JPH054373B2 - - Google Patents

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention provides interleukin-2 and interferon-γ inducers (hereinafter referred to as IL-2 and interferon-γ inducers, respectively).
It is called IFN-γ. ), particularly relates to IL-2 and IFN-γ inducers made from Nanbangisel seeds and methods for producing the same. [Prior Art] Interferon-inducing agents are substances that act on human and animal cells to induce interferon. Various natural substances that have interferon-inducing effects are known, but most of them are It has strong side effects. Therefore, a substance with IFN-inducing activity was isolated from the extract of the Chinese herbal medicine Dangki treated with hot water (Japanese Patent Application Laid-open No. 53-32107), and a substance with IFN-inducing activity was similarly isolated from the Chinese herbal medicine Mulberry White Bark. Several reports have been made, such as that of separation (Japanese Unexamined Patent Publication No. 53-99313). [Problems to be Solved by the Invention] The present invention is directed to Aeglnetia Indica L;
Extracted substances from the seeds of AIL) have significant IL-2,
The object of the present invention is to provide the same and to provide a means for separating and purifying the effective fraction. [Means for solving the problem] Nanbangisel is an annual leafless green plant belonging to the Moray family, the genus Nanbangisel.
It parasitizes the roots of sugarcane and other plants, and contains countless dust-like seeds inside the fruit. The interleukin-2 and interferon-γ inducers of the present invention are characterized in that they are isolated from the seeds of Nanbangisel, and distilled water, particularly distilled water, is extracted from the seeds of Nanbangisel with phosphate buffered saline. It is characterized by the fact that it is extracted from the seeds of Nanbangisel with water-saturated butanol, and the obtained aqueous layer is treated with pronase,
The precipitate-removed liquid is collected by ultrafiltration, or extracted with water-saturated phenol from Nanbangisel seeds, the resulting aqueous layer is treated with potassium acetate and ethanol, and the precipitate-removed liquid is compared with distilled water. After dialysis and removing RNA from the dialyzed fluid,
The resulting aqueous layer was further treated with potassium acetate and ethanol, and the precipitate removed solution was dialyzed against distilled water.
This method is characterized by concentrating the dialysis fluid. [Operation] The extract of the present invention can be extracted from the seeds of Nanbangisel with distilled water, especially phosphate buffered saline, or by the method of Morrison et al. Morrison & Leive.Fractions of
Lipopolysaccharide from Escherichia coli
0111：B4 Prepared by Two-o Extraction
Procedures.J.Biol.Chem. 250 ; 2911−2919,
(1975), significant IL-2 and IFN
-It was discovered that it has the ability to induce γ.
In addition, as a method for extracting this type of high-molecular polysaccharide, a similar method is used by Westphal et al.
Erf−orschung von Lipopolysacchriden
grammnegativ−er Bakterien, Angew.Chem .
66; 407-417, 1954).
Stronger IFN− than the extracted material by the method of Morrison et al.
Shows γ-inducibility. Although the structure and composition of the polysaccharide extracted by the present invention are not yet fully understood, when extracting the seeds of Nanbangisel with water-saturated phenol using the method of Westfahl et al.
Polysaccharides with almost no protein components bound to them are obtained, whereas extraction with water-saturated n-butanol using the method of Morrison et al. extracts polysaccharides in which lipid A, which is a lipid, is bound by proteins. It seems likely that The extracted substance of the present invention is water soluble and n-butanol insoluble, as described below.
It does not contain RNA (by Mezbaum method using orcinol reaction and ultraviolet absorption), and its molecular weight is thought to be in the range of 100,000 to 200,000. [Example 1] Preparation of phosphate-buffered saline extract (hereinafter referred to as crude extract) of Nanbangisel seeds 40 mg of Nanbangisel seeds were mixed with 0.1 molar phosphate-buffered saline (pH 7) containing no calcium ions or magnesium ions. .2) Add 1 ml and grind in a mortar. 15000xg of this crushed material,
Centrifuge for 15 minutes at 4°C, and the resulting supernatant
It was sterilized by passing through a millibore filter with a diameter of 0.45 μm to obtain an extract of the present invention. [Example 2] An equal volume of water-saturated n-butanol was added to the pulverized seeds of Nanbangisel, and the mixture was stirred at 0°C for 15 minutes. This mixture was centrifuged at 35000xg for 20 minutes and the separated lower aqueous layer was collected. The butanol layer and precipitate were further extracted with butanol twice. The aqueous fraction thus obtained was further centrifuged to remove insoluble matter, pronase, a proteolytic enzyme, was added to a final concentration of 20 μg/ml, and the mixture was treated at 37° C. overnight. This pronase treatment produced a white precipitate consisting of pronase-containing proteins, and this precipitate was removed by centrifugation.
Next, an ultrafiltration membrane (Amicon, USA) with a cut-off molecular weight of 3500 and a core side of 3000 was applied at a pressure of 1.5 kg/
cm ² to obtain the extract of the invention. The yield of Nanbangisel seeds was determined by uronic acid determination.
It was 45mg per gram. Next, a method for extracting polysaccharides from Nanbangisel seeds by the method of Westfahl et al. will be described as Example 3. [Example 3] An equal volume of water-saturated phenol was added to the pulverized seeds of Nanbangisel, and the mixture was stirred at 0° C. for 30 minutes, and then centrifuged at 200×g for 10 minutes to separate the aqueous layer. Phenol mixed in the aqueous layer was extracted and removed with ether, and ether dissolved in the aqueous layer was removed by bubbling nitrogen gas. Add 2 potassium acetate to the phenolic extract thus obtained.
% (w/w), 10 times the volume of 95% ethanol was added, and the precipitate formed after standing at 4° C. overnight was dissolved in distilled water. Potassium acetate was added to this solution at a ratio of 2% as above, and then mixed with an equal volume of ethanol and allowed to stand at 0° C. for 30 minutes. Then centrifuge at 5000xg for 20 minutes, mainly
The precipitate containing RNA was discarded. Furthermore, add 6 to the supernatant.
After adding twice the volume of ethanol and allowing it to stand for 1 hour, the resulting precipitate was centrifuged at 5000xg for 20 minutes and collected.
It was dissolved in distilled water and dialyzed against distilled water. This dialysis fluid contains polysaccharides and a small amount of RNA, so in order to destroy the RNA,
~40 μg of bovine pancreatic RNA degrading enzyme (Pancreatic
RNase) was added and treated at 38°C for 1 hour. After the treatment, 2% potassium acetate and 6 times the volume of ethanol were added, and the resulting precipitate was collected by centrifugation, dissolved in distilled water, and centrifuged at 200xg. The obtained supernatant was dialyzed against distilled water, and the dialyzed solution was concentrated to obtain a specimen according to Example 3. The yield was determined to be 38 mg per 1 g of Nanbangisel seeds by uronic acid determination. Chemical analysis of the polysaccharide fractions prepared in each of the above Examples and Example 3 was performed as follows. The quantitative determination of uronic acid is carried out by the method of Bitter et al. (Bitter, T., and Ewins, R.), which uses a carbazole reaction.
Amodified carbazole reaction for uronic
acids.Biochem.J. 81 ; 43, 1961.) and
RNA is prepared by Mezbaum's method using orcinol reactions {Mezbaum, W.Color reactions of
Nucleic acid components；In The nucleic acid components
acids. (Chargaff, E., and Davidson, J.,
editors). Academic Press, New York. 1 ;
283-305, 1939.} and ultraviolet absorption as indicators. In addition, protein quantification was performed using the method of Follin et al. (Follin, O., and Clocalteau, V.
and tryptohane determination in proteins.J.
Biol. Chem. 73 ; 627-650, 1927.) and ultraviolet absorption as indicators. (IL-2 and IFN-γ activity search) Preparation of sample for search Density gradient centrifugation from heparinized human peripheral blood (density, 1.077, Bo¨yum, A. Isolation of mono-
n-uclear cells and granulocytes from
human blood.Scan.J.Clin.Invest. 21 ; 77-89,
Peripheral blood mononuclear cells (hereinafter referred to as PBMCs) were
) and using leukocyte proliferation medium (RPM1-1640) containing 10% fetal bovine serum.
A cell suspension of 10 ⁷ /ml was prepared. To 1 ml of this PBMC suspension, 1 ml of each fraction prepared in Example 1, Example 2, and Example 3 was added, and the mixture was incubated at 37°C for 24 hours in air containing 5% carbon dioxide.
Cultured for 48 hours. After centrifuging this culture supernatant at 500xg for 15 minutes, a sterilized sample was obtained by passing it through a millibore filter, and IL-2 and IFN as described below were obtained.
−γ activity search. IL-2 activity measurement method CTLL cells, whose cell proliferation is dependent on IL-2,
In a 96-well microplate, 5 x 10 ³ CTLL cells were suspended in 500 μl of growth culture medium containing 10% test sample and implanted in each hole, and incubated in a 5% carbon dioxide incubator.
Cultured at 37°C for 24 hours. Then ³ H- on each hole
50 μCi of thymidine (Amersyam, UK) was added and the culture was continued for a further 16 hours, and the amount of ³ H-thymidine taken up was measured using a liquid scintillation counter. In this case, the IL-2 titer was calculated using recombinant IL-2 (manufactured by Shionogi) at a final concentration of 5.0.
CTLL cells were treated with standard preparations prepared at concentrations of 2.5, 1.25, 0.63, and 0.31 units/ml.
It was calculated based on the amount of ^3H -thymidine incorporated. IFN-γ activity measurement method Human amnion-derived FL cells (10 ⁶ ) were implanted in a 60 mm diameter plastic Petri dish and cultured at 37°C for 2 days in a 5% carbon dioxide incubator to prepare FL monolayer cells. Next, add 0.8 ml of the minimum essential medium to 0.2 ml of the test specimen, and add 0.5 ml of the 5-fold dilution of the test specimen.
FL monolayer culture cells were treated for 12 hours at 37°C. Vesicular stomatitis virus (Vesicular Stomatitis vi-rus) was then used in this treated FL monolayer culture cell.
The black-forming ability of VSV (hereinafter referred to as VSV) was calculated by comparing it with the VSV black-forming ability in untreated FL monolayer cultured cells. IFN titer is expressed as the highest dilution of the test preparation required to reduce VSV black-forming ability by 50%. IFN typing method The typing of IFN contained in the test specimen is as follows:
Add 0.2 ml of anti-IFN-α/β antibody (National Institutes of Health (NIH) INC. USA) to 0.2 ml of the test specimen, and add anti-IFN-γ antibody (Interferon Sciences INC.) to 0.2 ml of the specimen. .US) Add 0.2 ml of minimal essential medium and incubate for 30 minutes at 37°C.
0.6 ml was added to give a total volume of 1 ml. This specimen 0.5
FL monolayer culture cells were treated with ml at 37℃ for 12 hours,
The VSV black reduction rate was measured in the same manner as above. In addition, anti-IFN-α/β antibodies or anti-IFN-γ antibodies are
100 units of IFN-α or IFN-β, or
It was prepared to have a potency to neutralize 100 units of IFN-γ and used for testing. Chromatography of Extract The extract prepared in Example 2 was subjected to column chromatography (Sephadex G-200 Pharmacia,
Sweden, column size: φ 0.9 cm
Fractions were collected. For each fraction,
Inoculate 1× ¹⁰ PBMC/ml into each hole of a 24-well plate, add 0.1 ml of each fraction, and incubate for 24 hours.
Cultured at 37°C. After that, the culture supernatant was collected and IFN
activity and IL-2 activity were measured. Next, a method for measuring the IL-2 inducing ability of the crude extract of Nanbangisel seeds prepared in Example 1 will be described. The culture supernatant prepared by culturing the crude seed extract of Nanbangisel prepared in Example 1 and PBMC for 24 hours and 48 hours was measured by the method for measuring IL-2 titer described above. Figure 1 is a quantitative curve created using standard IL-2 (recombinant IL-2), and the horizontal axis is the standard IL-2.
-2 (unit/ml), the vertical axis represents the amount of ³ H-thymidine incorporated. Count numbers indicating the incorporation of ^3H -thymidine in the culture supernatants prepared by culturing for 24 hours and 48 hours above (unit: DPM)
was measured, and IL-2 (units/ml) corresponding to the counted number was determined from FIG. [Table] Table 1 confirms that the crude extract of seeds of Nanbangisel according to the present invention has IL-2 inducing ability. Next, a method for measuring the IFN production ability of the crude extract of Nanbangisel seeds prepared in Example 1 will be described. The crude extract of Nambanghisel seeds prepared in Example 1 and PBMC were collected after culturing for 24 or 48 hours, and the obtained supernatant was diluted 10 times to prepare FL monolayer culture cells. Process VSV
Black-forming ability was measured. Further, the black reduction rate is calculated by the following formula. Table 2 shows the results of two experiments. (1 - number of blacks in sample treatment/number of blacks in untreated control) x 100 [Table] Table 2 confirms that the crude extracted sample of Nanbangisel seeds has the ability to produce IFN. be done. In particular, the 24-hour standard compared to the 48-hour standard
IFN activity is high. Next, we will show a method for typing IFN induced from a crude extract of Nanbangisel seeds. Based on a crude extract of Nanbangisel seeds
A 5-fold dilution of the culture supernatant obtained by treating PBMC for 24 hours was used in this experiment. Experimented twice,
The measurement results are shown in Table 3. [Table] The numerical values in the table indicate black numbers and indicate the average value of two plates. Values in parentheses are untreated controls.
Antibody treatment against VSV black number on FL monolayer culture cells or treatment with untreated test IFN preparation
The percentage decrease in the number of VSV blacks on FL monolayer cultured cells is shown. This experiment confirms that the crude extract of Nanbangisel seeds has the ability to induce IFN-γ. In addition, a crude extract of Nanbangisel seeds was subjected to the above chromatography, and each fraction of
IL-2 and IFN induction ability were searched, and uronic acid (OD530nm, carbazole reaction) and protein (ODD280nm) were quantified, and the results are shown in FIG. In this measurement, the IFN titer indicates the VSV black reduction rate (%) for a 5-fold diluted product. As can be seen from Figure 2, IL in each fraction
-2, IFN induction ability, uronic acid content, and protein content show the same tendency, and from this it is assumed that the crude extract of the present invention is a single substance. Next, 500 μg each of the fractions prepared in Example 2 and Example 3 from Nanbangisel seeds.
(by uronic acid determination) using PBMCs for 24
After time stimulation, the IL-2 titer was measured for the culture supernatant. PBMC is A, B, C, D, E,
Prepared from 6 people of F. The measurement results of IL-2 titer are shown in Table 4 below. The unit is unit/ml. [Table] As can be seen from Table 4, Example 2 and Example 3
All the extracts prepared by Example 2 have IL-2 inducing ability, but the extract according to Example 2 has a stronger ability to induce IL-2.
It is clear that it possesses IL-2 inducing ability. Further, Table 5 below shows the measurement results of the IFN-γ titers of the extracts prepared in Examples 2 and 3.
For the measurement, first, each polysaccharide fraction prepared according to Example 2 and Example 3 was used to incubate PBMC for 24 hours.
The culture supernatant obtained by the treatment was diluted 5 times, this diluted specimen was divided into anti-IFN-γ treated and untreated specimens, and this specimen was then used to treat the FL monolayer culture. to form a VSV black and
This is a comparison of the number of VSV blacks in FL monolayer cultured cells treated with IFN-γ or untreated. still,
The numerical value indicates the reduction rate (%). [Table] As can be seen from Table 5, both the samples extracted in Example 2 and Example 3 have stronger IFN-γ
It can be seen that it has inductive ability. Next, PBMC was extracted using the polysaccharide fraction prepared in Example 2.
The culture supernatant obtained by stimulating IL-
2, and the results of measuring the IFN titer are shown in FIG. The IFN titer was measured by the black reduction rate of VSV using a 5-fold dilution of the PBMC supernatant.
The closed circles indicate the change in IL-2 activity, and the open triangles indicate the change in IFN titer. As can be seen from FIG. 3, both IL-2 and IFN titers increase depending on the amount of the polysaccharide of the present invention. Next, the results of a toxicity test on the polysaccharide prepared in Example 2 of the present invention will be explained. The polysaccharide prepared in Example 2 was injected into the peritoneal cavity of 10 Balb-C mice at 1 mg and 3 mg of 500 μg of uronic acid, but no abnormality was observed even after 28 days. [Effects of the Invention] The present invention provides interleukin-2 and interferon-γ inducer isolated from seeds of Nanbangisel, and also provides interleukin-2 and interferon-γ inducer isolated from seeds of Nanbangisel. Alternatively, the seeds of Nanbangisel are extracted with water-saturated butanol, the resulting aqueous layer is treated with pronase, and the precipitate removed liquid is collected by ultrafiltration, or the seeds of Nanbangisel are extracted with water-saturated phenol. The resulting aqueous layer was treated with potassium acetate and ethanol, and the precipitate removed solution was dialyzed against distilled water.
After removing RNA, the resulting aqueous layer is further treated with potassium acetate and ethanol, the precipitate removed solution is dialyzed against distilled water, and the dialyzed solution is concentrated to produce IL-2 and IFN- The γ-inducing agent has significant IL-2 and IFN-γ inducing activity, and is expected to be useful for the prevention and treatment of various viral infections in humans and animals.

[Brief explanation of the drawing]

Figure 1 shows a quantitative curve created using standard IL-2 (recombinant IL-2), and Figure 2 shows a crude extract of Nanbangisel seeds that was subjected to chromatography, and IL-2 in each fraction of the sample was subjected to chromatography. -2, and IFN
Search results for inducibility and uronic acid (OD530nm,
Figure 3 shows the results of quantification of protein (ODD280nm) (carbazole reaction) and protein (ODD280nm). Figure 3 shows the results of IL-2 and IFN It is a figure showing the result of measuring titer.

Claims (1)

[Scope of Claims] 1. Interleukin-2 and interferon-γ inducer isolated from seeds of Nanbangisel. 2. Interleukin-2, which is extracted from Nanbangisel seeds with distilled water;
and a method for producing an interferon-γ inducer. 3. The method for producing interleukin-2 and interferon-γ inducers according to claim 2, wherein the distilled water is phosphate buffered saline. 4. Interleukin-2 and interferon-γ, which are extracted from Nanbangisel seeds with water-saturated butanol, the resulting aqueous layer is treated with pronase, and the precipitate removed liquid is collected by ultrafiltration. Method for producing an inducer. 5 Extracted from Nanbangisel seeds with water-saturated phenol, the resulting aqueous layer was extracted with potassium acetate,
The precipitate removed solution was dialyzed against distilled water, RNA was removed from the dialyzed solution, the resulting aqueous layer was further treated with potassium acetate and ethanol, and the precipitate removed solution was dialyzed against distilled water. interleukin-2 and interferon-γ, which are characterized by dialyzing against the dialysis fluid and concentrating the dialyzed fluid.
Method for producing an inducer.