KR0163209B1 - Process for purification of high molecular weight-urogastron - Google Patents

Abstract

The present invention relates to a method for purifying a high molecular weight-urogastron having a molecular weight of 36,000 or more having gastric acid secretion effect and anti-ulcer action present in urine.

In particular, the present invention is complicated by the production of a multi-step column chromatography process in the conventional purification method, in order to improve the problem that the activity of urogastron is very low, urine of human or animal using an adsorbent The present invention relates to a method for purifying a high activity of 36,000 or more high molecular weight-urogastron in a simple process without using any column chromatography process by sequentially performing adsorption, ethanol-separation precipitation, extraction, ultrafiltration, and acetone deposition. .

Description

Method for Purifying High Molecular Weight-urogastron

The present invention relates to a method for purifying a high molecular weight-urogastron having a molecular weight of 36000 or more having an anti-ulcer effect and gastric acid secretion effect present in urine, and more specifically, adsorption of human or animal urine with an adsorbent, By sequential ethanol-separation, extraction, ultrafiltration and acetone deposition, high activity high molecular weight-urogastron (hereinafter referred to as HMW-UG) is purified in a simple process without using any column chromatography process. It is about how to.

Urogastrone, which is known to be present in human or animal urine, exists in various types such as molecular weight of 36,000 or more and 6,000. HMW-UG with a molecular weight of more than 36,000 is a glycoprotein containing carbohydrate fraction and has been known to have excellent gastric acid secretion inhibitory effect and anti-ulcer effect and is used as a therapeutic agent for gastric and duodenal ulcer. Such HMW-UG is known to have urogastron having up to five times higher activity in maternal urine than in the normal urine (YAKUGAKU ZASSHI 89,215-221 (1969)).

In the conventional method of separating and purifying HMW-UG, the urogastron fraction adsorbed on a suitable adsorbent is dissolved in ammonium acetate buffer, followed by DEAE-cellulose column chromatography, carboxymethyl-cellulose column chromatography, and then pH. Washing with 3.8 ammonium acetate buffer to separate urogastron using Sephacryl S-200 column chromatography and reverse phase chromatography (Chales D. Mount et al., Archives of Biochemistry and Biophysics, Vol. 225, No. 1, May 15, pp 1-7, 1987), or urine, was stirred with Bio-Rex 70 ion exchange resin and filtered through a glass filter to collect the resin. Protein fractions adsorbed with ammonium acetate buffer are separated and purified by sulfur-EGF-sepharose column (Kenichi Tsukumo et al., Biochermical and Biophysical Research Communications, Vol. 145, No. 1, May 29, 1987).

However, the above-described method for purifying HMW-UG is not only complicated because the process is manufactured through a multi-stage column chromatography process, and the activity of HMW-UG is very low even after purification through such process. Due to the problem, the price of the product was increasing. Currently, commercially available HMW-UG-containing products are manufactured by extraction and purification in pregnant horse urine.

Accordingly, the present inventors have intensively studied to find a method for purifying HMW-UG having high activity from human or animal urine by a simple process. As a result, the extraction is a simple process without using any column chromatography process in the purification process. By introducing a new ultrafiltration and acetone deposition process, and combined use, the present invention was completed by developing a purification method of HMW-UG having a high activity in a much simpler process than the conventional purification method.

That is, according to the present invention, the urine of a human or an animal (including a pregnant woman) is adsorbed using an adsorbent, ethanol-fractionated precipitation, extracted under pH 8.0-10.0 and ultrafiltered with a filtration membrane of molecular cut off of 30,000. It relates to a method for purifying HMW-UG by dipping in acetone.

The present invention is described in detail as follows.

The method of the present invention which effectively purifies HMW-UG by using a combination of extraction, ultrafiltration and acetone deposition processes, which is a simple process, consists of the following five steps.

(a) adsorption with adsorbent

(b) fractional precipitation with 35-45% ethanol

(c) extraction at pH 8.0-10.0

(d) Ultrafiltration with a molecular cut off of 30,000 filtration membranes.

(e) acetone deposition.

Referring to the HMW-HG purification method according to the present invention in more detail step by step as follows.

(a) step

The pH of the collected human or animal urine was first adjusted to pH 4.0-5.0, the protein contained in the urine was stabilized and cooled to prevent rot, and then the adsorbent was used to contain HMW-UG. Separate fractions.

Adsorbents are primarily used to separate protein fractions, including HMW-UG, from chilled urine. As the adsorbent, benzoic acid, silica gel, and kaolin are known as adsorbing the protein fraction including HMW-UG and not adsorbing the remaining impurity proteins. However, benzoic acid is easily adsorbed even in an acid solution, and benzoic acid is easily separated. This is preferable for the present invention. The reason for adjusting the pH by glacial acetic acid is that the pI value of HMW-UG is about 4.0-5.0, so that the net charge should be zero in order to adsorb it. Benzoic acid-adsorbed precipitates can be separated by centrifugation.

(b) step

Since the precipitate obtained in step (a) may contain benzoic acid and other impurities, it is washed with acetone, dried and fractionated to ethanol again.

Acetone washing removes benzoic acid and water, and this process is preferably repeated three times in terms of yield. After washing with acetone, it is dried and ethanol fractional precipitation is carried out to remove the impure protein. Here, HMW-UG has a property that the concentration of ethanol is settled at about 35-45%, so the concentration of ethanol is preferably 35-45%. The precipitate obtained in the fractionation process by ethanol is separated by centrifugation and dried to obtain the zorogastronone powder.

(c) step

Jourogastron powder obtained in step (b) is extracted with distilled water or buffer while maintaining the appropriate pH. At this time, the pH of the extract is preferably 8.0-10.0, after extracting by stirring for about 30 minutes after pH adjustment, and left at 4 ℃ for at least 12 hours and then centrifuged to obtain a more purified extract (Supermatamt). At this time, by adjusting the extract to pH 4.5 ± 0.5 with glacial acetic acid to further remove the impurities contained can be obtained more purified urogastron.

(d) step

The supernatant obtained in step (c) is subjected to ultrafiltration to separate low molecular weight urogastron and high molecular weight urogastron. The HMW-UG to be separated has a molecular weight of about 36,000 or more, and low molecular weight urogastron has a molecular weight of about 6,000, so it can be easily separated by ultrafiltration with a molecular membrane of molecular cut off (30,000). This ultrafiltration process can be repeated 5-10 times to obtain a higher yield of HMW-UG.

(e) step

Retentate obtained by ultrafiltration in step (d) is centrifuged after immersion in acetone. In particular, HMW-UG is fractionated and precipitated at 75-85% of acetone. Thus, precipitation of HMW-UG can be obtained by adding 4-5 times acetone to the residual liquid volume obtained in step (d). In this way, finally purified HMW-UG can be obtained.

As described above, since the HMW-UG purification method according to the present invention does not use any column chromatography process used in the conventional HMW-UG purification method, purification is easy, and adsorption using sorbent, ethanol-separation precipitation, By using a combination of ultrafiltration and acetone deposition, HMW-UG can be separated and purified in high yield as can be seen in the following examples and test examples.

The present invention will be described in more detail with reference to the following Examples. However, the present invention is not limited thereto.

Example 1

2 L of glacial acetic acid was added to 2,000 L of pregnant women with urine, and the pH was adjusted to 4.5 and left at 4 ° C. for 24 hours. 70 kg of benzoic acid (benzoic acid) was added to 140 L of acetone solution, and the dissolved solution was added to the cooled urine. After standing for 12 hours, centrifugation was performed to obtain a precipitate.

The precipitate was washed with acetone and dried, and then stirred in 15 L of 35% ethanol buffer for 3 hours to completely dissolve it, followed by centrifugation at 10,000 rpm to obtain a precipitate, which was dried to obtain zorogastron powder.

1,000 g of urogastron-fraction powder was added to 20 L of purified water, completely dissolved by stirring for 30 minutes while adjusting to pH 8.5 ± 0.5 with 5N sodium hydroxide solution, and then left for 12 hours, and centrifuged at 10,000 rpm for 10 minutes to give a supernatant solution. Got. The supernatant was ultrafiltration up to 2 L with a molecular weight cutoff of 30,000 filtration membranes, followed by ultrafiltration with up to 8 L of distilled water. This process was repeated five times to completely remove the salt, and then 10 L of acetone was added to the filtered residue, followed by centrifugation at 10,000 rpm to obtain a precipitate, which was dried at 60 ° C. to obtain HMW-UG75 g.

Example 2

2 L of glacial acetic acid was added to 2,000 L of normal human urine to adjust the pH to 4.5 and then left at 4 ° C. for 24 hours. 70 kg of benzoic acid (benzoic acid) was added to 140 L of acetone solution, and the dissolved solution was added to the cooled urine. After standing for 24 hours, centrifugation was performed to obtain a precipitate.

The precipitate was washed with acetone and dried, and then dissolved in 15 liters of 1.000 gdmf 40% ethyl alcohol buffer for 3 hours to completely dissolve. The precipitate obtained by centrifugation at 10,000 rpm was washed with 2 liters of ether and dried to obtain zorogastrone. A powder was obtained.

1,000 g of Zorogastron powder was dissolved in purified water 20LDP SJGRH, 5N sodium hydroxide solution, adjusted to pH8.5 ± 0.5 for 30 minutes, completely dissolved, and then left for 12 hours, centrifuged at 10,000 rpm for 10 minutes to obtain a supernatant. . The supernatant was ultrafiltered to 2 L with a molecular weight cut off of 30,000 filtration membranes. This process was repeated five times to completely remove substances having a molecular weight of 30,000 or less, and then 10L of acetone was added to 2L of the residue to obtain a precipitate, and the precipitate was dried under reduced pressure to obtain HMW-UG73g.

Example 3

2,000L each of pregnant and urinary urine was adsorbed to benzoic acid in the same manner as in Example 1 to obtain a precipitate, which was washed with acetone and dried, dissolved in 40% ethyl alcohol buffer, and centrifuged to obtain a precipitate. 1,000 g of precipitates from pregnant urine and normal urine were taken, dissolved in 20 L of distilled shoe, stirred for 30 minutes while adjusting to pH 8.5 ± 0.5 with 5 N sodium hydroxide solution, and prevented for 12 hours. The supernatant obtained by centrifugation was glacial acetic acid. The mixture was adjusted to pH4.5 ± 0.5 and left for 5 hours, followed by centrifugation to remove the precipitate. The precipitated solution was ultrafiltered with a molecular weight off filter membrane of 30,000 to completely remove substances having a molecular weight of 30,000 or less, and then 10L of acetone was added to 2L of residue to obtain a precipitate, which was dried to obtain HMW-UG72g. (Pregnancy urine) and 70 g (normal urine) were obtained, respectively.

Gastric acid secretion inhibitory activity and anti-ulcer effect of HMW-UG obtained in Examples 1, 2 and 3 were evaluated by the following test example.

[Test Example 1]

Gastric acid secretion inhibitory activity

Group 5 was prepared from 10 healthy rats with a body weight of 180 ± 5g, and fasted and watered for 24 hours, followed by pyloric ligation under ether anesthesia. In each of the four groups, HMW-UG powder 0.8 purified from Example 1 was used. A solution of mg, 0.4mg, 0.2mg, 0.1mg dissolved in 0.5mgl of saline solution and 0.5mgl of saline solution in another group (control group) were injected intravenously in the tail and euthanized to head and neck after 2 hours. The contents of were taken into a test tube, centrifuged at 5,000 rpm, the supernatant was taken, and the amount of gastric juice was quantified. The amount of gastric juice obtained was converted into the weight-per-kg rats, and then the average value was calculated.

At this time, the amount of HMW-UG showing 70% inhibition of gastric juice amount at various doses was expressed as activity per kg (rat), and the results are shown in Table 1.

In Table 1 it can be seen that the unit activity is 0,2mg / kg showing a degree of inhibition of 70.5%.

[Test Example 2]

The unit activity of HMW-UG powders purified from Examples 2 and 3 was measured in the same manner as in Test Example 1, and the results and the total unit activity of the HMW-UG powders purified from each Example (Production / Unit Activity) ) Is shown in Table 2 below.

As can be seen in Table 2, HMW-UG purified from normal urine can be seen that its activity is very high, especially HMW-UG purified from gestational urine (Examples 1 and 3) has its activity. Is 2 to 3 times higher than that purified from normal urine.

[Test Example 3]

Antiulcer test

Ten healthy male rats, weighing 180 ± 5g, were fasted for 24 hours in a group of 23 ± 5% constant temperature and humidity room. 5 ml orally was administered. Under anesthesia, the first group was injected with a solution of 0.4 mg of HMW-UG powder purified from pregnant urine of Example 3 in 0.5 ml of physiological saline, and 0.5 ml of physiological saline was injected into the other group (control). After 7 hours, euthanasia with head and neck abdomen was opened, and the stomach was removed, and 7 ml of formalin solution was injected. After 10 minutes, the ulcer size of the gastric mucosa was measured by a stereoscopic microscope (10 times) on a filter paper. Ulcer size of the urogastron group and the control group was tested by the Student T-testfh significance test. The ulcer index of the HMW-UG-administered group should show statistical significance (P0.05) compared to the control group, and the results are shown in Table 3.

[Test Example 4]

HMW-UG powder purified from Example 1, 2 and Example 3 was tested in the same manner as in Test Example 3 to evaluate the significance, the results are shown in Table 4.

Claims (3)

Adsorption of human or animal urine with an adsorbent, ethanol-fractionation, extraction at pH 8.0-10.0, ultrafiltration using a molecular membrane of 30,000 filtration membranes, gastric acid characterized by sequentially performing acetone sedimentation Purification method of high molecular weight-urogastron (HMW-UG) having secretion inhibition and anti-ulcer action. The method of purifying high molecular weight-urogastron (HMW-UG) according to claim 1, wherein the urine of human is pregnant urine. The method according to claim 1, after the extraction at pH 8.0-10.0, further comprising a precipitation removal step at pH 4.0-5.0 high molecular weight-urogastron with gastric acid secretion and anti-ulcer action (HMW-UG Purification method).