JPS62116519A - Cell metabolism activating substance extracted from placenta, extraction thereof and method for inactivating hepatitis virus - Google Patents

Abstract

NEW MATERIAL:A water-soluble protein originated from placenta obtained by normal delivery of man or cattle. The human-originated substance has an estimated molecular weight of 92,000 and an isoelectric point of about 5.5-6 and the bovine-originated substance has an estimated molecular weight of 80,000 and an isoelectric point of about 5-5.5. USE:A cell metabolism activating substance. It is used as a hepatotonic agent, remedy for gastric and duodenal ulcer, immunoactivator, etc. PREPARATION:A water-soluble protein originated human placenta can be produced by homogenating a human placenta at a low temperature, adding a phosphoric acid buffer solution containing NaCl and having low ionic concentration to the placenta, leaving the mixture to stand for 6hr - a whole day and night, centrifuging the mixture, adding 40% saturated ammonium sulfate to the supernatant liquid, separating the supernatant liquid, adding 60% saturated ammonium sulfate to the liquid, separating the precipitate by gel-filtration and purifying by ion exchange chromatography. The activity of the protein can be stabilized and viruses are inactivated by adding the water-soluble protein to 60% saturated ammonium sulfate solution and heat-treating the precipitate.

Description

[Detailed Description of the Invention] [A] Purpose of the Invention The present invention provides a novel water-soluble protein extracted from the placenta after normal parturition (birth) of humans or cows, a method for extracting the same, and a method for producing a hepatitis virus. Regarding inactivation methods.

(Industrial Application Fields) Placenta extract is known to have tissue metabolism activation effects and tissue regeneration and repair abilities, and has been used as a liver strengthener, anti-ulcer agent, and damage repair agent.

The novel water-soluble protein according to the present invention has a molecular weight of 92,000 when extracted from human placenta, and between 80,000 and 25,000 when extracted from bovine placenta.
It has a remarkable effect on activating cell metabolism. That is, the water-soluble protein according to the present invention promotes the proliferation and spread of fibroblasts, and when extracted from human placenta, promotes the spread of macrophages and promotes phagocytosis. Therefore, the novel water-soluble protein according to the present invention can be used directly or as a combination agent, for example, as a pharmaceutical agent, such as a liver tonic, a gastric or duodenal ulcer agent, a damage repair agent, an immunostimulant, and the like. In addition, in recent years, in cancer treatment, it is known that conventional anticancer drugs have a strong toxic effect on normal cells and reduce the immune function of the body, and substances that strengthen the immune system are attracting attention. It is thought that it is useful to use water-soluble proteins in combination in cancer treatment.

(Prior art) The placentas of various sources (animal species and genera) are used as starting materials, and the extracts obtained from these by various extraction methods or their active ingredients are separated and used as pharmaceuticals and cosmetics. Its use has been known for a long time. Among them, if we investigate the applications and extraction methods for water-soluble components, we will find, for example, the known publications shown in Table 1.

1 Table 1 Publications (Published by the Japan Patent Office) Also, when we examine the extracts of water-soluble components from placenta as shown in Table 1, we find that The common feature is that organic solvents, strong acids, or strong alkalis are used. Furthermore, other methods such as using proteolytic enzymes are also known.

The pharmacological effects of these extracts are known to be anti-ulcer effects and tissue metabolism activation effects, and as pharmaceuticals, they are available in the form of injections and oral preparations as liver tonics, gastric or duodenal ulcer treatments. It is used as an agent. Furthermore, in cosmetics, it is used in the form of an external application for the purpose of preventing skin aging (preventing wrinkles), preventing age spots (C inhibiting melanin pigment production).

In other words, it has long been known that there are substances in the placenta that have the ability to regenerate and repair tissues, and to determine their action or effect, tissue pieces such as guinea pig skin are used, and extracts or ingredients extracted from this are used. A method has been adopted in which the amount of oxygen consumed is determined using a Warburg pressure probe or the like.

(Problems to be Solved by the Invention) The present inventors have focused on the above-mentioned tissue metabolism activation effect or tissue regeneration and repair ability regarding placenta extracts, and also sought to re-evaluate extracts obtained by these conventional extraction methods. The test was started based on a more accurate test method.

The therapeutic effects of placenta-derived extracts are known to have the above-mentioned effects based on clinical reports using them, but nothing has been found to prove this from the ability to increase M at the cellular level. there was.

The present inventor focused on the important function of the placenta, which accelerates the growth of a fertilized egg by 100 million times during its 10-month in-utero life.
We developed a quantitative search method for direct proliferation effects on cells, and based on this, we searched for placenta-derived cellular metabolic activators.

The search method used by the inventor is as shown in the next section.
This revealed that none of the extracts shown in Table 1 had a high ability to proliferate cells. One of the causes of this was found to be due to the conventional extraction method and processing method for extract extraction. In other words, this is related to the fact that this substance is unstable to heat and organic solvents.

In other words, the anti-ulcer effect, tissue metabolism activating effect, etc. of conventional placenta extracts or extract components in clinical trials that have been known so far have been shown to be at least due to direct cell growth promotion. It was thought that the effect was obtained by activating tissue metabolism through a different mechanism of action, rather than a therapeutic effect.

Therefore, the present inventors have been wondering what kind of extraction method should be used to find substances that have a direct proliferation-promoting effect on cells from the placenta, and whether extracts or We continued our research on why the isolated components have little or no cell growth promoting effect, and as a result, we were able to obtain an extract that has a high cell growth promoting effect.

(Comments regarding measurement of cell proliferation-promoting effect, etc.) There is no way to accurately express cell proliferation-promoting effect, even with growth factors that are generally known today. Various methods have been proposed, and the inhibition of proliferation rate is quantitatively shown by cell number, protein amount, etc.

The present inventor has developed a method for quantitatively expressing the cell proliferation effect of a target substance by suppressing the cell proliferation rate of a control to near zero, if not killing it. More specifically, this method was achieved by adjusting the amount of fetal bovine serum (FBS) added.

On the other hand, the present inventor has investigated the cell growth promoting effect of this active substance in various cell lines, which are different from normal cells. Therefore, here, we decided to search using primary m cells taken from normal skin. That is, cells were aseptically separated from the skin of a guinea pig, and cells that had not mutated and were subcultured for a certain period of time were used to express the degree of cell proliferation of the target substance. The method is described below.

Skin cells include epidermal cells, LA, and fibroblasts, and for example, fibroblasts collected from guinea pigs (Hartley strain, female) thrive in Eagle's MEM medium supplemented with 5% tallow serum (CFBS). The multiplication time is approximately 48 hours, as shown in Figure 3.

These cells were established into a cell line approximately one year after collection.

Therefore, in order to express the cell proliferation rate of the target substance of the present invention,
In order to obtain cells that are as close to normal as possible, by cryopreserving cells that have been expanded and subcultured within 6 months after collection,
They were sequentially used to separate target substances and to study stabilization methods against heat treatment.

The method is as follows: First, 4 to 5 x 10'
Cells are seeded and Eagle's MEM medium supplemented with low concentration of FBS is changed every 2-3 days. The number of m cells is
Measurements are taken every 2-3 days using a Bürkertürk hemocytometer. Incidentally, at this time, the amount of protein and the amount of nucleic acid may be measured at the same time.

The target substance is adjusted according to the amount of protein (WI Hama). Add 1/10th the amount per medium each time the medium is replaced, and for the control (Funtrol), add only the same amount of physiological saline. .

In addition, the present inventors investigated the effect on macrophages, which are cells of the immune system, aseptically collected from the abdominal cavity of mice, using adhesion to the culture container and spreadability as indicators. did.

[Mouth] Structure of the Invention The present invention provides that the molecular weight of the water-soluble protein extracted from the placenta is
Extracted from human placenta after normal delivery, 92,000
The amount of cell metabolism activating substances is 80,000 and 25,000 when extracted from the placenta of cows after normal birth, and the amount of ammonium sulfate in the extraction process. A method for inactivating viruses from water-soluble proteins (substances that activate cellular metabolism), which is characterized by heat-treating precipitates that precipitate at 60% saturation concentration.

(Means for Solving the Problems) The gist of the present invention is as described above, but in order to show it more specifically, examples and effects will be described below.

Incidentally, in presenting the embodiments, the basic points according to the present invention are as follows.

(b) Human or bovine placenta is used as the starting material in the extraction method. For human placenta or bovine placenta, use a fresh placenta after normal delivery (birth), which has been frozen or stored at low temperature. In addition, if necessary, the placenta of livestock animals such as pigs can be used, but it is selected depending on the therapeutic or medicinal use, cosmetics, etc. of the animal species to be used. Just do it.

(b) Although the examples show the simplest method under the specified extraction conditions, the key points of operation in the extraction process are:
It is necessary not to apply heat, avoid extraction operations using organic solvents, and avoid using strong acids, strong alkaline decomposition, and various proteolytic enzymes. In other words, these treatment operations should not be used because they deactivate the activity of the target substance. According to this, conventional biological components are extracted by a combination of various methods, such as centrifugation, salting-out fractionation, gel filtration, dialysis, low temperature or vacuum concentration, and freeze-drying. You can get it. That is, the feature of the extraction method from the placenta according to the present invention is that it does not use any solvents, strong acids, strong alkalis, enzymes, etc. used in conventional extraction operations of placenta extracts or isolated components during the process. There is a particular thing.

[Example-1] Here, a human placenta after normal delivery or a cow placenta after normal delivery is cut into pieces without pretreatment of washing, and homogenized using a homogenizer or the like at a low temperature. To this, water or a buffer solution, such as a low ionic concentration phosphate buffer containing sodium chloride, is added, stirred, and left for 6 hours to 1 day and night, and then centrifuged to obtain a supernatant. When the starting material is human placenta, the supernatant should be at a concentration of 40% saturation, while when the starting material is bovine placenta, the supernatant should be at a concentration of 20%. After adding ammonium sulfate to a degree of saturation, centrifugation is performed to collect the supernatant, and then to this supernatant, ammonium sulfate is added again to a concentration of 60% saturation. and collect the resulting precipitate.

After the precipitate is dissolved in a small amount of water or physiological saline,
Perform desalination operation.

Desalting operations include gel filtration, ultrafiltration, and dialysis, and any of these methods may be used. , when human placenta is used as a starting material by dialysis for more than one day and night, it has a molecular weight of 92,000.
A liquid containing water-soluble protein was obtained.

On the other hand, when cow placenta is used as the starting material, 80,000 and 20,000
A liquid containing a water-soluble protein having a molecular weight of 15,000 was obtained.

[Example 2] When considering the use of the extract obtained in Example 1, which has a high cell proliferation promoting effect, sufficient consideration must be given to inactivating viruses. For example, heat treatment at 60°C for 110 hours is said to be effective in inactivating hepatitis viruses. When the freeze-dried liquid obtained in Example 1 was allowed to dissolve in water or physiological saline, it was heat-treated at 60°C for 10 hours. It was found that the cell proliferation promoting effect was also 100% inactivated. Furthermore, this novel water-soluble protein is characterized in that its cell proliferation promoting effect is completely inactivated by heat treatment at 60°C for 130 minutes.

Therefore, the present inventors have conducted various studies on stabilization methods that can withstand long-term treatment at high temperatures as a means to maintain the cell proliferation promoting effect of this substance and to inactivate only the hepatitis virus. entered in. And as a means of doing so,
For example, glucose, 77/-su% ima, Si! ! sugar,
Disaccharides such as maltose are added and heat treated,
The present inventors conducted further investigation and found that the protein was similarly inactivated. It is presumed that the protein undergoes severe denaturation and is thereby inactivated, and as a countermeasure to this, the protein structure is changed in advance in a highly concentrated salt solution, etc., and then heated in a precipitated state. Based on the idea that it would be possible to minimize irreversible denaturation of proteins, we completed a novel heat treatment method as shown in Example 3. This method is of course an advantageous method that can also be used as a heat stabilization method for various conventionally known components derived from blood and organs.

[Example 3] The liquid obtained in Example 1 was freeze-dried, suspended in a 60% saturated ammonium sulfur solution, or its precipitate was collected and heat-treated at 60°C for 10 hours. provided.

According to this method, it was found that the hepatitis virus was inactivated and that 80 to 90% of the proliferation promoting effect on cells remained.

4 and 5 show the results of measuring the cell proliferation effect of the target substance after treatment in Example 3 in comparison with that obtained in Example 1.

In other words, the method shown in Example 3 is a means to prevent the cell proliferation effect from being inactivated by heat treatment.

In addition, Examples 2 to 3 are based on known methods of inactivating hepatitis viruses in serum and protein preparations derived from various organs.
The results are based on the assumption that the process was carried out at 60°C for 110 hours, but according to the precipitation or suspension method using a highly concentrated salt solution, the temperature could be raised to a very high temperature. Of course, it is also possible to shorten the time.

[Example 4] Here, the protein obtained in Example 1 or 3 is an extract that has a direct metabolic activation effect on cells, which is the objective of the present invention, and it can be used as it is as a conventional placenta extract. It can be used in known fields of use (medicine, cosmetics, etc.), but if necessary, it can be further purified by fractionation using a combination of gel filtration and ion chromatography.

As carriers used for gel a filtration, Sephadex,
Examples include agarose and Sephacryl, and using Sephadex as an example, water-soluble proteins obtained from human placenta are first fractionated. [1] Resin: Sephadex G-200 Column size: 30X sso
mm Solvent: O, 01M phosphate buffer (0,14M
Contains NaCl) pH 7.4 Flow rate 4 cm/l+r The active fraction obtained in [1] above is further fractionated using an ion exchange resin. Supports used for ion exchange include Sephadex, Sepharose,
Examples include cationic and anionic exchangers such as Sephacryl, and DEAE-Sephadenx is used as an example for fractionation.

[2] Resin: DEAE-Sephadex A-25 Column size: 20X 2001ffQ Solvent: O, LM-0
, 05N) Liss hydrochloric acid buff (pH 7,4) Elution conditions: NaCl concentration 0 → 0.5M The fraction obtained in [2] above that flows out at NaCl concentration 0.1 → Q, 15M was transferred to Sephadex G- 200, etc., and the resulting active fraction is collected to obtain a water-soluble protein with a molecular weight of 92,000. Furthermore, in this purification method, the hepatitis virus was not inactivated in the one obtained in Example 1, so
The water-soluble protein with a molecular weight of 92,000 obtained here is placed in a 60% ammonium sulfate saturated solution to precipitate it, and the precipitate is heated to inactivate the hepatitis virus. The desired ability to activate cell metabolism is stably maintained.

On the other hand, the fractionation of water-soluble protein obtained from bovine placenta obtained in Example 1 or 3 was carried out under the following conditions in accordance with the method for water-soluble protein obtained from human placenta. So, I worked on refining it one by one.

(1) IM visit: Sephadex G-200 Column size: 31x370mm Solvent: 0.01M phosphate buffer (contains 0.14M NaCl) pH 7.4 Flow rate: 2cm
/hr [2] Resin: DEAE-Sephadex A-50 Column size: 20x200nn Solvent: 0.1M-0.05N Tris-HCl buffer (pH 8.0) Obtained by [2] above, NaCl concentration 0.05→
The two fractions flowing out at 0, IM and 0.2-0.25M were further gel-filtered using Sephadex G-200, etc., and the active fractions obtained thereby were fractionated, with molecular weights of 80,000 and 25,000. Get 1,000 soluble proteins.

In addition, regarding the product extracted and purified from this cow's placenta,
Viruses can be inactivated by placing the virus in a 60% ammonium sulfate saturated solution to precipitate it and heating the precipitate in the same manner as described above. In addition, in the treated product, the desired ability to activate cell metabolism is stably maintained without being deactivated.

The yield was about 100 to 200 square meters from 1 kg of human placenta, and about 150 to 300 square meters from 1 kg of cow placenta.

[C] Effects of the Invention The present invention resides in a novel water-soluble protein obtained from human or bovine placenta. This protein has a high direct metabolic activation effect on cells.

Comparing conventionally known placenta extracts or extracts with their extraction conditions, conventionally known extracts or extracts do not contain organic solvents, strong acids, strong alkalis,
Drugs such as proteolytic enzymes have been used, but the result is that these drugs have deactivated the cell proliferation-promoting substances in the placenta. Furthermore, a feature of the conventional method is that the placenta or homogenized placenta is washed as a pretreatment for the extraction operation, but the result is that woody substances are washed away. Of course, the present invention + 1, which has been inactivated even by heat treatment, is a cell growth promoting substance present in human or bovine placenta, which is added to cells to directly and quantitatively determine their proliferation ability. This was proven by the number of In addition, placenta extract by conventional extraction method,
As a result of pursuing the cause of why the ionized substance does not show a cell proliferation effect, they discovered that the chemical used for extraction and the heat treatment process had deactivated it, and completed the present invention. I was able to do it.

[Brief explanation of drawings]

FIG. 1 is an elution curve obtained by gel filtration using Sephadex G-200 of a heat-treated precipitate precipitated with 60% ammonium sulfate. In FIG. 1, □ (solid line) indicates water-soluble protein extracted from human placenta, and ---- (dotted line) indicates water-soluble protein completely extracted from cow placenta. Figure 2 shows aldolase (molecular weight 158,000) - A in the figure, bovine serum albumin (molecular weight 67,000) - B in the figure, and α-chymotrypsinogen A (molecular weight 25,000).
) - This is a standard line based on Kav values prepared using C1 as a marker protein in the figure. FIG. 3 shows the growth curve of the cells used in the present invention. Figure 4 shows the water-soluble proteins extracted from human placenta.
This figure shows the cell proliferation promoting effect before and after heat treatment. In the figure, A is the cell growth curve of the extract before heat treatment, the cell growth curve of the extract after heat treatment, and C is the cell growth curve of the control group (physiological saline addition group). It is a curve. FIG. 5 shows the cell proliferation promoting effect of water-soluble protein extracted from bovine placenta before and after heat treatment. In the figure, A is the cell growth curve due to addition of the extract before heat treatment, C is the cell growth curve of the extract after heat treatment due to addition, and C is the cell growth curve of the control group (physiological saline addition group). be. Figure 6 shows the water-soluble proteins extracted from human placenta.
It is an elution curve by DEAE Sephadex A-25. Figure 7 shows the D of water-soluble protein extracted from cow placenta.
It is an elution curve by EAE Cefazo7x A-50. Figures 8 and 9 show two active fractions of cefazo7x G-2, fractionated with DEAE Sephadex A-50, of water-soluble proteins extracted from bovine placenta.
Figure 10 is a microscopic diagram of the state of macrophages when water-soluble proteins extracted from human placenta were added to mouse peritoneal macrophages. In the figure, a shows a product to which bovine serum has been added, and a shows a product to which water-soluble protein extracted from human placenta according to the present invention has been added.
mg/m, 30 μg/m1. b shows that the cell spread is more active than in a.

Claims (3)

[Claims] (1) When the water-soluble protein extracted from placenta is extracted from human placenta after normal delivery, its estimated molecular weight is 92,000, and its isoelectric point is 5.5 to 6.0. If the water-soluble protein is extracted from the placenta of a cow after a normal birth, the estimated molecular weight is 80,000, and the isoelectric point is 6.5-7. 0, an estimated molecular weight of 25,000, and an isoelectric point of around 5.0 to 5.5. (2) The starting material is a human placenta after normal delivery or a cow placenta after normal delivery, which is cut into pieces and homogenized at low temperature, followed by a low ionic concentration phosphate buffer containing sodium chloride. is added and left to stand for 6 hours to overnight, then centrifugation is performed to collect the resulting supernatant, and then the supernatant is compared to the supernatant if the starting material is human placenta after normal delivery. , 40%
Ammonium sulfate is added to saturate the concentration, while when the starting material is placenta after normal calving of a cow, 2.
Add ammonium sulfate to a concentration of 0% saturation, collect the supernatant, and add 60% to the supernatant.
Estimation of water-soluble protein derived from placenta after normal human delivery, purified by adding ammonium sulfate to saturating concentration and purifying the resulting precipitate by a combination of gel filtration and ion exchange chromatography. The molecular weight is 92,000 and the isoelectric point is around 5.5 to 6.0, or the estimated molecular weight of a water-soluble protein derived from the placenta after normal calving of a cow is 80,000 and the isoelectric point. 6.5 to 7.0, an estimated molecular weight of 25,000, and an isoelectric point of about 5.0 to 5.5. Method of manufacturing activated substances. (3) The starting material is human placenta after normal delivery or cow placenta after normal delivery, which is cut into small pieces and homogenized at low temperature. After adding the equilibration solution and leaving it for 6 hours to 1 day and night, collect the supernatant obtained by centrifugation, and then
When the starting material is human placenta after normal delivery, 40
Ammonium sulfate is added to a concentration of % saturation, while when the starting material is placenta after normal calving of a cow,
Add ammonium sulfate to a concentration of 0% saturation,
The supernatant liquid is separated, and ammonium sulfate is added to the supernatant liquid to a concentration of 60% saturation, and the precipitate is heat-treated, or the precipitate is further gelled. Water-soluble protein purified by a combination of filtration and ion exchange chromatography is added to a solution of ammonium sulfate at a concentration of 60% saturation to precipitate it, and the precipitate is heat-treated.
A virus inactivation method according to claim 1, in which the activity of a cell metabolism activating substance is stable.