JP3261140B2 - Composition for promoting interferon production and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a composition for promoting the production of interferon isolated from royal jelly and a method for producing the same.

[0002]

2. Description of the Related Art Interferon (hereinafter abbreviated as IFN)
Is a protein produced by stimulated animal cells such as viruses and nucleic acids. It does not act directly on the virus, but induces an antiviral state on cells that have come into contact with IFN, thereby promoting the growth of many types of viruses. Block. That is, IF
N is known to have an antiviral protein by synthesizing an antiviral protein in contacted cells, and this protein exerts an antiviral effect by inhibiting the transcription and translation steps of viral genetic information. It is thought to function as part of the mechanism.

[0003] As described above, IFN not only has the activity of inhibiting the growth of viruses, but also has the activity of inhibiting the growth of cells (particularly tumor cells) and the activity of natural killer cells (NK cells).
It is a substance that exhibits a variety of physiological activities, including an activating effect and various effects on immunocompetent cells, and is greatly involved in maintaining homeostasis in living bodies. For this reason, in the field of pharmaceuticals and foods, various attempts have been made to utilize the above-mentioned physiological activity of IFN to prevent and treat various diseases or maintain health.

[0004] It is known that there are three types of IFN, α-type, β-type and γ-type. IFN
-Α is a protein having a molecular weight of 16,000 to 26,000 and having no sugar chain, which is produced by leukocytes, established B lymphoblasts and the like. I
FN-β is a glycoprotein having a molecular weight of 22,000 to 23,000, which is produced by fibroblasts, epithelial cells, and the like. IFN-
γ is a glycoprotein with a molecular weight of 20,000-25,000 produced by T lymphocytes in the presence of macrophages.

[0005] The production of IFN from cells is triggered by the cells being given a certain stimulus,
As such an IFN production-inducing factor, in addition to viruses, various factors such as bacteria and rickettsiae, extracts thereof, polynucleotides, and double-stranded RNAs are known. For example, IFN-α is induced by Sendai virus and Newcastle disease virus, and IFN-β is synthesized double-stranded RNA, poly I: C (Po
lyI: C, a polynucleotide copolymer consisting of inosinic acid and cytidylic acid).
It is known that the production of FN-γ is induced by stimulation of a mitogen (a blastogenesis factor), a diphtheria toxin or an antigen.

On the other hand, the present inventors have found that by adding an IFN production-inducing factor and royal jelly to animal cells and culturing them, the amount of IFN production is significantly increased.
Based on this fact, an IFN production enhancing substance comprising royal jelly and a method for enhancing IFN production using the same have already been proposed (see Japanese Patent Application Laid-Open No. 2-129128).

[0007]

As described above, it is already known that royal jelly contains an IFN production promoting substance, but what kind of components contained in royal jelly can provide the above-mentioned activity? Was not yet well understood. For this reason, it has been difficult to separate the component having the IFN production promoting activity from the royal jelly to increase the activity.

Accordingly, an object of the present invention is to separate a component having a strong IFN production promoting activity from royal jelly,
An object of the present invention is to provide a composition for promoting IFN production whose activity has been enhanced and a method for producing the same.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies to achieve the above object. As a result, royal jelly contains not only an IFN production promoting factor but also an inhibitory factor, and the inhibitory factor is DEAE. Since the promoter is not adsorbed and all the promoting factors are adsorbed, the activity can be increased by separation by ion exchange chromatography using DEAE. Further, the active fraction separated by DEAE is subjected to gel filtration chromatography. When applied, three ultraviolet peak fractions are obtained, and it is found that the combination of specific peak fractions enhances IFN production promoting activity, and the present invention is completed based on these facts. Reached.

Namely, the composition for promoting IFN production of the present invention
The supernatant is obtained by suspending royal jelly in a buffer solution, sonicating, and then centrifuging, or the supernatant obtained by further dialyzing the supernatant, and ion-exchange chromatography using DEAE. A fraction having interferon production promoting activity is collected by chromatography, and this fraction is subjected to gel filtration chromatography to obtain three ultraviolet absorption peak fractions, a peak fraction A containing a glycoprotein having a molecular weight of 251 kDa as a component. A peak fraction B containing a glycoprotein having a molecular weight of 36 kDa as a component and a peak fraction C containing a neutral polysaccharide having a molecular weight of about 10,000 as a component. It is characterized by consisting of two or more peak fractions.

The method for producing a composition for promoting IFN production according to the present invention comprises suspending royal jelly in a buffer solution, sonicating the suspension, and centrifuging the supernatant, or the supernatant obtained by centrifugation. The solution obtained by further dialysis is subjected to ion exchange chromatography using DEAE to collect a fraction having an interferon production promoting activity , and this fraction is subjected to gel extraction.
Filtration chromatography, molecular weight 251 kD
a peak fraction A containing the glycoprotein a as a component and a molecular weight of 36 k
A peak fraction B containing a glycoprotein of Da as a component and a molecular weight of about 1
And a peak fraction C containing a neutral polysaccharide as a component.
At least two or more peak fractions C as essential components
The upper peak fraction is collected.

Hereinafter, the present invention will be described in more detail.

[0013] Royal jelly, which is a raw material of the composition for promoting IFN production of the present invention, is a substance secreted from the salivary glands of honeybees and is a special feed for growing queen bees. Giving this royal jelly to a female bee larva will result in a queen bee, but without it will result in a normal worker bee. The main components of royal jelly are water 50-60%, protein 18.0%, carbohydrate 15.0%, fat 5.8%,
It has 1.5% ash and thiamine as vitamin B group,
Contains riboflavin, pyridoxine, nicotinic acid, pantothenic acid, biotin, folic acid, etc. Royal jelly is commercially available from various companies as a health food and can be easily obtained.

[0014] The IFN production promoting activity from royal jelly
When separating and purifying the components , it is preferable to prepare a royal jelly extract. This extract is, for example, royal jelly suspended in a suitable buffer, sonicated under low temperature conditions, centrifuged to collect the supernatant,
It can be prepared by dialysis as needed. In addition, by performing dialysis for 12 hours or more, the IFN production inhibitory substance is removed, so that the IFN production promoting activity can be increased.

[0015] Ion exchange chromatography using DEAE can be performed by a conventional method. That is, after equilibrating the column filled with DEAE with an appropriate buffer (for example, phosphate buffer), the royal jelly extract is added to the column packed with DEAE, and the non-adsorbed fraction is eluted with the buffer. Next, the adsorbed fraction is eluted with a buffer (eluent) in which the concentration of a solute (eg, NaCl) is gradually increased, and a fraction having IFN production promoting activity is collected from the fraction.

Gel filtration chromatography has a molecular weight of 1
It can be carried out by a conventional method using a substance having a separation ability of × 10 ⁶ to 1 × 10 ⁴ (protein). For example, TSK
The active fraction separated by DEAE is added to the column packed with the gel, and an appropriate buffer (for example, phosphate buffer) is added.
And then fractionate. The ultraviolet absorbance (for example, A ₂₈₀ ) of each fraction is measured, and the peak fraction is collected.

By the above gel filtration chromatography,
Three ultraviolet absorption peak fractions, ie, a peak fraction A containing a glycoprotein having the largest molecular weight as a component, a peak fraction B containing a glycoprotein having a next largest molecular weight as a component, and a neutral polysaccharide having the smallest molecular weight Is obtained as a peak fraction C. The estimated molecular weight of each peak fraction is such that A is 251 kD
a, B is 36 k Da, C is 10,000 before and after. Then , by combining the peak fraction C with the peak fractions A and / or B, a composition having a higher IFN production promoting activity can be obtained.

The composition for promoting IFN production thus obtained can be further enhanced in its activity by performing a suitable heat treatment. In this case, the heat treatment temperature is 5
About 0 to 100 ° C. is preferable.

The composition for promoting IFN production can be dialyzed, concentrated, and dried to produce a product, if necessary. As a drying method, for example, a freeze-drying method is preferably employed.

[0020]

According to the production method of the present invention, royal jelly is suspended in a buffer solution, sonicated, and then centrifuged, or a supernatant obtained by further dialyzing the supernatant. and by subjecting the ion-exchange chromatography using DEAE, formed with IFN production promoting activity
Since the fraction is selectively adsorbed on DEAE and can be eluted with an eluent in a specific concentration range, a fraction having an increased IFN production promoting activity can be obtained. In addition,
Fraction having IFN production promoting activity by gel filtration chromatography
Three ultraviolet absorption peaks
The UV fraction can be obtained
When the components are A, B, and C in order of decreasing molecular weight,
Combining fraction C with peak fractions A and / or B
As a result, a composition having an increased IFN production promoting activity can be obtained.
Can be.

[0021] Incidentally, in a preferred embodiment of the present invention, the upper
When the active fraction separated by the above method is heat-treated, the activity can be further increased.

The composition for promoting IFN production of the present invention comprises the three ultraviolet absorption peak fractions A and B obtained by subjecting the fraction having IFN production promoting activity adsorbed to the DEAE to gel filtration chromatography. Among C, since it is composed of at least two or more peak fractions containing peak fraction C as an essential component, it is possible to provide a composition having an increased IFN production promoting activity.

The composition for promoting IFN production of the present invention can be used for the same uses as those described in JP-A-2-129128. In other words, by directly ingesting the human body as a pharmaceutical or food, the IFN production in the body is enhanced, the immunity against infection with pathogens such as viruses, tumors, etc. is increased, and the prevention or treatment effect of these diseases is expected. be able to.
In addition, by adding this composition for promoting IFN production to a culture system of animal cells, the amount of IFN production can be significantly increased, which can contribute to the industrial production of IFN. By administering IFN thus produced to humans, it is possible to enhance immunity against infections by tumors and pathogens such as viruses, and to prevent or treat these diseases. Further, this IFN can be used as an experimental sample for elucidating a virus infection mechanism, a carcinogenesis mechanism, and the like.

[0024]

[Examples] (1) Method for measuring IFN-β production promoting activity (A) Cell culture method Cells used: Human osteosarcoma cells MG-63 (manufactured by Dainippon Pharmaceutical Co., Ltd.) were placed in a MEM-5% FCS medium [MEM Medium 9
5% and FCS 5% mixed medium] for 2 weeks before use.

Reagents used: PolyI: C (Polyinosinic-polyc)
ytidylic acid) (manufactured by SIGMA) Cycloheximide (CH) (manufactured by WAKO) Actinomycin D (AMD) (manufactured by SIGMA)

Culture method: MEM-5% FCS medium containing 3 × 10 ⁵ human osteosarcoma cells MG-63 was dispensed into each well of a 24-well plate by 500 μl.
Incubate for 24 hours.

The culture supernatant was removed, and a MEM-2% FCS medium [MEM medium 98] prepared so that the final concentration of each of PolyI: C and cycloheximide was 5 μg / ml.
% And FCS 2% mixed medium] in each well of 275 μl
Dispense each. Here, the final concentration means a concentration in a state of being added to each hole, and is the same in the following description.

Further, for promoting IFN production for activity measurement
25 μl of an aqueous solution prepared so that the final concentration of the composition is 25 μg / ml is added to a predetermined well. As a control group, 25 μl of only a phosphate buffer (NaPB) is added to a predetermined well.

In this state, the cells are cultured at 37 ° C. for 4 hours in the presence of 5% CO ₂ . Further, actinomycin D is added to each well to a final concentration of 4 μg / ml. afterwards,
Incubate for an additional hour at 37 ° C. in the presence of 5% CO ₂ .

After removing the culture supernatant and washing twice with phosphate buffered saline (PBS), the cells were washed with MEM-2% FCS.
500 μl of the medium was added to each well, and the cells were cultured at 37 ° C. for further 43 hours in the presence of 5% CO ₂ .

The culture supernatant thus obtained was measured for the amount of IFN-β produced by the following measurement method. The IFN production promoting activity was higher than that of the control group by I
Judgment was made based on whether the amount of FN-β production increased.

(B) Method for measuring IFN-β titer The amount of IFN-β contained in the IFN-β-containing sample was quantified by ELISA.

Measurement reagent Solid-phase antibody: Mouse monoclonal anti-Hu-IFN-β immunogl
obulins (Yamasa Shoyu Co., Ltd., 260740) Primary antibody: Rabbit polyclonal anti-Hu-IFN-β immunog
lobulins (LEE-2, 22-0101-31) Secondary antibody: Goat anti-rabbit-immunoglobulin peroxida
Block Ac when using se conjugate (BIO-RAD 172-1019)
e (trade name, manufactured by Dainippon Pharmaceutical Co., Ltd.) was diluted 3000 times. Substrate solution: 0.3mg / ml ABTS-0.006% H 2 O 2 -0.2M citrate b
uffer Reaction stop solution: 1.5% Oxalic acid Wash solution: 0.5% Tween 20-PBS (Tween 20 is a trade name, manufactured by Wako Pure Chemical Industries, Ltd., and PBS is prepared according to Dulbecco's PBS)

Measurement method 100 μl of primary antibody was placed in a 96-well plate (immunoplate).
Dispense 1 by 1 and leave at 4 ° C overnight. After washing the plate three times with the washing solution, 250 μl of 4-fold diluted Block Ace is dispensed and left at 4 ° C. overnight.

After washing the plate with the washing solution, the above culture supernatant containing IFN-β is added in an amount of 50 μl each, and the plate is allowed to stand at 4 ° C. for 16 to 20 hours. After washing the plate three times with the washing solution, 100 μl of the primary antibody is added thereto, and the mixture is incubated at 37 ° C. for 3 hours.

After washing the plate three times with the washing solution, 100 μl of the secondary antibody is added, and the plate is incubated at 37 ° C. for 1 hour.
After washing the plate three times with the washing solution, 100 μl of the substrate solution is added, and the mixture is incubated at 37 ° C. for 30 minutes.

The reaction stop solution was added in an amount of 100 μl each, and
The OD value is measured at 5-492 nm. The amount of IFN-β was quantified in parallel with the culture supernatant.

(2) Separation and purification of a component having an activity of promoting IFN-β production in royal jelly (A) Preparation method of royal jelly extract Royal jelly was prepared at a concentration of 40 mg / ml (766.4 μg protein / ml) at 10 mM Buffer solution (NaP
B, pH 7.0) and sonicate for 1 hour with ice cooling. This suspension was centrifuged at 10,000 × g at 4 ° C. for 20 minutes, and the supernatant was used as a royal jelly extract.

[0039] The above extract was used for dialysis by I
The effect of increasing FN-β production promoting activity was examined. That is,
The extract was dialyzed 0-24 hours against 10mM phosphate buffer 500 times, the dialyzed solution was sampled every 2 hours, while measuring the A ₂₈₀ absorbance, IFN-beta by the method described above Production promotion activity was measured. External dialysis solution
Replaced at 4, 8, and 12 hours.

Table 1 shows the results. Note that IFN-β
The production promoting activity was expressed as a relative activity with the IFN-β production amount being 1.00 when a royal jelly extract without dialysis was used.

[0041]

[Table 1]

As shown in Table 1, most of the _A280 light-absorbing substance migrated to the outer dialysis solution within 8 hours. Also,
The increase in IFN-β production promoting activity by dialysis was observed only after 24 hours. Therefore, it is considered that dialysis for at least 12 hours or more is required to increase the activity by dialysis.

For the subsequent ion-exchange chromatography, the above extract was dialyzed overnight against a 100-fold volume of 10 mM phosphate buffer.

(B) Separation of a component having INF-β production promoting activity by ion exchange chromatography DEAE TOYOPERAL 650M (column size: 1.5 × 15 cm, trade name, manufactured by Tosoh Corporation) was added to 10 mM phosphate buffer (pH
After equilibration in 7.0), royal jelly extract 20m
1 was added to the column. The non-adsorbed fraction was eluted using a 10 mM phosphate buffer (pH 7.0). The adsorption fraction is
Using a linear concentration gradient of 0 to 1000 mM NaCl / 10 mM phosphate buffer (pH 7.0), an elution speed of 1 m
It eluted at 1 / min. The adsorbed fraction was dialyzed against 10 mM phosphate buffer to remove NaCl.

The eluted fractions were measured for IFN-β production promoting activity by the method described above, and fractions eluted with approximately 300 mM NaCl having activity were collected. The relative activity of this active fraction with the IFN-β production in the case of using the royal jelly extract as 1.00 was determined to be 1.3.

(C) Separation of a Component Having INF-β Production Acceleration Activity by Gel Filtration Chromatography The active fraction obtained by the above ion exchange chromatography was concentrated 5 times by freeze-drying. TSK gel G3000SW
(Column size: 7.5 × 30 cm, trade name, manufactured by Tosoh Corporation) was added with 1 ml of the above concentrated solution, and fractionated at a flow rate of 1 ml / min using 100 mM phosphate buffer.

[0047] Thus for each fraction obtained, as well as measuring the A ₂₈₀ absorbance was measured protein concentration and sugar concentration. The result is shown in FIG. In the figure, the straight line represents the _A280 absorbance,-○-represents the protein concentration, and-△-represents the sugar concentration. Also, "669
“↓”, “440 ↓”, “67 ↓” and the like indicate the position where the molecular weight marker is eluted. In this case, as the reference material,
The following were used.

[0048] Thyrogloburin: 669 k Da Ferritin: 440 k Da Bovine Serum Alubuminn: 67 k Da Ovalbumin: 43 k Da Chymotrypusinogen A: 25 k Da

The upper column of the figure shows the results of measuring the IFN-β production promoting activity of the samples obtained from each fraction. The IFN-β production promoting activity was expressed as a relative activity with the control IFN-β production amount being 1.00. Here, “nondiluted” is a result using a sample collected from each fraction, and “× 10 diluted” indicates a result using a 10-fold diluted solution of this sample. This was performed in anticipation of a decrease in the amount of IFN-β produced by the inhibitor.

FIG. 1 shows that three A ₂₈₀ absorption peak fractions A, B and C were obtained. Peak fraction A, B
Contains both sugars and proteins, but it can be seen that the lowest molecular peak fraction C is a fraction containing only sugars. The predicted molecular weight of each fraction, A is 251 k Da, B is 3
6 k Da, a C 10,000 before and after.

Further, Table 2 shows the results of measuring the IFN-β production promoting activity when these fractions were used in combination, together with the results when each fraction was used alone. In addition, IF
The N-β production promoting activity was determined by the control I
The relative activity was indicated by setting the FN-β production amount to 1.00.
Since the respective fractions were mixed in equal amounts and added in a fixed amount, the concentration of each fraction in the mixture was も し く は or 1 /
It is 3.

[0052]

[Table 2]

As shown in Table 2, when used alone, peak fractions A and B showed almost no effect, and peak fraction C showed the strongest effect. Peak fractions A and B
In the case where was combined, a higher accelerating activity was obtained than the peak fraction C alone. However, the highest accelerating activity was obtained when peak fraction A or B was added to peak fraction C, and the activity was rather reduced when all peak fractions were mixed. This result indicates that multiple components are involved in the activity of royal jelly for promoting IFN-β production. Above all, the active substance was the peak fraction C mainly composed of sugar, and it was found that the activity was increased by the coexistence of other components.

(D) Polyacrylamide gel electrophoresis experiment of each active fraction For each active fraction shown in Table 2, Stacking Gel: 4.0% gel, 0.125M Tris, pH6.8 Separating Gel: 12% gel, 0.375 SDS-polyacrylamide gel electrophoresis was performed using M Tris, pH 8.8.

The gel after electrophoresis was stained by the following method. Silver staining: Performed by a conventional method using a silver staining set for electrophoresis (manufactured by Wako Pure Chemical Industries, Ltd.). PAS staining: Soak the gel in 7.5% acetic acid and leave at room temperature for 1 hour. Gel in 4% aqueous solution of 0.2% periodic acid
Soak for 45 minutes. Immediately soak the gel in Schiff reagent (manufactured by Wako Pure Chemical Industries, Ltd.) and refrigerate for 45 minutes. Decolorize at room temperature by changing 10% acetic acid 2-3 times.

FIG. 2 shows the results of silver staining of the protein. Also,
FIG. 3 shows the results of PAS staining of the sugar. In these figures, A, B, C, AB, BC, and ABC indicate the peak fractions and mixtures thereof.

Many protein bands were detected in the peak fractions A and B, but were not detected in the peak fraction C at all. Sugar was detected only in the vicinity of the 43K band, which is the main band of protein, and it was revealed that the type of glycoprotein contained in royal jelly was limited. No sugar band was detected in the peak fraction C in which the highest activity was observed. This result indicates that the active substance in the peak fraction C is a neutral polysaccharide having no charge in a slightly alkaline buffer used for electrophoresis.

(E) Heat treatment experiment The effect on the IFN-β production promoting activity when the peak fraction C was treated at various temperatures for 30 minutes was examined. Table 3 shows the results. In the table, the relative activity is expressed by setting the IFN-β concentration of the non-added group to 1.00.

[0059]

[Table 3]

Thus, it can be seen that the treatment at a high temperature of 50 ° C. or higher increases the activity of promoting IFN-β production. Although there is some variation in the measurement results, an increase in activity of 10 times or more is observed as compared with the non-added group.

[0061]

As described above, according to the present invention,
The royal jelly is suspended in a buffer, sonicated, and the supernatant obtained by centrifugation, or the solution obtained by further dialyzing the supernatant, is subjected to ion exchange chromatography using DEAE, Further, by subjecting the composition to gel filtration chromatography, a composition having an increased IFN production promoting activity can be obtained. Therefore, this I
By ingesting the composition for promoting FN production into the human body as a medicine or food, the production of IFN in the body is enhanced, and the immunity against infections by tumors and pathogens such as viruses is increased, thereby preventing or treating these diseases. The effect can be expected. Further, by adding this composition for promoting IFN production to a culture system of animal cells,
The production amount of IFN can be significantly increased, which can contribute to industrial production of IFN.

[Brief description of the drawings]

[1] The active fractions were adsorbed to DEAE of royal jelly, is a table further indicating the A ₂₈₀ absorbance of fractions when subjected to gel filtration chromatography, and protein concentration, and sugar concentration.

FIG. 2 is a diagram showing the results of silver staining of a gel obtained by subjecting each active fraction obtained in Examples of the present invention to polyacrylamide gel electrophoresis.

FIG. 3 is a diagram showing PAS staining results of gels obtained by subjecting each active fraction obtained in Examples of the present invention to polyacrylamide gel electrophoresis.

Front page of the continuation (58) investigated the field (Int.Cl. ^7, DB name) A61K 38/21 A61K 31/715 A61K 35/46 A61P 37/02 BIOSIS (STN) MEDLINE (STN) EMBASE (STN) CAPLUS ( STN)

Claims (3)

(57) [Claims] 1. A method in which royal jelly is suspended in a buffer and subjected to sonication and then centrifuged, or a supernatant obtained by further dialysis of the supernatant is subjected to ion-exchange using DEAE. A fraction having interferon production promoting activity is collected by exchange chromatography, and this fraction is obtained by subjecting the fraction to gel filtration chromatography to obtain three ultraviolet absorption peak fractions, each of which contains a glycoprotein having a molecular weight of 251 kDa as a component. Peak fraction A with a molecular weight of 36 kDa
A peak fraction B containing a glycoprotein as a component and a molecular weight of about 10,
A composition for promoting interferon production, comprising at least two peak fractions having a peak fraction C as an essential component among peak fractions C having a neutral polysaccharide as a component. 2. A suspension obtained by suspending royal jelly in a buffer and sonicating, and then centrifuging the supernatant or a supernatant obtained by further dialysis of the supernatant. A fraction having interferon production promoting activity was collected by exchange chromatography, and this fraction was collected.
Was subjected to gel filtration chromatography to give a molecular weight of 251.
a peak fraction A containing a kDa glycoprotein as a component and a molecular weight of 3
Peak fraction B containing a 6 kDa glycoprotein as a component, and a molecular weight
A peak fraction C containing about 10,000 neutral polysaccharide as a component;
At least two fractions containing peak fraction C as an essential component.
A method for producing a composition for promoting interferon production, comprising collecting peak fractions of at least one species . 3. The method for producing the composition for promoting interferon production according to claim 2 , wherein the fraction is heat-treated.