JPS6118722A - Stimulator for defensive ability of living body - Google Patents

Abstract

PURPOSE:The titled preparation that contains, as an active ingredient, polysaccharides isolated from ginseng. CONSTITUTION:Polysaccharides, preferably containing at least one monosaccharide selected from arabinose, galactose, and glucose and having more than 100,000mol.wt., which have been isolated from ginseng, are used to give the objective preparation stimulating biological defensive ability such as functional stimulation of reticuloendothelial system, resistance to microbism and antitumor ability. The preparation is in the form of dust, granules, tablets, capsules, syrup or injection solution and is given orally or parenterally. The dose is 0.1-1,000 mg/day/man based on the polysaccharide in oral administration once a day or in portions. The polysaccharide is extracted from genseng with water and the extract is precipitated by addition of an organic solvent. Then, the precipitate is subjected to chromatography with an ion-exchange resin or gel filtration agent.

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION The present invention relates to a biodefense enhancer containing a polysaccharide fraction isolated from medicinal ginseng as an active ingredient.

Among medicinal ginseng, Panax ginseng (Pan ginseng)
Ax ginseng C, A, MEYER) is also called Korean Ninojin, and it is well known that it has been used since ancient times as a tonic, tonic, anti-inflammatory, diuretic, and antidiabetic drug. Panax notoglnseng (Panax notoglnseng F, H, C) is also a type of medicinal ginseng.
The dried tuberous root of HEN) is called Tashichi, and Tashichi has been used since ancient times as a hemostatic, antitumor, analgesic, and anti-inflammatory agent, and in recent years has been served as a tonic and tonic, especially in China.

In addition, reports on the pharmacological effects of ingredients in medicinal ginseng have mainly been made on the ingredients of ginseng, such as anti-diabetic activity C JP-A-5! ;-1, 2.272/2004, etc.), and antitumor effects (Japanese Patent Application Laid-open No. 01, 2, etc.). However, there are almost no reports on glycoprotein components or polysaccharide components, and the current situation is that there is still room for further research.

By the way, as people grow older, their ability to resist various diseases generally decreases, making them more susceptible to yuzu diseases. The cause is a decline in various internal organs and metabolic processes, as well as a decline in the function of phagocytes, which process foreign substances and waste.
It is thought that the body's defense ability declines.

As a result of repeated research on the components of medicinal ginseng, the present inventor discovered that the polysaccharide fraction in medicinal ginseng has a biological defense ability, thereby completing the present invention.

SUMMARY OF THE INVENTION The present invention aims to provide a novel biological defense ability enhancing agent, and is based on the unexpected discovery even for those skilled in the art that the polysaccharide component in medicinal ginseng has this effect.

Therefore, the biological defense ability enhancer according to the present invention is characterized in that the active ingredient is a polysaccharide component of isolated medicinal ginseng.

Needless to say, the provision of the biological defense enhancer according to the present invention will make a significant contribution to measures against various diseases.

DETAILED DESCRIPTION OF THE INVENTION Medicinal ginseng The medicinal ginseng as used in the present invention refers to Aralliaceae (Arallaceae).
aceae), refers to plants belonging to the genus Panax, including Panax ginseng, Panax ginseng, and American ginseng (Panax qulnquafollum).
LINNE), Chikusetsu=y Schiff (Panax j
Examples include aponlcua C, A, MEYER). The root part is particularly favorable for obtaining the polysaccharide component, and in terms of obtaining efficiency, it is convenient to use the dry part. It is also possible to subject these plants of the genus Panacus to tissue culture by a conventional method and use the resulting culture.

Polysaccharide component and method for obtaining the same The polysaccharide component used in the present invention is a polysaccharide itself or substantially composed of a polysaccharide component having a nonuniform molecular weight.

Therefore, any type of constituent sugar residue, composition ratio, average molecular weight, structure, etc. can be used as long as it is a polysaccharide derived from medicinal ginseng, but preferably arabinose (
arablnoae), galactose
ose ) and glucose (gluaoa@)
A polysaccharide component having a molecular weight of 10,000 or more and containing at least seven types of sugars selected from the following as unit components is preferable.

Note that the polysaccharide used in the present invention follows the usual concept, that is, it has a nonuniform molecular weight and an average degree of polymerization of 1.
Refers to linear or branched macromolecules exceeding 000.

In order to obtain the polysaccharide component used in the present invention, any method capable of isolating the component from carrots may be used. That is, medicinal carrots are preferably crushed, then extracted by immersing them in water or a water-soluble organic solvent, and the organic solvent is added to obtain a precipitate. The desired product can then be obtained by subjecting the precipitate to chromatography using an ion exchange resin or gel filter. The raw material carrot may be subjected to a defatting operation, and the desired product may also be obtained by subjecting the precipitate in a casing to dialysis.

Biodefense Enhancing Agent The biodefense enhancing agent of the present invention may be composed of the polysaccharide component of medicinal ginseng itself or mixed with an appropriate excipient, binder, or diluent for formulation, such as powder, granules, or tablets. It can be administered orally or parenterally in the form of capsules, tablets, injections, etc. Further, other drugs may be mixed as necessary. The dosage may be adjusted depending on age, body weight, and symptoms, but for oral administration, it is usually administered to an adult for 7 days, at a dosage of 0.00% as a polysaccharide component. /~1ooott5t is desired and 10 A preferred example is one consisting of a polysaccharide component of medicinal ginseng and an auxiliary component in the formulation. Another preferred embodiment of the present invention is a unit dosage form for administering the above-mentioned daily dose in seven or more divided doses.

In addition, the toxicity of the polysaccharide component in the present invention is, for example, in the case of substance B in the experimental example described later, in ICR male mice (!
against). No deaths were observed with intravenous administration of 2 o o tru)/Kp, and lentinan, which is structurally similar to substance B (Kimura Ikube et al., Modern Igaku, 3 otsu, 70 do 1.t (/rito /)) and PS- (Krestin (Sankyo)), etc., are generally considered to be low in toxicity, as they are known to have low toxicity.

Enhancement of biological defense ability When foreign substances (bacteria, viruses, etc.) invade the living body, biological defense reactions such as phagocytosis by macrophages and antibody production by differentiation of lymphocytes appear to eliminate foreign invaders. A mechanism emerges. However, when the immune system is weakened and protective factors are decreased, aggressive factors (foreign substances) become dominant, making the body susceptible to various diseases such as opportunistic infections.

In the present invention, the action of enhancing biological defense function refers to the action of activating the biological defense reactions shown above to eliminate foreign substances from the living body, and has secondary antiviral, antitumor, antiparasitic, and antiviral effects. It concerns the elimination of bacterial action and other foreign antigens.

Experimental Example 1) Obtaining polysaccharide components Panax ginseng was added to 2 with water and sotol, and the extract obtained by digestion was concentrated under reduced pressure, and the resulting residue (yield/
After dissolving 1 and 1 in an appropriate amount of water, ≠ times the amount of ethanol was added to the solution, and the resulting precipitate was obtained at a yield of 73.g.

The ethanol precipitate was transferred to a column (21rga
otrrm) to obtain a fraction eluted with water (yield: 1/%).

Then, the fraction was transferred to DEAE-TOYOPEARL,
Column packed with 10M (Toyo Soda) (, z, rmmx
0.02M-NH11HCO5
By elution, substance A was obtained (yield θ, ≠j).

Substance A was further filled with preparative TSK gel taooOPWG (Toyo Soda).
n) and eluted with water to obtain a single substance B (yield: O1.2%).

λ) Properties and structure of substance B, TSK gel (for analysis) by high performance liquid chromatography (HLC)≠ooopw and 6θ00P
Gel permeability analysis using W (Toyo Soda) showed that it was single, had no ultraviolet (UV) absorption, was a white powder, and had a specific optical rotation ([α]kun0=+/j3.00). (C= o, rij,
H2O). 1. In the measurement of molecular weight by light scattering method. /NH of substance B showing ≠≠×706
Trimethylsilyl ester of 2S0I hydrolyzate (TM
Arabinose, galactose, and glucose were detected in gas chromatography (GLC) of the S) compound, and from the peak area ratio, the production ratio was o and t, respectively.
: r, o : yti, z.

/3 C-NMR (A7.,!'M output, Western: p2o)
As shown in Figure 5, in Nioki, a glycogen-like signal with α-1, glycan as the main chain and branches to α-1, Otsu (Photols Daiia et al.
100. /QJ (/ri r2)), a signal assigned to terminal α-arabinofuranose (C1:δ=/
/θ,1,C2:δ=zaλ,,z,c5:δ=77,
G, C11: δ=Zahiro, 7. C5: δ= g1, z
) etc. were recognized.

3) Carbon clearance of substance B (Carbon c
le-11ranee) Functional activity effect (1) Experimental animals ICR male mice were raised for a certain period of time, and then healthy J-week-old mice/group/θ mice were used in this experiment.

(2) Experimental method Carbon clearance method of Halpern et al.
al., J. Retjculo-endothe
1, Soc, /, 77 (/ri6≠)), the test solution at the concentration shown below was intraperitoneally administered to the above mice, and 1 hour later, a carbon suspension (Pelican Inc. (manufactured by Pelican)) was administered to the above mice. diluted with physiological saline) to r m
It was administered via the tail vein at a ratio of l/KP. 2 minutes and 7
After 0 minutes, 1.20 μm of blood was collected from the eye socket of the mouse using a micropive tube.

After hemolyzing the obtained blood by adding o, i% Na2CO3 solution rml, it was measured using a spectrophotometer (Takashuma Seisakusho).
The absorbance at 7 nm (OD6r5) was measured to determine the rate at which the carbon suspension disappeared from the blood.

(Dosage of test solution) G) Physiological saline (control) Genus J/KP (B) Substance B 10 try/Ky-+, 2 fRy
/ml (jml/に2)e→・yu0tsu/now→tgu/ml (・)←２)～
Hiroo silk/KP-+Za silk/mJ (・
) The statistical processing of the results was performed using Sta-Punt (S
student'a) was performed in the assay.

(3) Experimental results The relationship between the dose of substance B and the rate of disappearance of the carbon suspension was as shown in FIG. In addition, the value written above the bar graph in the drawing is the disappearance rate of the carbon suspension (
X10'rnin-'').

By administering substance B, enhancement of carbon clearance ability was observed in a dose-dependent manner, and particularly remarkable effects were obtained in the qomy/Kp administration group.

≠) Enhancement effect of substance A on infection resistance (1) Experimental animals ICR male mice were kept in an animal house for a certain period of time, and then 7 groups of 6 mice, which appeared to be healthy, were used in this experiment. .

(2) Experimental method A test solution with the concentration shown below was administered intraperitoneally to the above mice.
E, coil isolated from patients with urinary tract infection after hours of treatment.
0U-00 strain (r x 10Bcell/ml
) 0. / Inoculate rttl subcutaneously on the back of mice every 3 hours! It was determined whether the animal was alive or dead.

(Dosage of test solution) ←) Physiological saline (control) m/ / Kp (b)
Substance A, 2, 15'/KP-+0. ! My/ml
(rrrtl/ni9)hi") "'O"
i'/Kp-+, 21ny/d(tm//Kp)←)
-tpotq/Kp-+rmy/ml (rml/
Kp) (3) As for the experimental results, the survival rate of Substance A over time for co11-infected mice was consistent with that shown in FIG. In addition, in the graph (
Values in ) indicate the number of survivors out of 6 mice.

Administration of substance A caused a dose-dependent effect on line extension,
Of these, in the Hiroq/Kp administration group, only 7 cases died after the evaluation time, and the remaining 5 cases remained alive even after j days.
is.

Effect of Substance B on Antibody Production Ability (1) Experimental Animals After raising ICR male mice in an animal house for a certain period of time, J-week-old mice that appeared to be healthy were used in this experiment as 7 mice per group J. did.

(2) Experimental method Jerne et al.'s plaque method (Jerne et al.
,N.

K,, etal,, 5science, /IA0
．． 4t03(7Fl+3)), the above mice were sensitized by administering O3/ml of a sheep red blood cell (hereinafter referred to as 5RBC) suspension at /X 10Bcell/mA' through the tail vein, and then sensitized at 3 hours every 3 hours. A test solution at the concentration shown below was administered intraperitoneally over a period of days. The thymus of the sensitized Hiro was removed and the number of antibody-producing cells against 5RBC was measured.

(Dosage of test solution) G) Physiological saline (control) mL / ) P (B)
Substance B O,,2mj//? -+0-0gugu/rn
l (,r ml /9)hi → I,2g/
Now → Q, ≠1・l/go (!go/Kf)) substance E
1.oInf? /Kp-+2m9/ml (j ml/
Kp ) (e) 〃 ri■/ Kp→zada/m/
('') The results were statistically processed using Student's test.

(3) Experimental Results The relationship between the dose of substance B and the number of antibody-producing cells against 5RBC was as shown in the table below.

The number of antibody-producing cells against 5RBC was observed to increase inversely with the dose of substance B, especially at 0.2 m97 Kg.
In the treated group, a remarkable increase of about 3 times that in the control group was obtained.

6) In vitro macrophage activation effect of substance B (11 Experimental method) Thioglyconate medium 3- was administered into the peritoneal cavity of a BALB/C male mouse, and after 3 days, the mouse was exsanguinated, and Eagle's MEM medium was intraperitoneally administered! M was injected, and peritoneal exudate cells (hereinafter referred to as PEC) were collected. Thereafter, they were washed with phosphate buffered saline (hereinafter referred to as "PBS"), and the toi
10'Ce1ls/! in Dulbecco's modified Eagle MEM medium supplemented with tallow serum. Adjusted to rLl. Pour 0.2 ml/cell suspension into the flat-bottom tube of the well.
We dispensed 11 each and 3 hours after dispensing, 1? 7℃/!
The cells were also cultured under r% CO2 to remove floating cells. Furthermore, test solutions of substance B, washed with PBS- and adjusted to concentrations of 10, 30 and 100 μI/- in Dulbecco's modified Eagle MEM medium supplemented with 10 T tallow serum, were added at 0.
After culturing for φg hours, 20μ of the medium was dispensed into each well. The glucose content was measured using a glucose C-test kit (Wako).

In addition, statistical processing of the results is performed using Stude
nt's) was carried out using the -test, and 9 was obtained.

(2) Experimental Results The relationship between the amount of substance B added and the amount of glucose consumed by PEC was as shown in the table below.

Addition of substance B promoted glucose consumption in a dose-dependent manner, especially 5Op9/rru5,/0 (71
6! Significant promotion was observed in the /lnl addition group.

- Test *2 p < o, oi **: p (0,001 7) Antitumor effect on substance Seven groups of animals were used in this experiment.

(2) Experimental method Subcutaneously in the axillary region of the above mouse/X 10BCell/
ml Sarcoma %lIO cell suspension 0. /ml was administered and transplanted. After transplantation, the test solution with the concentration shown below was administered intraperitoneally every J hours for 10 days, while /
Every week! The M tumor coefficient (major axis (chu) x short axis (mamo)) was measured over a week. moreover,! After a week, the tumor was removed and the tumor weight was measured.

(Dosage of test solution) (a) Physiological saline (control) 3ml/? (B)
Substance A 2. ! m9/Kg-*0. ! m9/ml (
Evening ml/Ky) G"3' 10m9/Ni9
-+, 2m9/ml (tt) //
m Tv/ni9→g da/hemp (〃) (3) Experimental results The effect of substance A on reducing the antitumor coefficient on Sarcoma/10 cells is consistent with that shown in Figure 3, and! The tumor weight after a week was as shown in the table below.

In the group to which Substance A was administered to Jjm9/Kp, a decrease in the tumor index began to be observed at week λ, and almost regressed at week ≠. Furthermore, in the evening, the gold side was completely healed and regressed.

♂) Effect of substance B on inducing antitumor macrophages (1)
Experimental method After raising DBA/2 male mice in an animal house for a certain period of time,
Substance B was administered to 7,975 apparently healthy animals aged 7 to 10 weeks.
Using BS-! Test solution 10 prepared to Fm97ml
9 (PBS-only was used as a control) was administered intraperitoneally to m1/, PEC were collected 1, 3, and 3 days later, and macrophages were isolated according to the method of Kumagai et al. (Immunology Experimental Procedures, 211.73). ~24L7ri (/ri7ri)).

Isolated macrophages were treated with RP supplemented with to% tallow serum.
MI #l) medium was prepared to 10 Cs1l/-.

Pour 0.2 ml of the cell suspension into the T-well flat-bottom tube (v-).
/well 1, and after culturing for 7 hours, remove floating cells,
Washed with PBS-. Next, 10% beef fat serum added R
PMI /1. 105 Cal is using I10 medium
Target cells prepared at /m' (mastocytoma P at 1
5 cells) were aliquoted in O, Co/we IL and cultured for an hour. 3 hours before the end of culture, add μCi/3H-thymidine.
After adding W (Ill) and completing the culture, the cells were collected using a glass filter, and the 3H-thymidine/
The amount was measured using a liquid syntin-silon counter, and the multicellular damage index was calculated using the following formula.

Control (cpm): Amount of 3H-thymidine uptake when cultured with unstimulated macrophages Sample (cpm): Amount of 3H-thymidine uptake when cultured with macrophages activated with the sample (2) Experimental results Addition of substance B The changes over time in the macrophage cytotoxicity index were as shown in the table below.

Macrophages obtained 1 day after addition of substance B were found to exhibit strong cytotoxicity against mastocytoma ptit cells, and after 3 days and ! Although the activity of macrophages obtained after a day gradually decreased, a cytotoxicity index of about 30% was obtained.

Therefore, it was revealed that compound B has a strong macrophage activating effect.

(1) Direct action of substance B on cultured cells (1) Experimental method As mouse normal cells, 3T3-Swiss Alpino cell line CG, Todara, etal, J, Ce11
．． Biol, /7°2 Tata (ldit3)) and Sarco r/10 cell line (J,
Nut, Cancer In5t.

/3. /2 types of cells (l0!3)) were grown in D'MEM medium (Nissui) supplemented with FBS (GIBCO). j rul/vre
11. 5. J well flat bottom V-to (-Z Cm"
, aliquoted into Buns (5) and heated at 37°C/j% CO
2/ Cultured in the evening under humid conditions. After that, remove the medium and add substance B in advance! 16. with D'MEM medium (Nichisui) supplemented with Chi FBS (GIBCO). so and 2yo)
Test solution prepared to a concentration of tg/ml (for control,
Addition of FBS (GIBCO) D'M instead of substance B
EM medium (Nissui) added) respectively! 2 per concentration
The cells were added to each well and cultured for tg. then o, in)
The cells were treated with Trypan blue and the number of viable cells was measured (Mcllman, W.
F., J.

Immunology,, 79.4Ur (/9'j
7) ) (2) Experimental results Addition amount of substance B and increase rate of cells after culture for a long time (chi)
The relationship was as shown in Figure μ).

JTJ-Swiss Alpino cell line and sarcoma ir
Regarding cell lines, substance B had no effect on cell proliferation at any concentration.

Therefore, it was confirmed that there was no direct effect on cultured cells.

to) Evaluation of experimental results From the above experimental results, the polysaccharide component isolated from medicinal ginseng is effective as a nonalcoholic agent in terms of biodefense ability to promote reticuloendothelial system function, bacterial infection resistance, and antitumor ability. It can be said that there is.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the effect of substance B on enhancing carbon clearance ability. FIG. 1 is a graph showing the effect of substance A on enhancing resistance to E. coli infection. FIG. 3 is a graph showing the antitumor effect of substance A on sarcoma iro cells. Figure ≠ is a graph showing the direct effect of substance B on cultured cells. FIG. 5 is a reproduction of the 13C-NMR spectrum of substance B.

Claims (1)

[Scope of Claims] 1. A biological defense enhancer containing an isolated polysaccharide component of medicinal ginseng as an active ingredient. 2. Medicinal ginseng is Panax notog
inseng F. H. The biological defense ability enhancer according to claim 1, which is CHEN). 3. The biodefense enhancer according to claim 1 or 2, wherein the polysaccharide component contains at least one type of sugar selected from arabinose, galactose, and glucose as a unit component. 4. The biological defense ability enhancer according to claims 1 to 3, wherein the polysaccharide component has an average molecular weight of 100,000 or more. 5. The biological defense ability enhancing agent according to claims 1 to 4, wherein the biological defense ability is an ability to promote reticuloendothelial system function. 6. The biological defense ability enhancer according to claims 1 to 4, wherein the biological defense ability is bacterial infection resistance. 7. Claim 1, wherein the biological defense ability is antitumor ability
The biological defense ability enhancer according to item 4.