms of medicinal iron preparations and iron-reinforced foods.

Claims (10)

Preparation method of iron-transferrin extract comprising each of the following steps: (Iii) Plasma isolated from blood is subjected to ion exchange chromatography using a batch protein fractionator consisting of a reaction vessel, a glass filter and an extraction vessel to obtain a transferrin-containing fraction. ; (Ii) filtering and lyophilizing the transferrin containing fractions to obtain a concentrated transferrin extract powder; And, (Iii) Ferric chloride (FeCl ₃ ), iron sulfide (FeSO ₄ ), ferric citrate, ferric lactate, ferrous citrate or ferric lactate in a diluted solution of transferrin extract powder. (ferrous lactate) iron in a weight ratio of 0.5 to 100% relative to the transferrin extract, dialyzed and sterilized, and then lyophilized to obtain an iron-transferrin extract in powder form. The method of claim 1, The blood is characterized in that the blood or blood Method for preparing iron-transferrin extract. delete The method of claim 1, Ion exchange chromatography is carried out using an anion exchange resin Sepharose (Sepharose), Sephadex (Sephadex) or Sephacel (Sephacel) Method for preparing iron-transferrin extract. The method of claim 1, Transferrin fraction is characterized in that extracted with phosphate or tris buffer solution of pH 6.0 to pH 11.0 Method for preparing iron-transferrin extract. The method of claim 1, Transferrin extract powder is characterized in that it is diluted in distilled water to 1 to 30% concentration Method for preparing iron-transferrin extract. delete delete The method of claim 1, Dialysis is performed for 12 hours to 96 hours in distilled water using a dialysis membrane of MWCO (molecular weight cut off) 2 to 10 kDa Method for preparing iron-transferrin extract. Iron preparation comprising an iron-transferrin extract prepared by the method of claim 1 as an active ingredient.