JPH0259840B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is a novel dental fluid transport promoting substance obtained from bovine salivary glands, particularly parotid glands, and a method for producing the same. When a rabbit hypothalamus extract is administered to parotidectomized rats, fluid transport from the pulp of the tooth to the dentin and odontogenic cell tubules is no longer observed; It has already been shown that body fluid transport is observed when administered with substances. From this, it is thought that hypothalamic factors act directly on the parotid gland, and that promotion of fluid transport from the pulp of the tooth to the odontogenic cell tubules of the tooth depends on the parotid gland. Steinman et al. conducted research on caries-inducing substances and the development of dental caries in rats, and isolated a substance that promotes tooth fluid transport from pig parotid gland. [Endocrinology, 83 , 807]
(1968); Endocrinology, 106 , 1994 (1980)]. Furthermore, Steinman et al. reported that administration of this active substance to rats improves the supply of nutrients to the teeth, promotes tooth development and strengthening of tooth structure, and suppresses the incidence of dental caries. The present inventors also found that the salivary glands of cattle, especially the parotid gland,
As in the case of pigs, based on the expectation that there would be a substance that promotes tooth fluid transport (hereinafter referred to as a "dental fluid transport promoting substance"), this substance was introduced into the parotid gland of a cow. As a result of conducting research to determine whether the substance exists, the existence of the substance was confirmed.As a result of further research on the separation and purification of the substance, a method was discovered that could isolate an extremely pure tooth fluid transport promoting substance. This led to the elucidation of its physical and chemical properties. The tooth body fluid transport promoting substance provided by the present invention is a protein having the following physicochemical properties. (1) Properties: White powder (2) Molecular weight: Within the range of 25,000 to 35,000 as measured by disk electrophoresis on SDS-polyacrylamide gel. (3) Isoelectric point: PH is within the range of 5.8 to 6.6. (4) Rf value: 0.26~ as measured by electrophoresis on 10% polyacrylamide gel
It is within the range of 0.34. (5) Ultraviolet absorption spectrum: Maximum absorption wavelength (λmax) =
Approximately 280 nm Measurement conditions: 0.80 mg of sample dissolved in 1 ml of physiological saline, optical path length 1
cm, measured using a Hitachi EPS-3T spectrophotometer. (6) Absorbance: E ¹ % _280on is within the range of 12 to 17. Measurement conditions: Same as the measurement conditions for ultraviolet absorption spectrum. (7) Solubility: Soluble in water (physiological saline) Insoluble in acetone. (8) Color reaction: Ninhydrin reaction Positive Biuretz reaction Positive Miron reaction Positive Sakaguchi reaction Positive Moritshu reaction Positive Anthrone reaction Positive Elson-Morgan reaction Positive (9) Partition coefficient (Kav value) Between the gel layer and liquid layer in gel filtration The distribution coefficient is calculated using the following formula. Kav＝Ve－Vo／Vt－Vo Vt＝Total volume of gel bed Ve＝Eluent volume Vo＝Volume of solvent outside gel particles Sephadex G-100 as gel filtration material
(manufactured by Pharmacia Fine Chemicals) and a 0.05M phosphate buffer with a pH of 7.2 is used as the eluent, the Kav value of the protein of the present invention is about 0.40. As mentioned above, the tooth fluid transport promoting substance extracted from pig parotid gland by Steinman et al. has a molecular weight of 8100, an isoelectric point of PH7.5, and an ultraviolet absorption spectrum as measured by disc electrophoresis on SDS-polyacrylamide gel. Although the substance has no maximum absorption wavelength (λmax) and therefore has an absorbance of 0 at E ¹ % _280on , the physical and chemical properties of the substance of the present invention indicate that at least the molecular weight and ultraviolet absorption characteristics are similar to those of pig parotid glands. It is clearly different from the tooth body fluid transport-promoting substance extracted from , and is considered to be a new substance that has not been described in conventional literature. The salivary glands of mammals, especially the parotid gland, contain salivary gland hormones, which are proteins and have effects such as promoting hard tissue growth, activating mesenchymal tissue, lowering serum calcium, and increasing white blood cells. It has various excellent physiological activities and is widely used as a medicine. However, the present inventors have confirmed that this salivary gland hormone has no effect on body fluid transport to rat dentin odontogenic cell tubules. Therefore, it is clear that the dental fluid transport promoting substance present in the bovine parotid gland is a substance different from the salivary gland hormone in terms of its action. That is, the tooth fluid transport promoting activity of the substances of the present invention and salivary gland hormones was measured by the following method: Acriflavine hydrochloride as a fluorescent substance was intraperitoneally administered to 5-week-old rats at a rate of 5 mg per 100 g of body weight. Immediately after administration, the substance of the present invention or salivary gland hormone was intravenously administered in a volume of 0.1 ml, and the animals were decapitated 16 minutes later. Within 1 minute after decapitation, the mandible was extracted and frozen. A molar cut perpendicular to the occlusal plane was prepared using a frozen microtome, and the migration of fluorescent substances to the dentinal odontogenic cell tubules was observed under a fluorescence microscope. As a result, a case where the transfer was sufficiently carried out is judged as positive (+~), and a case where the transfer was insufficient or not carried out at all is judged as negative (-). Table 1 below shows the results of a comparison of the effects of the tooth body fluid transport promoting substance of the present invention extracted from bovine parotid gland and salivary gland hormones on promoting body fluid transport to the teeth.

[Table] The tooth body fluid transport promoting substance of the present invention is produced by using bovine salivary glands, such as parotid glands, as a raw material and following the steps described below: (a) Aqueous extract of bovine salivary glands is made acidic to a pH of 4.5 to 5.5. (b) passing the resulting supernatant through a molecular sieve until the molecular weight is 5000.
50,000, and (c) subjecting the fraction to isoelectric point fractionation using an ion exchanger to collect a fraction with an isoelectric point of PH5.8 to 6.6. It can be manufactured through Water extraction of the bodies of bovine salivary glands can be carried out by methods known per se. For example, a fresh gland body taken from a cow's salivary gland is finely chopped, about 4 times the volume of water is added, and the pH is neutralized (PH6.5 ~ 7.5), preferably after adjusting the pH to 7.0, perform extraction while stirring. Stirring is generally carried out under cooling, preferably between 0 and
It is advantageous to carry out the reaction at 5° C. for several hours, usually 2 to 3 hours, with the addition of an appropriate preservative (for example toluene). Once the extraction is complete, pass the mixture through a sieve (about 10 to 20 meshes) to separate the aqueous extract. The residue remaining on the sieve can be discarded as is, or if necessary, the same extraction operation as described above may be repeated for the residue a desired number of times (usually one or two more times). When the pH of the aqueous extract from which the residue has been removed is adjusted to 4.5 to 5.5, preferably 5.0, with an inorganic acid such as hydrochloric acid, a precipitate is formed, which is then cooled (preferably in a refrigerator; kept at about 0 to 5°C). It is desirable to allow the mixture to stand for several hours to one day to further complete the precipitation. Centrifuge the precipitate (e.g. 8000 rpm for 20 minutes)
Then, separate and collect the supernatant. The collected supernatant liquid is subjected to molecular sieve chromatography using gel particles or molecular sieving operation using a flat membrane or hollow fiber membrane such as an ultrafiltration membrane or an ultrafiltration fiber. Molecular sieve operation by molecular sieve chromatography is used by filling a column with gel particles such as dextran and polyacrylamide. As the dextran gel used, for example,
Sephadex G-50, G-75 and G-100
(manufactured by Pharmacia Fine Chemicals), and polyacrylamide gels such as Bio-Gel P-
30, P-60 and P-100 (Bio-Rad
Laboratories, Inc.) can be advantageously used. There are no particular restrictions on the particle size of these gel particles, but usually
A thickness of 40 to 120μ is suitable. Further, as a buffer solution that can be used for development, it is preferable to use a buffer solution in which gel particles are equilibrated, such as a 0.1M acetate buffer solution (PH5.0). The operation can be performed by a method known per se, and the desired fraction can be easily obtained. In addition, when using an ultrafiltration membrane or ultrafilter, an upper limit ultrafiltration membrane or an ultrafiltration fiber with a molecular weight cut off of 30,000 to 100,000, preferably about 50,000 and a molecular weight cutoff of 1000 to 10,000, preferably 5,000 This is carried out by ultrafiltration using a lower limit ultrafiltration membrane or a lower limit ultrafiltration fiber. Examples of ultrafiltration membranes include:
Diaflo XM-50 (retention limit 50000) and YM-5
(manufactured by Amicon 5000), for example, Hollow Fiber H1P50 (retention limit
50000) and H1P5 (5000, manufactured by Amicon), etc. can be used. When using an ultrafiltration membrane for separation, put the supernatant obtained above into a 202 type stirred cell (manufactured by Amicon) loaded with XM-50, pressurize with 2 kg/cm ² of nitrogen gas, and filtrate. do. This filtrate is placed in the same cell as described above loaded with YM-5 and concentrated while pressurized with nitrogen gas to obtain a fraction with a molecular weight cut-off of about 5,000 to 50,000. When using an ultrafiltration fiber, load the supernatant obtained above into a DC4 type ultrafiltration device.
By circulating the hollow fiber at high speed,
Fractions similar to those obtained using an ultrafiltration membrane can be obtained. In this way, an active ingredient-containing liquid having a molecular weight cut-off of 5,000 to 50,000 can be obtained. The molecular weight fraction obtained in this way is 5000~
50,000 active ingredient-containing liquid can be subjected to isoelectric point fractionation using an ion exchanger, but in order to purify the active ingredient-containing liquid, it is freeze-dried and then extracted with water, or the concentrated liquid is An operation can be carried out in which ammonium sulfate is added to produce a precipitate at a saturation of 70 to 80%, the precipitate is dissolved in water, and then desalted by dialysis. The desalting operation can be easily carried out by a method known per se, for example, by filling the solution in a cellophane tube and using distilled water as the external dialysis fluid. The concentrated solution is further purified, if necessary, by isoelectric point fractionation by electrophoresis, chromatography using a hydrophobic adsorbent, an anion exchanger, or porous glass, either alone or in combination of two or more. be able to. The method of isoelectric focusing by electrophoresis is carried out by a method known per se, that is, by applying an electric current to form a stable PH gradient in a free solution or a suitable gel, and applying the present invention to the PH gradient. In this method, the active substance is migrated to a PH layer that is the same as its isoelectric point, the target substance is concentrated in the PH layer, and then extracted from the concentrated fraction. As a free solution or gel, a density gradient solution of sucrose or a polyacrylamide gel can be used, and the target substance can be
48 for the free solution to concentrate in the PH layer.
In the case of gel, it is necessary to apply electricity for about 6 hours. The object of the present application can be obtained from within the layer with a pH of 5.8 to 6.6. A method using a hydrophobic adsorbent includes, for example, octyl-cellulose CL-6B or phenyl-cephalose CL-6B (Pharmacia
Fine Chemicals) etc. are packed in the column.
Approximately 10 to 20 inorganic salts such as ammonium sulfate and common salt
Purification can be carried out by adding the concentrated solution obtained in the above step to a solution containing %. The method using an anion exchanger is a weak alkaline solution, preferably 10-50mM Tris buffer (PH8.5-9.5).
After contacting the active substance dissolved in a buffer solution such as ion exchanger with an anion exchanger to adsorb the target substance, the above buffer solution or PH in which inorganic salts such as sodium chloride, potassium chloride, etc. are added to increase the ion concentration is acidified (PH Approximately 4~
It can be purified by elution using the acetate buffer described in 5). In the method using porous glass, the concentrated liquid containing the active substance is made acidic, preferably pH 4.5 to 6.0, and then brought into contact with porous glass, and then made alkaline, preferably pH 4.5 to 6.0, by adding an amino acid such as glycine or proline. The target substance can be eluted using a solution with a pH of 7.5 or higher. In addition to these purification steps, the following isoelectric focusing method using an anion exchanger can also be repeatedly performed. The molecular weight cutoff obtained as above is 5000~
The solution containing 50,000 active ingredients is then subjected to isoelectric point fractionation using an ion exchanger. For the isoelectric point fractionation using an ion exchanger, chromatofocusing, which separates proteins using the difference in isoelectric point, can be used. As the ion exchanger used for chromatofocusing, for example, PBE94 anion exchanger (manufactured by Pharmacia Fine Chemicals) can be used. To perform chromatofocusing, the PBE94 anion exchanger described above is equilibrated with a starting buffer. The buffer used here can be a buffer used for conventional anion exchangers, such as
Examples include 0.025M Tris buffer (PH8.3). Fill the column with the equilibrated anion exchanger,
Next, the molecular weight cutoff 5000 obtained by the above molecular section operation
~50,000 active substance-containing solution is slightly alkaline, preferably pH 7.5-8.5, is added to the column and eluted using the eluent. Polybuffer (a buffer containing several types of buffer ions to provide uniform buffering capacity over a wide pH range) is used as the eluent. The polybuffer used is Polybuffer 74 or Polybuffer 96 (Pharmacia
(manufactured by Fine Chemicals), etc. The pH of the polybuffer when dropping it onto the column
5.0, as the polybuffer flows through the column, a pH gradient is formed within the column, and proteins are eluted in order from those with higher isoelectric points, and the target substance of the present application is contained in the eluate with a pH of 7.0 to 7.4. It is.
The eluate thus obtained can be subjected to the above-mentioned ultrafiltration membrane, ultrafiltration fiber, or dialysis, desalted, and then freeze-dried to obtain the desired tooth fluid transport promoting substance. . The tooth fluid transport promoting substance thus obtained is useful for the prevention and treatment of dental caries. That is, in dental caries, lactic acid, which is also a metabolite of microorganisms, remains in dental plaque caused by the action of microorganisms, and acid corrosion progresses, and caries progresses not only to the enamel layer but also to the dentin. There are many factors involved in the development of dental caries, but even when the dentin is finally caried, the tooth has the ability to protect itself by forming secondary dentin. At this time, the supply of nutrients is essential for the functional growth of dentin, and it is thought that substances that enhance tooth body fluid transport act to promote the formation of secondary dentin. In order to use the tooth fluid promoting substance according to the present invention as such an anti-caries agent or a therapeutic agent, it is necessary to add the tooth fluid promoter to a pharmaceutical preparation solution (distilled water for injection, physiological saline, phosphate buffer, glycine buffer, veronal buffer, etc.). An injection solution can be prepared by adding and dissolving a body fluid promoting substance. Furthermore, in order to improve moldability, adjuvants such as sodium chloride, glycine, lactose, mannitrate, sorbitol, sucrose, hydrolyzed starch, and dextran can be added to this injection solution to form a freeze-dried preparation. moreover,
Dental fluid transport substances can also be added to commonly used dentifrices and used as anti-caries and therapeutic agents. Furthermore, this substance can be expected to promote not only teeth but also the hard tissues of living organisms in general, especially ossification. Example 1 1 kg of bovine parotid gland was minced, 4 times the volume of water was added, and the mixture was stirred for 2 hours for extraction, followed by centrifugation and separation into precipitate and supernatant. Add twice as much water to the precipitate, repeat the extraction and centrifugation, combine the resulting supernatant with the previous one, and adjust the pH to 5.0 by adding 1N hydrochloric acid. The precipitate was removed by centrifugation at 8000 rpm for 20 minutes, and the supernatant was PHified with 1N sodium hydroxide.
It was set to 7.0. This solution was passed through an ultrafiltration fiber HIP50 (manufactured by Amicon) with a molecular weight cutoff of 50,000 using a DC4 type ultrafiltration device (manufactured by Amicon).
The obtained filtrate was concentrated using an ultrafiltration fiber HIP5 (manufactured by Amicon) with a molecular weight cutoff of 5000. After ammonium sulfate was dissolved in this concentrated solution to 80% saturation, it was left standing at about 4° C. overnight and centrifuged. Water was added to the resulting precipitate to extract the active ingredient. The extract was equilibrated with 0.1M acetate buffer (PH5.0) using a Sephadex G-75 column (Pharmacia
Gel manufactured by Fine Chemicals, diameter 5 cm, height 90 cm)
and eluted with the same buffer, the active fraction was collected and lyophilized to obtain the active substance. The active substance was then dissolved in 0.025M Tris buffer (PH8.3) and PBE94 equilibrated with the same buffer.
Polybuffer 74 (PH5.0: Pharmacia
Fine Chemicals), and active peaks were collected and concentrated. Purify by chromatography again using PBE94 gel column, and apply ultrafiltration membrane PM-10 with a molecular weight cutoff of 10,000.
(manufactured by Amicon) and freeze-dried to obtain a tooth fluid transport promoting substance (8.1 mg). This substance shows a single band in polyacrylamide gel disc electrophoresis, and the molecular weight measured by SDS-polyacrylamide gel disc electrophoresis is approximately
The Rf value determined by electrophoresis using 30,000, 10% polyacrylamide gel was 0.28. Furthermore, the amino acid analysis values obtained from the results of hydrolysis using 6N hydrochloric acid at 110°C for 24 hours were as shown below. Constituent amino acids (number of residues per 100 residues) Lysine 7.54, Histidine 4.27, Arginine
3.63, aspartic acid 12.50, threonine
5.17, serine 4.52, glutamic acid 9.31, proline 8.09, glycine 7.92, alanine 6.78, valine 7.48, methionine 1.05, isoleucine
1.92, leucine 10.57, tyrosine 3.10, phenylalanine 4.35, tryptophan 0.77 It showed activity in promoting body fluid transport to the teeth at 70 ng/Kgi.v. in rats. Example 2 Sephadex G- obtained in the same manner as Example 1
The lyophilized product of the 75 active fraction was added to 0.01M sodium phosphate buffer (PH6.8) containing 1.0M ammonium sulfate.
Octyl-Sepharose CL-6B column (Pharmacia Fine Chemicals gel, diameter 2.6) was prepared by equilibrating this solution with the same buffer.
cm, height 20 cm), wash with the same buffer solution and allow it to pass through. After dialyzing this non-adsorbed liquid, it was concentrated and then 2% amphoteric carrier (BioLite PH3-10, Bio
Isoelectric focusing was carried out using a 4% polyacrylamide gel containing 4% polyacrylamide gel (manufactured by Rad Laboratories).
The active part of the isoelectric focusing gel was extracted with water, desalted using ultrafiltration membrane PM-10 (manufactured by Amicon), and purified by chromatofocusing in the same manner as in Example 1. A lyophilized product (5.6 mg) was obtained.
In rats, a dose of 90 ng/Kgi.v. showed activity in promoting body fluid transport to the teeth. Example 3 Cefdex G-75 obtained in the same manner as Example 1
The lyophilized active fraction was dissolved in 0.05M Tris buffer (PH
9.0) and equilibrated the solution with the same buffer.
DE52 (diameter 5.6 cm, height 20 cm), washed with the same buffer, and eluted with 0.05 M Tris buffer (PH 9.0) containing 0.5 M NaCl. This eluate was desalted using an ultrafiltration membrane PM-10 (manufactured by Amicon), and then subjected to chromatofocusing in the same manner as in Example 1 to obtain a purified lyophilized product. The yield of active substance obtained was 6.8 mg, which was
At 70ng/Kgi.v., it showed activity in promoting body fluid transport to teeth. Example 4 Cephadex G- obtained in the same manner as Example 1
The lyophilized product of the 75 active fraction was dissolved in 0.05M acetate buffer (PH
CPG-10 dissolved in 5.0) and equilibrated with the same buffer
Column (Electronucleonics 2000Å,
(120 to 200 meshes, diameter 2.6 cm, height 20 cm), washed with the same buffer, and eluted with 0.5 M glycine solution (PH9.0). This eluate was desalted using an ultrafiltration membrane PM-10 (manufactured by Amicon) and purified by chromatofocusing in the same manner as in Example 1 to obtain the desired lyophilized product (7.5 mg). Obtained. In rats, a dose of 80 ng/Kgi.v. showed activity in promoting body fluid transport to the teeth.

Claims (1)

[Claims] 1. Obtained from bovine salivary glands, has a molecular weight of 25,000 to 35,000 and an isoelectric point of PH5.8 to 35,000, as measured by disk electrophoresis on SDS-polyacrylamide gel.
6.6, the Rf value measured by 10% polyacrylamide gel electrophoresis is 0.26 to 0.34, the maximum absorption wavelength (λmax) of the ultraviolet absorption spectrum is approximately 280 nm, and the absorbance E ¹ % _280on is 12 to 17. Substances that promote tooth fluid transport. 2 (a) A process of acidifying the aqueous extract of bovine salivary glands to pH 4.5 to 5.5 and removing the resulting precipitate, (b) Passing the resulting supernatant through a molecular sieve until the molecular weight is 5000.
50,000, and (c) subjecting the fraction to isoelectric point fractionation using an ion exchanger to collect a fraction with an isoelectric point of PH5.8 to 6.6. A method for producing a tooth fluid transport promoting substance, characterized in that: 3. The method according to claim 2, wherein the fraction having a molecular weight of 5,000 to 50,000 obtained in step (b) is subjected to salting out using ammonium sulfate, water extraction, and desalting treatment, followed by step (c). .